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TEPZZ 899 6 A_T(11) EP 2 899 262 A1

(12) EUROPEAN PATENT APPLICATIONpublished in accordance with Art. 153(4) EPC

(43) Date of publication: 29.07.2015 Bulletin 2015/31

(21) Application number: 13838905.1

(22) Date of filing: 21.03.2013

(51) Int Cl.:C12M 3/00 (2006.01) C12M 1/34 (2006.01)

C12N 5/0793 (2010.01)

(86) International application number: PCT/JP2013/057976

(87) International publication number: WO 2014/045618 (27.03.2014 Gazette 2014/13)

(84) Designated Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TRDesignated Extension States: BA ME

(30) Priority: 19.09.2012 JP 2012205561

(71) Applicant: Japan Science and Technology AgencySaitama 332-0012 (JP)

(72) Inventors: • URISU Tsuneo

Okazaki-shiAichi 444-0005 (JP)

• WANG Zhi-hongNagoya-shiAichi 466-0064 (JP)

• UNO HidetakaToyota-shiAichi 470-0335 (JP)

• SAITOH MihoNagoya-shiAichi 466-0802 (JP)

• NAGAOKA YasutakaOkazaki-shiAichi 444-0864 (JP)

(74) Representative: Cabinet Plasseraud52, rue de la Victoire75440 Paris Cedex 09 (FR)

(54) FORMATION AND USE OF NEURONAL NETWORK, AND NEURON SEEDING DEVICE

(57) Provided are a culture device for neuronal net-work formation that makes it possible to construct a neu-ronal network while restricting movement of neuronsgrown in the culture medium, and a means for the usethereof. A culture device for neuronal network formationin which is formed a cell fixation part surrounded by aplurality of projections on a flat substrate that can be filledwith cell culture medium, and in which (1) there are es-tablished spaces having widths such that the neuron cells

cannot pass through between the plurality of projections,(2) the inner diameter is such that the cell fixation partcan house the cells of from one to a number of neurons,(3) the substrate surface, which is the bottom of the cellfixation part, is coated by an extracellular matrix-formingsubstance and/or provided with minute through-holes forsuctioning culture medium by a suction device on thelower side of the substrate surface.

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Description

Technical Field

[0001] The present invention relates to the formationand use of a neuron network, and to a neuron seedingdevice. More specifically, the invention relates to a cul-turing device for formation of a neuron network, thatthrough culturing forms a neuron network via synapticjunctions between axons of neurons and dendrites of oth-er neurons, and to a method of using the device to formsuch a neuron network. In addition, the invention relatesto high throughput screening technology, planar patch-clamp technology and neuron imaging technology usingsuch a neuron network.[0002] Furthermore, the invention relates to a neuronseeding device for efficient seeding of neurons in a plu-rality of selected regions (cell plating sections) of a cul-turing device for formation of a neuron network or a planarpatch-clamp device utilizing the culturing device.[0003] According to the invention, the term "neuron"includes, firstly, various types of neurons such as centralneurons and peripheral neurons. The neurons are pref-erably in a state yet without axonal or dendritic processes.The term "neuron" also encompasses, secondly, cellscapable of differentiating into neurons, such as iPS cellsand ES cells, and more preferably neural stem cells thatare en route to differentiation to neurons from iPS cellsor ES cells. In addition, the term "neuron" includes, third-ly, cells having a property of forming an intracellular net-work, and cells capable of differentiating into cells havinga property of forming an intracellular network.[0004] Also, the term "neuronal cell body" refers to thecell body section excluding the processes such as theaxons and dendrites of the neuron.[0005] Furthermore, the terms "selected region" and"cell plating section" refer to the region on a plate in whichthere are set the neurons which are to be the center ofneuron network formation, or the target of current/voltageapplication for ion channel current measurement or var-ious types of imaging, and "selected cells" refers to theneurons in the selected region (cell plating section).

Background Art

[0006] For the purpose of research or for practical use,the prior art has proposed keeping neurons in culturemedium (particularly liquid medium) to construct neuronnetworks in vitro, with the neurons in a live state.[0007] In NPL 1, for example, there is described for-mation of a region surrounded by a plurality of protrusionson a Si plate bearing a transistor, as shown in Fig. 2,placement of a large ganglion, as an aggregate of pe-ripheral neurons, of L. stagnalis, therein and detection ofvariations in the potential of the neurons. Also, NPL 2discloses a neurochip wherein a plurality of roughly cir-cular enclosures known as "cages" (approximately 9 mmheight) such as shown in Fig. 3, are formed on a plate,

neurons are situated in the space at the center sectionof each cage, and the axons of neurons are caused toelongate toward neurons in adjacent cages, through sev-eral tunnels provided in the cage.

Citation List

Non-patent literature

[0008]

NPL 1: G. Zeck, et al., PNAS 98 (2001) 10457-10462NPL 2: J. Erickson, et al., J. Neurosci. Methods, 175(2008) 1-16

DISCLOSURE OF THE INVENTION

Problems to be Solved by the Invention

[0009] According to research by the present inventors,the following problems are encountered when formingneuron networks using mammalian neurons, for exam-ple.[0010] That is, in order to construct a neuron networkhaving a satisfactory network-like form, it is necessaryto plate the neurons in the prescribed selected region(the regions that are to constitute the node points of thenetwork). However, mammalian neurons in a live statein liquid medium are active and can potentially migratein a random fashion, requiring restrictions on their migra-tion.[0011] Nevertheless, a problem has been encounteredwhen using a structure such as shown in Fig. 1, for ex-ample, wherein circular recesses (5 mm depth) connect-ed by narrow grooves to guide elongation of axons onthe plate are provided and neurons are placed in the re-cesses, in that while this restricts migration of the neu-rons, most of the neurons situated in the recesses dieafter 2 to 3 days, making it impossible to form a satisfac-tory neuron network.[0012] This problem is explained by the presumptionthat neurons are highly sensitive to the culturing condi-tions and the conditions near the cells, and that for for-mation of a neuron network it is necessary to facilitaterecognition of adjacent neurons by each other. In theexperimental structure described above, each of the neu-rons are accommodated in a recess, and it is difficult forthe cells to recognize each other because of the leveldifference of 5 mm at the plate surface between the ad-jacent neurons. This is assumed to be the reason thatformation of a neuron network is inhibited, the death rateof neurons is increased and the synapse formation isunderdeveloped.[0013] NPL 1 places nerve tissue in a region surround-ed by a plurality of protrusions, but the object is the giantganglion of L. stagnalis that does not have active mobility.Also, the gaps between the plurality of protrusions easilyexceed 50 mm, and it is completely impossible to restrict

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ordinary neuronal migration. Furthermore, the structureis on a transistor plate with numerous irregularities havingheights exceeding 5 mm.[0014] Next, in NPL 2, the neurons in each cage aremutually surrounded by irregular structures known as"cages", each having a height of approximately 9 mm. Inaddition, while the cage is provided with tunnels havinga width of 10 mm and a height of 1 mm for axon elongation,it is difficult for neurons in the cages to mutually recognizeadjacent neurons through such narrow tunnels. There-fore, NPLs 1 and 2 do not solve the problems alluded toabove, nor do they suggest a solution means.[0015] Methods of forming neuron networks based onapplication of high throughput screening are importanttechniques in terms of how neuron seeding is accom-plished. For example, when a network is formed having100 measurement points, with 25 points of cell platingsections surrounding each of the measurement points,this requires seeding of a prescribed number of neuronalcell bodies in a short period of time, usually within 1 hour,at a total of 2500 cell plating sections.[0016] However, NPLs 1 and 2 do not disclose a neu-ron seeding system for efficient seeding of neurons atmultiple cell plating sections. Also, in a method whereina tool such as an ordinary pipette or a pipette with a met-rological function, or a microinjector, is used to seed neu-rons in individual cell plating sections by manual opera-tion, firstly, it is difficult to accomplish proper seeding infine cell plating sections and secondly, the seeding effi-ciency is extremely inferior, and therefore such a methodis non-realistic.[0017] A demand therefore exists for a device for neu-ron seeding that allows seeding of cells without damagein multiple cell plating sections in a short period of time,and in an essentially simultaneous manner. The devicemust be constructed in such a manner as to not inhibitformation of a neuron network in the planar direction ofthe apparatus plate.[0018] It is therefore a first object of the present inven-tion to provide a culturing apparatus for formation of aneuron network and a method of forming a neuron net-work, that can solve the problems mentioned above. Itis a second object of the invention to provide a planarpatch-clamp device, a high throughput screening tech-nique and a neuron imaging technique utilizing the neu-ron network.[0019] Furthermore, it is a third object of the inventionto provide a neuron seeding device for efficient seedingof neurons in a plurality of cell plating sections of a cul-turing device for formation of a neuron network or a planarpatch-clamp device utilizing the culturing device.

Means for Solving the Problems

(Construction of first invention)

[0020] The construction of the first invention designedto solve the aforementioned problems is a culturing de-

vice for formation of a neuron network, wherein cell plat-ing sections surrounded by a plurality of protrusions areformed on a flat plate that can be filled with a cell culturemedium, the cell plating section satisfying the followingconditions (1) to (3).

(1) gaps are set between the plurality of protrusionswhich defines the cell plating sections, wherein thegaps are wide but do not allow a neuronal cell bodyto pass through.(2) the inner diameter of the cell plating sections de-fined by the plurality of protrusions is of sizes capableof accommodating one to several neuronal cell bod-ies. The term "several" means 2 to 10, preferably 2to 6 and more preferably 3 to 5.(3) the plate surface forming the bottom of each cellplating section comprises at least one constituent ofthe following (a) and (b).

(a) It is coated with an extracellular matrix-form-ing substance.(b) fine through-holes for suction of medium bya an aspirator provided below the plate surfaceare provided, wherein the hole diameters beingsuch that the neurons cannot pass through.

[0021] For the first invention, the phrase "one to severalneuronal cell bodies" means at least one and no morethan 10, and preferably at least one and no more than 5neuronal cell bodies.

(Construction of second invention)

[0022] The construction of the second invention de-signed to solve the aforementioned problems is a cultur-ing device for formation of a neuron network, wherein theculturing device of the first invention corresponds to anyone of the following (1) to (3).

(1) cell plating sections are formed as selected re-gions on the plate, and one to several neurons areplaced in the cell plating sections as the selectedcells for the cell plating sections, while other neuronsare simply seeded on the plate.(2) multiple cell plating sections are formed on theplate with appropriate gaps between them, and oneto several neurons are placed in the cell plating sec-tions while one cell plating section is used as a se-lected region in which the neuron is placed.(3) a culturing device for high-throughput analysis ofa neuron network, wherein the cell plating sectionsare dispersed at appropriate locations so that severalor many units of neuron networks corresponding to(1) or (2) can be formed on the plate.

[0023] The second invention will now be explainedbased on the conceptual drawings Fig. 4(a) and Fig. 4(b).Fig. 4(a) shows an essential portion plan view of the plate

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of a culturing device according to (1) of the second in-vention, wherein the neuron 11 shown at the center (ac-tually one to several neurons) is situated in a selectedregion that is the cell plating section 13 surrounded bymultiple (6) cylindrical protrusions 12, with the surround-ing neurons 11 simply being seeded on the plate. A net-work is also formed by these neurons 11.[0024] Fig. 4(b), on the other hand, shows an essentialportion plan view of the plate of a culturing device ac-cording to (2) of the second invention, wherein multiplecell plating sections 13 are formed mutually separatedby gaps on the plate in an appropriate manner, a neuron11 (actually one or more neurons) is situated in each ofthe cell plating sections 13, and one of the cell platingsections 13 is used as the selected region. A network isalso formed by these neurons 11.[0025] In a culturing device according to (3) of the sec-ond invention, a plurality of or many neuron network unitssuch as shown in Fig. 4(a) or Fig. 4(b) are dispersed atappropriate locations on the plate, so that they are mu-tually independent network units.

(Construction of third invention)

[0026] The construction of the third invention designedto solve the aforementioned problems is a culturing de-vice for formation of a neuron network according to thefirst invention or second invention, the culturing devicefor formation of a neuron network being a planar patch-clamp device designed for a neuron network, wherein:

(1) the plate is an electrical insulating plate,wherein fine through-holes are formed so as to passthrough both sides of the plate surface which com-pose the bottom of the cell plating section of the elec-trical insulating plate, (2) liquid pool sections andelectrode sections are provided on the neuron net-work-formed side, as the first surface side of the finethrough-holes, and on the second surface side whichis the opposite side, respectively, wherein the liquidpool sections that are to hold the conducting liquidas the cell culture medium, and the electrode sec-tions are disposed to be electrically conductive tothe conducting liquid of the liquid pool section, and(3) the liquid pool sections on the first surface sideare liquid pool sections for neurons plated in the cellplating sections.

(Construction of fourth invention)

[0027] The construction of the fourth invention de-signed to solve the aforementioned problems is a cultur-ing device for formation of a neuron network, wherein ina planar patch-clamp device according to the third inven-tion, the electrode sections on the first surface side andsecond surface side comprise the following constituents(a) to (c).

(a) an electrode receptacle, wherein at least a portionof the receptacle wall that is in contact with the con-ducting liquid, when the conducting liquid is intro-duced into the liquid pool sections, is composed ofan inorganic porous material.(b) an electrode having a precious metal chloride(NmCl) layer formed on a surface layer section ofthe precious metal (Nm), and housed in the electrodereceptacle.(c) an electrode solution filled into the electrode re-ceptacle, wherein the precious metal chloride(NmCl) and an alkali metal chloride are dissolved atsaturated concentration.

(Construction of fifth invention)

[0028] The construction of the fifth invention designedto solve the aforementioned problems is a culturing de-vice for formation of a neuron network according to anyone of the first invention to fourth invention, wherein theculturing device for formation of a neuron network is usedfor a purpose according to any one of the following (A)to (C).

(A) Use for measurement and analysis of neuronalion channel current in a neuron network.(B) Use for imaging analysis, including at least Caimaging analysis, imaging analysis with synapto-physin or synapsin labeling as synaptic site markers,imaging analysis with MAP2 as a dendrite marker,and imaging analysis with FM1-43 or FM4-64 whichlabels endosomes or exosomes.(C) Use in high throughput screening systems forneuron networks.

(Construction of sixth invention)

[0029] The construction of the sixth invention designedto solve the aforementioned problems is a culturing de-vice for formation of a neuron network according to thefifth invention wherein, when the culturing device for for-mation of a neuron network is to be used for imaginganalysis according to (B), the culturing device comprisesone or more of the following constituents (D) to (F).

(D) a photodetector for detecting light emitted byneurons is set above the plate.(E) an irradiating device that irradiates light onto theneurons or plate surface is set above the plate.(F) the irradiating device of (E) is equipped with anoptical focusing system for irradiation of light only ona prescribed single neuron.

(Construction of seventh invention)

[0030] The construction of the seventh invention de-signed to solve the aforementioned problems is a methodof forming a neuron network under culturing for any de-

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sired research purpose, by means of a culturing devicefor formation of a neuron network according to any oneof the first invention to sixth invention.[0031] The method of forming a neuron network com-prises:

(1) a step of seeding neurons on said flat plate thatis filled with cell culture medium,(2) a step of placing and/or plating one or severalneurons in each cell plating section by an extracel-lular matrix-forming substance in the cell plating sec-tion, and/or by suctioning liquid medium from the finethrough-holes at the bottom of the cell plating sec-tion, and(3) a step of forming synaptic junctions between neu-rons by axons or dendrites, wherein while movementof the neurons plated in the cell plating section isrestricted by the plurality of protrusions, the presenceof mutually adjacent neurons is recognized acrossthe gap sections of the plurality of protrusions.

(Construction of eighth invention)

[0032] The construction of the eighth invention de-signed to solve the aforementioned problems is a methodof forming a neuron network under according to the sev-enth invention, wherein during seeding of the neurons instep (1), glial cells are also seeded at sections other thanthe cell plating sections.

(Construction of ninth invention)

[0033] The construction of the ninth invention designedto solve the aforementioned problems is a neuron seed-ing device for seeding of neurons in multiple cell platingsections each surrounded by a plurality of protrusions onthe apparatus plate, wherein the neuron seeding deviceis set on the flat apparatus plate that can be filled withcell culture medium in a culturing device for formation ofa neuron network or in a planar patch-clamp device uti-lizing such a culturing device,wherein a board-shaped device body that can be set onthe apparatus plate has a size covering the multiple cellplating sections and the flat bottom of the device body isin contact with the apexes of the plurality of protrusionsat the multiple cell plating sections,the device body is provided with (1) a suspension supplyport for externally supplying of a neuron suspension hav-ing neurons suspended at a fixed density, (2) a pluralityof fine suspension flow channels extending in a branchedmanner from the suspension supply port, inside the de-vice body, and (3) a suspension injection port for injectingof neuron suspension into each cell plating section,opened at the bottom of the device body at the end ofeach suspension flow channel.

(Construction of tenth invention)

[0034] The construction of the tenth invention de-signed to solve the aforementioned problems is a neuronseeding device according to the ninth invention, whereinthe plurality of suspension flow channels are set to havesubstantially the same lengths, and the design is suchthat when the neuron seeding device is placed on theapparatus plate of the culturing device for formation of aneuron network or the planar patch-clamp device, theindividual suspension injection ports are located to pre-cisely correspond with the individual cell plating sections.

(Construction of eleventh invention)

[0035] The construction of the eleventh invention de-signed to solve the aforementioned problems is a neuronseeding device according to the ninth invention or tenthinvention, wherein the board-shaped device body com-prises an upper board provided with the suspension sup-ply ports and a lower board provided with the suspensioninjection ports, joined in a closely bonded state, withgrooves composing the suspension flow channels beingformed on at least one of the bonding surfaces of theupper board and lower board.

(Construction of twelfth invention)

[0036] The construction of the twelfth invention de-signed to solve the aforementioned problems is a neuronseeding device according to any one of the ninth inven-tion to eleventh invention, wherein the culturing devicefor formation of a neuron network is a culturing devicefor formation of a neuron network according to the firstinvention, and/or the planar patch-clamp device accord-ing to any one of the ninth invention to eleventh inventionis a planar patch-clamp device according to the third in-vention.

(Construction of thirteenth invention)

[0037] The construction of the thirteenth invention de-signed to solve the aforementioned problems is a neuronseeding device according to any one of the ninth inven-tion to twelfth invention, wherein the device body furthercomprises second suspension flow channels for injectionof the neuron suspension into regions other than the cellplating sections of the apparatus plate of the culturingdevice for formation of a neuron network or the planarpatch-clamp device.

Effect of the Invention

(Effect of the first invention)

[0038] The culturing device for formation of a neuronnetwork of the first invention comprises cell plating sec-tions surrounded by a plurality of protrusions, formed on

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a flat plate, and therefore the neurons placed therein havetheir random movement restricted by the plurality of pro-trusions. Random movement of the neurons is thereforerestricted.[0039] However, the insides and outsides of the cellplating sections are on the same flat plate surface, withno level differences (irregularities) between them. Fur-thermore, wide gaps are set between the plurality of pro-trusions of the cell plating sections, though not wideenough to allow passage of the neuronal cell bodies, andthe gap sections are spaces that are open above, insteadof narrow tunnel spaces such as disclosed in NPL 2. Con-sequently, the neurons situated in the cell plating sec-tions can easily recognize adjacent neurons with whichthey are to form a network, and it is possible to create asatisfactory neuron network based on formation of syn-aptic junctions between axons and dendrites utilizing thegaps between the protrusions.[0040] In addition, since the inner diameters of the cellplating sections defined by the plurality of protrusionshave sizes allowing accommodation of one to severalneuronal cell bodies, one to several neuronal cell bodiesare situated and plated in each cell plating section. Gen-erally speaking, neurons (especially iPS cells and thelike) can survive more stably for longer periods when ina state of aggregates of several cells (clusters). On theother hand, if a single neuron is placed in the cell platingsection, signal transfer between neurons will be simpli-fied and analysis of the network function will be easier.According to the first invention, one to several neuronalcell bodies are situated and plated in each cell platingsection, thereby creating a satisfactory balance betweenboth of the aforementioned requirements.[0041] When a culturing device for formation of a neu-ron network according to the first invention is used, neu-rons can survive for long periods of 4 weeks or longer,and an active neuron network can be maintained.[0042] Furthermore, according to the first invention, anextracellular matrix exhibiting adhesive force for neuronsis coated onto the plate surface composing the bottomof the cell plating section, and/or fine through-holes forsuction of cell culture medium by an aspirator providedbelow the plate are provided with hole diameters that donot allow passage of neurons. Thus, one to several neu-ronal cell bodies are securely situated and plated in thecell plating sections that are to serve as nodes of themesh-like neuron network. With these features, the firstinvention can solve the aforementioned problems of theinvention.

(Effect of the second invention)

[0043] According to the second invention there is pro-vided a culturing device for formation of a neuron net-work, used to form a neuron network as shown in Fig.4(a) or a neuron network as shown in Fig. 4(b), and aplurality or many of such neuron network units may beformed on the plate. Thus, a culturing device for high-

throughput analysis of a neuron network is provided.

(Effect of the third invention)

[0044] A planar patch-clamp device allows multipointmeasurement by constructing a plurality of patch-clampdevices on a solid plate having electrical insulating prop-erties, such as a silicon chip, and it has fine through-holesfor measurement of ion channel current at each of thecell placement locations of each patch-clamp device. Ina planar patch-clamp device of the third invention, the"fine through-holes for suction of cell culture medium byan aspirator provided below the plate", as specified by(3)(b) of the first invention, are used as fine through-holesfor measurement of the ion channel current.[0045] Also, a substance such as adenosine 5-[β-thio]diphosphate, uridine 5-triphosphate trisodium salthydrate or EGF, which has a neuron-attracting effect,may be mixed with the cell culture medium on the secondsurface side to attract neurons into the cell culture me-dium on the second surface side, thereby guiding theneurons to the fine through-holes to more reliably keepthe neurons in the fine through-holes during the culturingperiod.[0046] Since a conventional planar patch-clamp de-vice does not have a cell culturing function, a problemhas been encountered in that it has not been applicablefor cells that require culturing, such as neurons. In otherwords, because the lifetime of cells to be measured is asshort as 1 hour or even 30 minutes or less under non-culturing conditions, the device only has limited applica-tion for innovative drug screening, and it has been difficultto apply for functional analysis of cells wherein a pipettepatch clamp is employed. In addition, it has been difficultto successfully carry and trap cells at the locations of thefine through-holes provided in the plate.[0047] According to the third invention, however, a cul-turing device for formation of a neuron network accordingto the first invention or the second invention is used asa planar patch-clamp device for a neuron network, sothat a planar patch-clamp device can be provided that isdesigned for a neuron network of neurons that requireculturing, and that results in a notably extended lifetimefor the neurons to be measured. Furthermore, by usinga culturing device for formation of a neuron network spec-ified by (3) of the second invention, it is possible to ac-complish high-throughput screening in a neuron network.

(Effect of the fourth invention)

[0048] Incidentally, the following issue arises with aplanar patch-clamp device according to the third inven-tion.[0049] Specifically, when an extracellular matrix-form-ing substance is adhered to the peripheries of the finethrough-holes for measurement of ion channel current(the cell plating sections), as according to (3)(a) of thefirst invention, a slight gap is formed between the neuro-

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nal cell membrane and the plate surface at each finethrough-hole periphery, thereby lowering the "seal resist-ance". The current flowing through this gap is added asleak current to the ion channel current, and fluctuationstherein contribute to noise. When the seal resistance islowered, therefore, noise current becomes significanteven with respect to slight fluctuations in the appliedmembrane potential, and it becomes difficult to accurate-ly measure the ion channel current.[0050] For accurate measurement of cellular ion chan-nel current it is effective and essential to counter noisecurrent not only when the seal resistance of the planarpatch-clamp device is low but also when the seal resist-ance is not low. Effective measures against noise currentinclude not only increasing the seal resistance but alsominimizing fluctuations in the applied membrane poten-tial at the electrode side.[0051] Furthermore, when the planar patch-clamp de-vice electrode used is an electrode having a preciousmetal chloride (NmCl) layer (for example, an AgCl layer)formed on the surface layer section of the precious metalNm (for example, silver Ag), fluctuations in the appliedmembrane potential are mainly due to fluctuations in theinterface potential between the surface of the AgCl/Agelectrode and the solution surrounding it, or fluctuationsin the liquid-liquid interface potential.[0052] Thus, by dipping an AgCl/Ag electrode in anelectrode solution which is a saturated solution of AgCland an alkali metal chloride (for example, KCl) (the KClconcentration being about 100-150 millimoles) in theelectrode receptacle of the electrode section, and con-tacting these with a conducting liquid such as a cell cul-ture solution (the KCl concentration being about a fewmillimoles) through the receptacle wall formed of an in-organic porous material, as according to the fourth in-vention, the interior and exterior of the electrode recep-tacle are brought to an electrically conductive state. How-ever, since the liquid itself cannot appreciably passthrough the pores of the inorganic porous material, mix-ture of the electrode solution in the electrode receptaclewith the conducting liquid outside the electrode recepta-cle is minimal enough to ignore. As a result, a constantand large difference in KCl concentration is maintainedbetween the interior and exterior of the electrode recep-tacle, the AgCl/Ag electrode interface potential and theliquid-liquid interface potential are constant, and fluctu-ations in the applied membrane potential do not occur.

(Effect of the fifth invention)

[0053] According to the fifth invention, a culturing de-vice for formation of a neuron network according to thefirst invention to fourth invention may be used for (A)measurement and analysis of the neuronal ion channelcurrent in the neuron network, (B) imaging analysis thatincludes at least Ca imaging analysis, imaging analysiswith synaptophysin or synapsin labeling as synaptic sitemarkers, imaging analysis with MAP2 as a dendrite mark-

er, and imaging analysis with FM1-43 or FM4-64 whichlabels endosomes or exosomes, or (C) a high throughputscreening system for a neuron network.

(Effect of the sixth invention)

[0054] According to the sixth invention, when a cultur-ing device for formation of a neuron network accordingto the fifth invention is used for imaging analysis accord-ing to (C), because it further comprises one or more el-ements from among (D) a photodetector, (E) an irradiat-ing device and (F) an optical focusing system, effects areobtained such as, firstly, that it is possible to accomplishanalysis without inhibiting the neuron network function,since measurement can usually be carried out in a non-contact and non-destructive manner, secondly, that theoptical measurement allows analysis at high speed, andthirdly, that it is possible to exactly excite a single neuronand precisely analyze it with the (F) optical focusing sys-tem, even when multiple neurons (cell clusters) are sit-uated in the cell plating section.

(Effect of the seventh invention)

[0055] According to the method of forming a neuronnetwork according to the seventh invention, steps (1) to(3) mentioned above are carried out using a culturingdevice for formation of a neuron network according toany one of the first invention to sixth invention.[0056] Thus, when neurons are seeded on a flat platefilled with cell culture medium, the extracellular matrix-forming substance in each cell plating section, or suctionof cell culture medium through the fine through-holes atthe bottom of the cell plating section, allows one to sev-eral neuronal cell bodies to be reliably placed and platedin the cell plating section. More specifically, cell bodiesmay be placed and plated on the fine through-holes ofthe cell plating section.[0057] In addition, since the inner diameter of each cellplating section roughly corresponds to a size allowingaccommodation of one to several neuronal cell bodies,one to several neurons are reliably situated in each cellplating section. These neurons have their random move-ment restricted by the plurality of protrusions forming thecell plating section. Furthermore, since the inside andoutside of the cell plating section are on the same flatplate surface and there is no level difference (irregulari-ties) between them, the neurons disposed on the cellplating section can easily recognize adjacent neuronsthat are to form the network, through the gaps betweenthe plurality of protrusions composing the cell plating sec-tion. Thus, the neurons can be kept in an actively livestate while forming a satisfactory neuron network basedon formation of synaptic junctions between axons anddendrites utilizing the gaps between the protrusions.[0058] Given these aspects, the seventh invention al-lows formation of a satisfactory neuron network betweenindividual neuron clusters of several neurons placed and

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plated in each cell plating section. With this method, theneurons form a stable network with approximately 100%probability, and culturing can be continued for prolongedperiods of 4 weeks and longer. It is therefore a highlyuseful technique for producing a high throughput screen-ing element for a neuron network.

(Effect of the eighth invention)

[0059] According to the eighth invention, when neu-rons are seeded by step (1) of the seventh invention, theneurons are seeded both inside the selected regions andoutside the selected regions while glial cells are seededoutside the selected regions. Thus, since glial cells arepresent near the synapse and in contact with the neurons,as known from publications such as "F.W. Pfrieger et al.,Science 277(1997) 1684-1687", this increases matura-tion of the neuron network and allows construction of aneuron network with a more homogeneous spatio-tem-poral function.

(Effect of the ninth invention)

[0060] According to the ninth invention there is provid-ed a neuron seeding device for efficient seeding of neu-rons in multiple cell plating sections of a culturing devicefor formation of a neuron network or a planar patch-clampdevice utilizing the culturing device. Thus, for formationof a neuron network based on application of high through-put screening, there is provided means for solving theimportant issue of how the neuron seeding is accom-plished.[0061] The board-shaped device body of the devicecomprises (1) a suspension supply port for external sup-ply of a neuron suspension, (2) a plurality of fine suspen-sion flow channels extending in a branched fashion fromthe suspension supply port, and (3) a suspension injec-tion port opened at the bottom of the device body at theend of each suspension flow channel. Thus, even whena large number, in the hundreds, of cell plating sectionsare provided on the apparatus plate , it is possible to sup-ply the neuron suspension to all of the cell plating sectionsin a short period of time.[0062] The method of supplying the neuron suspen-sion to the suspension supply port is not restricted, butin consideration of fluid friction resistance in the multiplefine suspension flow channels, supply of the neuron sus-pension is preferably carried out with a tool or deviceallowing injection of liquid into the suspension supply port(for example, an injector or microsyringe, or a smallpump-type injector), under a pressurized state. Since inmost cases the size of the injection port of the tool ordevice for liquid injection will be larger than the inner di-ameter of the suspension supply port, in such cases aconnecting pipe with a tapered tip end may be fitted atthe injection port of the liquid inject tool/device, allowingthe tapered tip end to be inserted into the suspensionsupply port. The tapered section of the tip end of the

connecting pipe may be provided by fitting a small stain-less steel nozzle-shaped pipe member, for example.[0063] The neurons injected into the cell plating sectionhave their flow out of the cell plating section halted bythe plurality of protrusions of the cell plating section, andtherefore stop within the cell plating section. On the otherhand, the suspension medium flows out between the plu-rality of protrusions of the cell plating section. Conse-quently, the neurons are seeded into the cell plating sec-tion in an intact state with minimal stress. Furthermore,since seeding of the neurons is accomplished all at once,seeding of the neurons in the multiple cell plating sectionson the apparatus plate is completed essentially simulta-neously, and within a very short period of time (aboutseveral tens of seconds).[0064] Also importantly, the neuron seeding device issetup in the culturing device for formation of a neuronnetwork or the planar patch-clamp device, on a flat ap-paratus plate that can be filled with cell culture medium.Thus, the construction is such that the neuron suspen-sion is injected from above the cell plating section. Con-sequently, there is no hindrance to formation of a neuronnetwork in the planar direction on the apparatus plate.[0065] Incidentally, while it is possible to anchor theneuron seeding device on the apparatus plate of the cul-turing device for formation of a neuron network or theplanar patch-clamp device, if it is set in a detachable man-ner and removed after seeding of the neurons, it will notimpede supply of oxygen or carbon dioxide gas duringculturing of the seeded neurons and will not interfere withmeasurement of the network or introduction of chemicalsolution from above.

(Effect of the tenth invention)

[0066] According to the tenth invention, the multiplesuspension flow channels are set to substantially thesame length, and therefore seeding of the neurons in themultiple cell plating sections on the apparatus plate iscompleted accurately and simultaneously. Stated differ-ently, if the liquid volume of the neuron suspension sup-plied from the suspension supply port is controlled afteradjusting the dispersion density of the neurons of theneuron suspension on the nodes, an effect is obtainedwhereby the injection rate of neuron suspension into thecell plating section (and therefore the number of neuronsseeded) can be controlled in essentially a precise man-ner, and whereby the number of neurons seeded in themultiple cell plating sections can be controlled to be es-sentially equal. These effects may be considered to bemajor effects for forming a neuron network that is pre-sumably to be used for high throughput screening.[0067] Also, since the design is such that when theneuron seeding device is placed on the apparatus plateof the culturing device for formation of a neuron networkor the planar patch-clamp device, the individual suspen-sion injection ports are located to precisely correspondwith the individual cell plating sections, seeding of neu-

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rons in the multiple cell plating sections is accomplishedin a precise manner. In this regard, when the neuronseeding device is placed on the apparatus plate of theculturing device for formation of a neuron network or theplanar patch-clamp device, the labeling marks for posi-tioning of both may be provided on either or both theapparatus plate and device body, and it is particularlyeffective when the device body is made of a highly trans-parent material since this will allow such labeling marksto be visible through it.

(Effect of the eleventh invention)

[0068] According to the eleventh invention, the board-shaped device body has an upper board provided with asuspension supply port and a lower board provided witha suspension injection port, with grooves forming sus-pension flow channels in at least one of the bonding sur-faces of the boards, thus facilitating the working stepsfor forming numerous fine, curved suspension flow chan-nels inside the device body. However, the method ofworking to form the suspension flow channels is not lim-ited to the one described here.

(Effect of the twelfth invention)

[0069] According to the twelfth invention, there are pro-vided specific and preferred embodiments of neuronseeding devices, wherein the culturing device for forma-tion of a neuron network is one according to the first in-vention, and/or the planar patch-clamp device is one ac-cording to the third invention.

(Effect of the thirteenth invention)

[0070] When neurons have been seeded only in thecell plating sections of the apparatus plate of the culturingdevice for formation of a neuron network or the planarpatch-clamp device, a neuron network is not satisfactorilyformed in most cases because of an insufficient overallnumber of individual neurons in the apparatus plate. Inorder to solve this problem, the device body of the thir-teenth invention further comprises second suspensionflow channels for injection of a neuron suspension intoregions other than the cell plating sections of the appa-ratus plate. Since it is thus possible to seed neurons byappropriately injecting a neuron suspension into regionsother than the cell plating sections of the apparatus plate,the neuron network can be formed in a particularly sat-isfactory manner.

Brief Description of the Drawings

[0071]

Fig. 1 shows a recess structure on a plate preparedas a prototype by the present inventors (comparativeexample).

Fig. 2 shows the structure on a Si plate as disclosedin NPL 1.Fig. 3 shows the structure of the cage disclosed inNPL 2.Fig. 4 is a pair of plan views conceptually showingthe essential portions of a culturing device for forma-tion of a neuron network according to (1) and (2) ofthe second invention.Fig. 5 is a cross-sectional view of the first example.Fig. 6 is an overview of the second example.Fig. 7 is an overview of the third example.Fig. 8 is an overview of the fourth example.Fig. 9 is an overview of the fifth example.Fig. 10 is an overview of the sixth example.Fig. 11 is an overview of a neuron seeding devicemain body according to the seventh example.Fig. 12 is an overview of first and second suspensionflow channel systems according to the seventh ex-ample.Fig. 13 shows the details of the essential portion ofthe first suspension flow channel system accordingto the seventh example.Fig. 14 shows the sizes during seeding of rat hippoc-ampal neurons.Fig. 15 shows the sizes during culturing of rat hip-pocampal neurons.Fig. 16 is an optical microscope photograph showinga separate experiment carried out with rat hippoc-ampal neurons.

Best Mode for Carrying Out the Invention

[0072] Embodiments of the invention will now be ex-plained, including their best modes. The technical scopeof the invention is not limited by these embodiments.

[Technical field of the invention]

[0073] The technical field of the invention is the tech-nical field of forming a neuron network while culturingneurons in a stable condition. Also, it is the technical fieldof measuring ion channel current on cell surfaces. It isadditionally the field of stimulating cells by injecting cur-rent or applying voltage. It is yet further the field of highthroughput screening technology of the type involvingmeasurement of ion channel current or stimulating cellsby injecting current or applying voltage. It is still yet furtherthe field of Ca imaging or various other types of imagingtechniques designed for neurons or neuron networks.

[Neurons and neuron networks]

[0074] Neurons comprise cell bodies that are the mainbodies of the cells, and axons and dendrites that elon-gated from the cell bodies. There are no restrictions onthe type of neurons, but firstly there may be mentionedneurons such as central neurons or peripheral neurons,and most preferably neurons in a state before processes

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such as axons and dendrites have developed. Secondly,there may be mentioned cells capable of differentiatinginto neurons, such as iPS cells and ES cells, and morepreferably neural stem cells that are en route to differen-tiation to neurons from iPS cells or ES cells. Thirdly, theremay be mentioned cells having a property of forming anintracellular network, and cells capable of differentiatinginto cells having a property of forming an intracellularnetwork. The neurons are preferably animal neurons,and most preferably neurons of mammals, which includehumans. The sizes of the cell bodies of the neurons willusually be less than 20 mm, and more specifically areabout 3 to 18 mm.[0075] The neuron network has as its basic structuralunit a pair of neurons, namely a trigger cell that emits asignal and a follower cell that receives it. The presentinventors have found that when the difference in theheights on the surface on which the trigger cells and fol-lower cells are present is about the size of the cells, thecell death rate increases.

[Culturing device for formation of neuron network]

[0076] In the culturing device for formation of a neuronnetwork according to the invention, cell plating sectionsare formed by a plurality of protrusions, on a flat platethat can be filled with a cell culture medium (most pref-erably a liquid medium).[0077] The "flat plate that can be filled with a cell culturemedium" has the construction described for the planarpatch-clamp device described below, for example. Thecell plating sections are a plurality or many set on theplate according to the structure of (1) to (3) of the secondinvention. However, since the base unit in the neuronnetwork is a pair of neurons consisting of a trigger celland a follower cell, when the structure is such that spon-taneous firing of a neuron is the trigger and ion channelcurrent is received by the follower cell, a cell plating sec-tion as the selected region can operate as a functionalanalysis element even at a single location. The mutualgaps between the cell plating sections as the selectedregions will differ depending on the type of neuron net-work and cannot be specified for all cases, but they maybe about 50 to 500 mm, for example.[0078] The shapes of the plurality of protrusions form-ing the cell plating section are not restricted, but are pref-erably fence-shaped or post-shaped, for example. Theheights of the protrusions are also not restricted, but theyare generally preferred to be heights of about 10 mm toeffectively restrict random movement of the neurons, andfor example, for mouse cerebral cortex or hippocampalneurons they are preferably heights of about 5 to 10 mm.[0079] The cell plating section satisfies the followingconditions (1) to (3).[0080]

(1) Gaps are set, between the plurality of protrusionsforming the cell plating sections, which are wide but

do not allow a neuronal cell body to pass through.The protrusion gaps are determined according to thesizes of the mammalian neuronal cell bodies, whichvary within about 3 to 18 mm, and it is difficult tospecify an absolute value for all cases. As one ref-erence point, if the cell body size is represented asX mm, the upper limit for the gaps is preferably nogreater than 0.9X mm and especially no greater than0.7X mm, and the lower limit for the gaps is preferablyat least 0.3X mm and especially at least 0.5X mm.When the top ends of the plurality of protrusions arepreferably not connected to each other as this willessentially form a tunnel structure. Synapse forma-tion may occur in the tunnels of a tunnel structure,but imaging of such synapses is not possible.

(2) The inner diameters of the cell plating sectionsformed by the plurality of protrusions are of sizescapable of accommodating one to several neuronalcell bodies. The inner diameters of the cell platingsections are appropriately set according to the sizesof the neuronal cell bodies and the number of cellbodies in the cell plating section. For example, if thecell body in each cell plating section is a single mam-malian neuronal cell body, the inner diameter of eachcell plating section is preferably about 10 to 25 mm.If the inner diameter of the cell plating section is ex-cessively greater than the size of the cell body, toomany cell bodies may become situated in a singleselected region, while if the inner diameter of the cellplating section more than 50% smaller than the sizeof the cell body, it may not be possible for the cellbody to be stably situated in the cell plating section.

(3) The plate surface forming the bottom of each cellplating section comprises at least one element of thefollowing (a) and (b).

(a) It is coated with an extracellular matrix-form-ing substance.

(b) There are provided fine through-holes forsuction of medium by an aspirator provided be-low the plate surface, the hole diameters beingsuch that the neurons cannot pass through.

[0081] Of these conditions for (3), the (a) extracellularmatrix-forming substance is one that exhibits adhesiveforce for neurons in order to plate the neurons at thebottom of the cell plating section, and examples for theconstituent materials include polylysine, collagen (typeI, type I and type IV), fibronectin, laminin, proteoglycan,(versican, decholin and the like), proteoglycan (aggre-can), link proteins, entactin, tenascin, proteoglycans[chondroitin sulfate proteoglycan, heparan sulfate prote-oglycan (perlecan and the like), keratan sulfate prote-oglycan and dermatan sulfate proteoglycan], hyaluronicacid (a type of glycosaminoglycan), elastin, fibrin, gelatin,

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Matrigel, and the like.[0082] The fine through-holes for suction of cell culturemedium specified by (b) allow suctioning of cell culturemedium with an aspirator on the lower side of the plate,thereby plating the one to several neurons situated in thecell plating section onto the bottom of the cell plating sec-tion, the hole diameters being of a size that the neuronsdo not pass through, such as about 1 to 3 mm.

[Planar patch-clamp device]

[0083] One example of effective use of the culturingdevice for formation of a neuron network is as a planarpatch-clamp device for a neuron network.

(Common planar patch-clamp device)

[0084] Several different membrane proteins are ar-ranged on the surface of cells composing an organism,and binding of chemical substances (signal transfer sub-stances such as ligands) to specific sites on the cell sur-face or electrical or optical stimulation (gate trigger)opens and closes the channels serving as openings formembrane proteins, to control transport of ions andchemical substances between the outside and inside ofthe cell membrane. The ion channels that carry out thiscontrol are membrane proteins that are important for bi-ological signal transfer, and measurement of electricalchanges in channel proteins, i.e. ion channel current, isone of the goals for functional measurement and devel-opment of function-related chemicals.[0085] The need to meet this goal has led to the de-velopment of techniques that employ pipette patchclamps, planar patch-clamps and the like. Pipette patchclamps have a drawback in that they cannot be appliedto high throughput screening by multipoint measurement.In contrast, a planar patch-clamp is a flat plate-like patch-clamp device that allows multipoint measurement of cellion channel current by constructing a plurality of patch-clamp devices on a solid plate such as a silicon chip, andit has fine through-holes for measurement of ion channelcurrent at each of the cell placement locations of eachpatch-clamp device.[0086] However, since a common conventional planarpatch-clamp device does not have a cell culturing func-tion, a problem has been encountered in that it has notbeen applicable for cells that require culturing, such asneurons. In other words, because the lifetime of cells tobe measured is as short as 1 hour or even 30 minutesor less under non-culturing conditions, the device onlyhas limited application for innovative drug screening, andit has been difficult to apply it for functional analysis ofcells wherein a pipette patch clamp is employed. In ad-dition, it has been difficult to successfully carry and trapcells at the locations of the fine through-holes providedin the plate.

(Planar patch-clamp device of the invention)

[0087] In contrast, a planar patch-clamp device of theinvention also has a neuron-culturing function, unlike aplanar patch-clamp device with a common constructionas described above, and it allows effective minimizationof noise current during ion channel current measurementand stable positioning of cells. That is, the characteristicconstruction of the device is such that cell-anchoringforce is applied to the opening for plating of the neuronat the fine through-holes provided on the plate, and aliquid pool section capable of current flow to an electrodeis provided on the surfaces on both sides of the through-holes in the plate, the liquid pool section being fillablewith a conducting liquid (for example, a cell culture solu-tion). With this planar patch-clamp device, it is possibleto easily trap neurons at the locations of the fine through-holes, and to measure ion channel activity over sufficienttime under cell culturing conditions.[0088] Specifically, the culturing device for formationof a neuron network in the planar patch-clamp device ofthe invention is constructed in a manner according to thefollowing (1) to (3).

(1) The plate is an electrical insulating plate, and finethrough-holes are provided connecting the surfaceson both sides of the electrical insulating plate.(2) The neuron network-forming side, as the first sur-face side of each fine through-hole, and the secondsurface side on the opposite side, each have a liquidpool section for holding of the conducting liquid, andan electrode section situated so as to allow conduc-tion to the conducting liquid of the liquid pool section.(3) The liquid pool section of the first surface side isthe liquid pool section for the neurons plated in thecell plating section.

(Main construction of planar patch-clamp device of the invention)

[0089] In the planar patch-clamp device of the inven-tion, therefore, fine through-holes are provided allowingcommunication between the first surface side (the sur-face side on which the cells are placed) and the secondsurface side, which are both surfaces of the plate withelectrical insulating properties.[0090] The plate with electrical insulating properties ispreferably a plate made of glass, ceramic, plastic or thelike. When a silicon plate is to be used, a preferred ex-ample is a silicon plate (SOI plate) having a laminatedstructure with a silicon layer on the first surface side, asilicon oxide layer in the middle and a silicon layer on thesecond surface side. Since a silicon plate having such alayered structure has a very highly insulating interlayerpresent between two silicon layers, it is possible to es-tablish a high resistance state during ion channel closureof the cell being measured, and to reduce backgroundnoise.

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[0091] The number of through-holes in the plate is notparticularly restricted, but it is preferably several to many,and for example, two to several dozen, or more. The innerdiameters of the fine through-holes are preferably innerdiameters such that liquid can pass through but the neu-rons cannot (for example, about 1 to 3 mm), althoughthere is no restriction to this range for the inner diameters.[0092] Also, both the first surface side and second sur-face side of the through-holes of the planar patch-clampdevice have a liquid pool section for holding of the con-ducting liquid, and an electrode section situated so as toallow conduction to the conducting liquid of the liquid poolsection.[0093] The construction of the liquid pool sections isnot restricted so long as it satisfies the condition of "hold-ing the conducting liquid while allowing conduction to theelectrode section to the conducting liquid", and they maybe formed, for example, by layering a spacer member orplate member on each of the first surface side and secondsurface side of the plate, and providing notched sectionsin the spacer member in the regions corresponding tothe through-holes of the plate, as shown in the first ex-ample.[0094] While not necessarily constituting a restriction,preferably the spacer member and plate member on thefirst surface side are made of optically opaque materials,and preferably the spacer member and plate member onthe second surface side are made of optically transparentmaterials.[0095] The liquid pool sections are constructed in afluid-tight manner themselves, while being provided withliquid flow channels or openable/closeable openings forintroduction and discharge of a conducting liquid (a con-ducting liquid that is cell culture medium in which neuronsare dispersed). Each liquid pool section on the first sur-face side of the plate has the top of the liquid pool sectioncovered with a covering member such as cover glass,and if necessary the covering member may be removedto open the liquid pool section.[0096] In the planar patch-clamp device, the first sur-face side and second surface side are provided with elec-trode sections having novel constructions, which will bedescribed below under "Electrode section structure inplanar patch-clamp device".[0097] Also in the planar patch-clamp device, prefera-bly the liquid pool section on the first surface side has aconstruction with a main pool for placement of the cells,and a secondary pool in which the electrode section onthe first surface side is situated, each formed of an opti-cally opaque material, and a narrow liquid flow channelconnecting the pools. Also, the liquid pool section on thesecond surface side is preferably connected to a liquidflow channel for introduction and discharge of a conduct-ing liquid, with the electrode section on the second sur-face side being situated in the liquid flow channel.[0098] Furthermore, the liquid pool sections on the firstsurface side correspond to the selected regions of theneuron. Thus, a plurality or many liquid pool sections on

the first surface side are set on the plate with suitablemutual gaps between them in the two-dimensional direc-tion, with cell plating sections being formed surroundedby a plurality of protrusions in each liquid pool section onthe first surface side.[0099] Likewise, a plurality of liquid pool sections arealso set on the second surface side at locations corre-sponding to the liquid pool sections on the first surfaceside, the liquid pool sections on the first surface side andthe second surface side being connected by the finethrough-holes of the plate. The liquid pool sections onthe second surface side are also connected to a liquidsuction device, and when negative pressure is appliedto the liquid pool section on the second surface side bythe liquid suction device, negative pressure is also ap-plied to the liquid pool section on the first surface sidethrough the fine through-holes. The fine through-holescorrespond to the fine through-holes for suctioning of cellculture medium at the bottom of the cell plating sectiondescribed as (b). Also, an extracellular matrix-formingsubstance with cell-anchoring force is adhered to the pe-riphery of the opening on the first surface side at the finethrough-hole. This corresponds to coating of an extracel-lular matrix-forming substance on the bottom of the cellplating section, described as (a).

[Electrode section structure in planar patch-clamp de-vice]

[0100] In the planar patch-clamp device, the electrodesections on the first surface side and second surface sidealso preferably comprise the following elements (a) to (c).

(a) An electrode receptacle of which at least a portionof the receptacle wall that is to contact with the con-ducting liquid introduced into the liquid pool section,is composed of an inorganic porous material.(b) An electrode having a precious metal chloride(NmCl) layer formed on a surface layer section ofthe precious metal (Nm), housed in the electrodereceptacle.(c) An electrode solution comprising the preciousmetal chloride (NmCl) and an alkali metal chloridedissolved to saturated concentration, filled into theelectrode receptacle.

[0101] The type of precious metal Nm in the electrodesection structure is not restricted, but is preferably silverAg or platinum Pt, with silver Ag being especially pre-ferred. Thus, the precious metal chloride NmCl is pref-erably silver chloride AgCl or platinum chloride PtCl, withsilver chloride AgCl being preferred. The alkali metalchloride is also not restricted, but is preferably potassiumchloride KCl. The inorganic porous material composingat least a portion of the receptacle wall is preferably po-rous glass or porous ceramic.[0102] Also, preferably the electrodes of the electrodesection satisfy the following (1) or (2).

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(1) A rod-shaped electrode protruding in the elec-trode receptacle, with a precious metal chlorideNmCl layer formed on the surface layer section of acore material made of a precious metal Nm.(2) A tubular electrode formed on the inner peripheralsurface of the wall section of the electrode recepta-cle, the bottom layer on the receptacle wall side beinga vapor deposition layer of a precious metal Nm, andthe surface layer contacting the electrode solutionbeing a vapor deposition layer of a precious metalchloride NmCl.

[Imaging analysis using culturing device for formation of neuron network]

[0103] A culturing device for formation of a neuron net-work or planar patch-clamp device according to the in-vention may be used for various types of imaging anal-ysis, including at least Ca imaging analysis, imaging anal-ysis with synaptophysin or synapsin labeling as synapticsite markers, imaging analysis with MAP2 as a dendritemarker, and imaging analysis with FM1-43 or FM4-64which labels endosomes or exosomes.

(Ca imaging analysis)

[0104] Ca imaging is a method in which a Ca probe (adye that bonds to Ca ion and emits fluorescence) is in-troduced into a neuron, and inflow of Ca ion into the cellbody, when an action potential is generated in the neuron,is captured as fluorescence, and it allows analysis of cel-lular ion channel current by observing the fluorescenceproduced during generation of an action potential or dur-ing propagation of an action potential.[0105] By thus forming a neuron network using neu-rons with an introduced Ca probe, and for example, con-ducting current injection or voltage application to a singleneuron within it, it is possible to perform measurementby Ca imaging of a plurality or many neurons.[0106] According to this method, a single neuron of theneuron network (a first neuron) is selected and stimulatedby current injection or voltage application to generate anaction potential, while simultaneously the action potentialis propagated to a surrounding adjacent neuron (secondneuron) through the neuron network, and the state ofpropagation from the second neuron to a third neuronadjacent to it may be measured by Ca imaging.[0107] Electrode stimulation is an example of a priorart method, but with this method it is difficult to selectivelystimulate single neurons, and analysis becomes com-plex. Also, although selective stimulation of single neu-rons is possible by stimulation using a micropipette elec-trode, as another prior art method, it is difficult to accom-plish multichannel measurement for high throughputscreening. The method of the invention allows the meas-uring device to be greatly downsized to facilitate mul-tichannel measurement.

(Imaging analysis by synaptophysin and synapsin)

[0108] Synaptophysin and synapsin are synapse ves-icle membrane proteins, used as markers of synapticsites, and by binding a dye to their antibodies and utilizingantigen-antibody reaction to bind the dyes to these pro-teins, it is possible to accomplish labeling of synapticsites.

(Imaging analysis by MAP2)

[0109] MAP2 is a dendrite marker, and adding a dyeto its antibody and conducting reaction allows labeling ofdendritic sites.

(Imaging analysis by FM1-43 and FM4-64)

[0110] FM1-43 and FM4-64 reversibly enter the cellmembrane without passing through the cell membrane,and emit fluorescence only when binding to the cell mem-brane, and can thus label endosomes and exosomes.They have the feature of allowing labeling while main-taining cellular biological function.

(Optical systems for imaging analysis)

[0111] When a culturing device for formation of a neu-ron network or a planar patch-clamp device according tothe invention is used for the various types of imaginganalyses mentioned above, the device preferably com-prises the following optical system elements.[0112] First, a photodetector for light emitted by neu-rons is set on the first surface side of the plate of thedevice. Also, an irradiating device for irradiation of laserlight or the like onto the neuron or plate surface is set onthe first surface side of the plate of the device. The irra-diating device most preferably is also equipped with anoptical focusing system to irradiate light only on specifiedsingle cells.[0113] By providing such an optical system element itis possible to carry out optical measurement in a non-contact, non-destructive manner, allowing analysis with-out inhibiting the function of the neuron network, whilealso allowing high-speed analysis and accurate analysisby excitation of single neurons in a precise manner withan optical focusing system.

[Neuron seeding device]

[0114] The neuron seeding device of the invention isa device for seeding of neurons in multiple cell platingsections surrounded by a plurality of protrusions on anapparatus plate, where a culturing device for formationof a neuron network or a planar patch-clamp device uti-lizing the culturing device, is set up on a flat apparatusplate that can be filled with cell culture medium.[0115] The "culturing device for formation of a neuronnetwork" is not limited in its construction so long as it has

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multiple cell plating sections formed surrounded by a plu-rality of protrusions on a flat apparatus plate that can befilled with cell culture medium. Likewise, a "planar patch-clamp device" is not limited in its construction so long asit is a device utilizing a culturing device for formation ofa neuron network and has multiple cell plating sectionsformed surrounded by a plurality of protrusions on a flatapparatus plate that can be filled with cell culture medium.Most preferably, however, the "culturing device for for-mation of a neuron network" is a culturing device for for-mation of a neuron network of the invention according tothe embodiment described above, and the "planar patch-clamp device" is a planar patch-clamp device of the in-vention according to the embodiment described above.[0116] In a neuron seeding device according to the in-vention, the device body has a board shape that can beset on an apparatus plate of the culturing device for for-mation of a neuron network or a planar patch-clamp de-vice. Generally speaking, "board shape" means a sheet-like or thick sheet-like form, and in most cases the planarshape will be quadrilateral (square or rectangular). Ac-cording to the invention, however, the specific form ofthe "board shape" is not restricted so long as it is a formthat can be set on the apparatus plate of the culturingdevice for formation of a neuron network or the planarpatch-clamp device, and at least the bottom is flat so thatthe bottom contacts the apexes of the plurality of protru-sions at the multiple cell plating sections on the apparatusplate, and so long as the bottom has a size covering themultiple cell plating sections when set on the apparatusplate.[0117] It is convenient if the "board-shaped" devicebody has a planar shape and size (area) basically cor-responding to the apparatus plate of the culturing devicefor formation of a neuron network or the planar patch-clamp device. The planar shape of the device body is notlimited to being quadrilateral, and may be circular, ellip-tical or some other irregular shape, and the thicknessmay be from about several mm to several cm, for exam-ple. The area of the bottom of the device body preferablycorresponds to the area of the apparatus plate, and forexample, it may be freely selected from about 2 to 3 cm2

to about several tens of cm2, or even larger.[0118] The device body area or the area of the appa-ratus plate corresponding to it is preferably set as appro-priate in consideration of factors such as the number ofcell plating sections on the apparatus plate and the de-gree of integration in micromachining described belowfor the device body. The constituent material of the devicebody is also not restricted, but preferred examples areinorganic materials such as glass, and organic materialssuch as plastics. It is particularly preferred to use a trans-parent material.[0119] A board-shaped device body preferably has anupper board provided with a suspension supply port anda lower board provided with a suspension injection port,both joined in a closely bonded state, with a groove form-ing the suspension flow channel formed on at least the

bonding surfaces of the upper board and the lower board.The cross-sectional shape of the groove may be semi-circular or quadrilateral. In this case, the groove essen-tially forms the suspension flow channel after the upperboard and lower board have been joined. When groovesare formed in both the upper board and lower board pre-cisely corresponding to semicircular cross-sections, theresult is that a circular cross-sectional suspension flowchannel is formed.[0120] However, the board-shaped device body maybe a single board so long as it can be worked to form aplurality of suspension flow channels inside it. Such work-ing can be accomplished by a stereolithographic method(three-dimensional stereolithography) using a photocur-ing resin, although the production efficiency is reduced.[0121] The device body comprises (1) a suspensionsupply port for external supply of a neuron suspensioncontaining neurons suspended at a fixed density, (2) aplurality of fine suspension flow channels extending in abranched fashion from the suspension supply port insidethe device body, and (3) a suspension injection port forinjection of the neuron suspension into the cell platingsection, opening into the bottom of the device body atthe end of each suspension flow channel.[0122] When the neuron seeding device is placed onthe apparatus plate of the culturing device for formationof a neuron network or the planar patch-clamp device,the individual suspension injection ports must be locatedto precisely correspond with the individual cell platingsections. Also, as mentioned above, for precise position-ing of the suspension injection port and the cell platingsection during placement of the neuron seeding device,it is preferred to display a positioning label (marker) onthe device body made of a transparent material, or onthe apparatus plate of the culturing device for formationof a neuron network or the planar patch-clamp device.[0123] The suspension supply port of (1) opens outonto the top side of the device body. Even if it opens outonto the side of the device body, however, it is usable solong as the suspension supply port slopes diagonallydownward toward the main body interior. Furthermore,assuming that the neuron suspension is to be suppliedin a pressurized state into the suspension supply port,when leakage of the neuron suspension from the sus-pension supply port after the neuron seeding procedurehas been completed is not a concern, the suspensionsupply port may open out in the horizontal direction onthe side of the device body.[0124] The suspension supply port may be providedat only one location of the device body, but when a con-siderably large number of fine suspension flow channelsof (2) and suspension injection ports of (3) have beenset, it is preferred to provide suspension supply ports atnumerous locations of the device body, appropriately dis-persed, from the viewpoint of facilitating communicationby the flow channel design. When suspension supplyports are provided at numerous locations, supply of neu-ron suspension to the ports may be accomplished using

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separate injectors or the like for each, or alternatively aconnecting pipe having a single pipe at the base end andbranching into multiple pipes at the distal end, may beconnected to a single injector or the like at the base end,and the multiple pipes at the distal end each connectedto suspension supply ports at the numerous locations.[0125] In either case, the inner diameter of each sus-pension supply port will usually be as small as from aboutseveral hundred mm up to a few mm, for example, andtherefore as stated above under "Effect of the Invention",a connecting pipe having a tapered tip end may be in-serted into the injection hole of a liquid injecting tool suchas an injector or microsyringe, and the tapered sectionat the tip end may be inserted into the suspension supplyport. The "tapered section at the tip end" of the connectingpipe may be, for example, a stainless steel insertion noz-zle formed into a conical shape that gradually narrowstoward the tip. Even when a "connecting pipe branchinginto multiple pipes at the distal end" is used to supply theneuron suspension to suspension supply ports at multiplelocations, it is possible to mount the insertion nozzle ateach of the tip sections of the branched pipes.[0126] The suspension flow channels of (2) above maybe, for example, fine flow channels with an inner diameterof about 50 mm to 500 mm. The cross-sectional shapesof the suspension flow channels may be circular, semi-circular, quadrilateral or the like. Basically, the suspen-sion flow channels are formed along essentially the pla-nar direction in the device body. The suspension flowchannels extend as multiple branches from the suspen-sion supply port, and the form of the branches will some-times be extensions as multiple branched suspensionflow channels directly from the suspension supply port,or a small number such as one, 2 or 3 main line suspen-sion flow channels from the suspension supply port, withbranch-line suspension flow channels connected in orderto multiple suspension injection ports branching out fromthese main line suspension flow channels.[0127] Also, the straight line distances from the sus-pension supply port to the individual suspension injectionports may depend on the positions where the suspensioninjection ports are set, and do not need to be the same.However, for the reasons explained in regard to the effectof the tenth invention, it is highly preferred for the lengthsfrom the suspension supply port of (1) to the suspensioninjection ports of (3) in the plurality of suspension flowchannels to be set so that they are substantially the same.This condition can be met if, for example, a by-pass sec-tion for adjustment of the flow channel length is purposelyset in a suspension flow channel that is a specified mainline, and/or in a suspension flow channel that is a spec-ified branch line.[0128] In regard to the suspension injection ports of(3), each suspension injection port is set so as to be po-sitioned at an individual cell plating section. The suspen-sion injection ports open out downward into the flat bot-tom of the device body. Also, as mentioned above, whenthe device body is set on the apparatus plate of the cul-

turing device for formation of a neuron network or theplanar patch-clamp device, the bottom of the device bodycontacts with the apexes of the plurality of protrusions ofthe cell plating section. The neuron suspension injectedthrough the suspension injection ports is thus reliably in-jected inside the cell plating section. For this reason, theopening diameters of the suspension injection ports arepreferably about the same as the inner diameter of thecell plating section defined by the plurality of protrusions,or just slightly smaller or slightly larger.[0129] The device body preferably further comprisesa second suspension flow channel system for injectionof neuron suspension into the regions other than the cellplating section of the apparatus plate of the culturing de-vice for formation of a neuron network or the planar patch-clamp device, for the reasons explained above in regardto the effect of the thirteenth invention.[0130] The second suspension flow channel systemmay have second suspension supply ports with similarstructures as those of (1) above, for example, providedat an appropriate number of locations on the board-shaped device body. In this case, the second suspensionsupply ports may penetrate to the bottom of the devicebody without passing through the fine suspension flowchannels, or they may communicate with the second sus-pension injection ports similar to (3) that open into thebottom of the device body after branching into a pluralityof second suspension flow channels similar to (2). In thesecond suspension flow channel system, the plurality ofsecond suspension flow channels do not need to be setto have substantially the same lengths.

Examples

[0131] Examples of the invention will now be de-scribed. However, the technical scope of the invention isnot limited by these examples.

[First example]

[0132] A device according to the first example is shownin Fig. 5. This device has a culturing device for formationof a neuron network, constructed as a culturing planarpatch-clamp device to serve as an ion channel biosensor.[0133] The electrical insulating plate 14 used in thisdevice was a silicon plate. The plate 14 is provided withfine through-holes 15 having diameters of 1 to 3 mm, thatallow communication between a first surface side (thetop end in the diagram) and a second surface side (thebottom end in the diagram). In Fig. 5, one fine through-hole 15 is provided at the center of the plate 14, but alarger plate may be used and a plurality or many finethrough-holes 15 provided, with the following construc-tion applying to each of the fine through-holes 15.[0134] A cell plating section 13 is formed surroundedby a plurality of protrusions 12 on the plate surface abovethe opening on the first surface side of the fine through-hole 15, with one to several neurons 11 being placed in

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the cell plating section 13 (only one neuron is shown inthe diagram for convenience).[0135] The plate 14 is sandwiched between the firstsurface side and second surface side by a pair of spacers16, 17. The constituent material of the spacers 16, 17 isnot restricted, but the spacer 16 on the first surface sideis preferably an elastic, optically opaque material, andfor example, silicon rubber, PDMS (polydimethylsi-loxane) or the like may be used. On the other hand, thespacer 17 on the second surface side is preferably anoptically transparent material.[0136] At the center section of the spacer 16 there isformed a notch as a large culturing space 18 for con-struction of a neuron network, and on the plate 14 surfacein this culturing space 18 there are formed one, severalor many cell plating sections 13 in which a neuron 11 isplaced (the diagram shows only a single cell plating sec-tion for convenience). Neurons 11 are also seeded atsections other than the cell plating section 13 on the plate14 surface.[0137] In the spacer 17, a notched section 19 which iscircular, for example, is formed at the section correspond-ing to the fine through-hole 15 of the plate 14, wherebythe opening in the second surface side of the fine through-hole 15 opens out into the notched section 19. Thus, thenotched section 19 may also be one, several or many,corresponding to the cell plating sections 13 and the finethrough-holes 15 (although only one notched section 19is shown in the diagram for convenience).[0138] In addition, the entirety of the plate 14 and thepair of spacers 16, 17 have a structure that is clampedby a pair of strong plates 20, 21. The material of the plates20, 21 is not particularly restricted so long as it is a ma-terial that can withstand autoclave sterilization at about120°C. However, the plate 20 on the first surface side ispreferably made of an optically opaque material. On theother hand, the plate 21 on the second surface side ispreferably made of an optically transparent material.[0139] In the construction described above, a notchedsection that is circular, for example, is provided at thecenter section of the plate 20 on the first surface side, ata location corresponding to the culturing space 18 of thespacer 16 on the first surface side, having a size similarto that of the culturing space 18. On the circumferenceof the notched section there may be formed a recess-likestep where the thickness of the plate is reduced, and acovering member (not shown) such as cover glass maybe placed on the step as a construction to allow openingand closing of the opening of the notched section in thespacer 16. A main pool 22 is thus constructed on the firstsurface side.[0140] On the other hand, a liquid pool section 23 isformed on the second surface side by using the plate 21to plug the opening of the notched section 19 at the spac-er 17 on the second surface side. The main pool 22 onthe first surface side and the liquid pool section 23 on thesecond surface side are connected via the fine through-hole 15.

[0141] The main pool 22 forms the first region of theliquid pool section on the first surface side. The main pool22 is connected with a secondary pool 25 forming thesecond region of the liquid pool section on the first surfaceside, through a narrow liquid flow channel 24 provided inthe spacer 16. The secondary pool 25 is formed by a holeformed through both the spacer 16 and the plate 20. Anelectrode section 28 on the first surface side, describedbelow, is situated in the secondary pool 25.[0142] A cell culture medium as conducting liquid isintroduced into and held in the liquid pool section on thefirst surface side composed of the main pool 22, liquidflow channel 24 and secondary pool 25. Neurons maybe dispersed in the conducting liquid. The conductingliquid used may be a buffering solution comprising 140mM NaCl, 3 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piper-azineethanesulfonic acid (HEPES), 2.5 mM CaCl2, 1.25mM MgCl2 and 10 mM glucose at pH 7.4 (with HCl), orcell culture medium such as Dulbecco’s modified Eagle’smedium (DMEM: Sigma) with addition of 10% (v/v) FBSand 1% (v/v) Glutamax™ (Gibco). The composition of theconducting liquid can be appropriately changed depend-ing on the type of neuron.[0143] Into the liquid pool section 23 on the secondsurface side there is introduced a buffering solution orcell culture medium, known as a pipette solution, whichmay be 40 mM CsCl, 80 mM CsCH3SO4, 1 mM MgCl2,10 mM HEPES, 2.5 mM MgATP, 0.2 mM Na2EGTA (pH7.4). Introduction of the conducting liquid into the liquidpool section 23 is accomplished through a tubular liquidintroduction flow channel 26, and discharge is accom-plished by a liquid discharge flow channel 27. In this ex-ample, PEEK tubes with an outer diameter of 1 mm andan inner diameter of 0.5 mm were used for the liquidintroduction flow channel 26 and liquid discharge flowchannel 27, but the constituent materials of these liquidflow channels may be other materials instead, so longas the materials can withstand autoclave sterilization atabout 120°C.[0144] In the liquid discharge flow channel 27 there isset an electrode section 29 on the second surface sideformed in the same manner as the electrode section 28on the first surface side (shown in outline fashion by adash-dot line). The construction of the electrode sections28, 29 will be described below. Normally, the electrodeof the electrode section 28 on the first surface side isgrounded, and a membrane voltage is applied to the elec-trode of the electrode section 29 on the second surfaceside.[0145] When the conducting liquid in which the neu-rons 11 are dispersed has been introduced into the mainpool 22, and the conducting liquid of the liquid pool sec-tion 23 is suctioned with an appropriate liquid-suction de-vice connected to a liquid discharge flow channel 27, theconducting liquid of the main pool 22 is also suctionedthrough the fine through-hole 15. By this procedure, it ispossible to effectively place a neuron 11 in the cell platingsection 13 shown in Fig. 5 (that is, the location of the

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opening of the fine through-hole 15).[0146] Furthermore, the suction pressure can form finepores in the cell membrane of the neuron 11 at the portioncontacting with the fine through-hole 15. For formationof such pores in the neuron 11 there may also be men-tioned a method of introducing a solution of a cell mem-brane pore-forming penetrating antibiotic such as nysta-tin or amphotericin B, from the liquid introduction flowchannel 26 into the liquid pool section 23 on the secondsurface side. When such pores are formed in the cellmembrane, the liquid pool section 23 on the second sur-face side is in a state of electrical conduction with theinterior of the neuron.[0147] On the other hand, as means for placing theneuron 11 in the cell plating section 13 (the open locationof the fine through-hole 15), an extracellular matrix-form-ing substance 30 with cell-anchoring force may be ad-hered to the periphery of the opening on the first surfaceside of the fine through-hole 15 of the plate 14.[0148] In this construction, when the prescribed ionchannel is being expressed on the neuron 11 and a stim-ulator that opens the ion channel is added to the liquidpool section on the first surface side, the ion channelopens and a channel current corresponding to the ap-plied voltage flows between the electrode section 28 onthe first surface side and the electrode section 29 on thesecond surface side. When a gap is present between thecell membrane of the neuron 11 and the plate 14 duringthis time, the seal resistance is lowered and leak currentis superimposed on the channel current.[0149] The membrane potential has induced voltageby electromagnetic waves present between the space,an interface potential between the electrode metal sur-face and the buffering solution surrounding it, and a liq-uid/liquid interface potential, superimposed in addition tothe voltage actually applied between the electrodes, andtherefore the leak current varies in accordance with thevariation in induced noise and interface potential. Thus,noise appears as fluctuation of the baseline with respectto the ion channel current.[0150] The detailed data will not be shown here, but ina pipette patch clamp that can easily provide seal resist-ance at or above the gigaohm level, the effect of baselinefluctuation noise is small enough to be ignored even witha relatively large fluctuation in membrane potential. How-ever, this fluctuation in membrane potential must be re-duced for a culturing planar patch-clamp with relativelylow seal resistance (up to 10 MΩ). For this example, astable electrode was developed having low fluctuation inmembrane potential. Such an electrode allows measure-ment to be carried out with significantly reduced fluctua-tion in membrane potential and minimal noise currenteven with low seal resistance.[0151] The structures of electrode sections 28, 29 onthe first surface side and second surface side will nowbe described, without a detailed diagram. The interior ofthe tubular electrode receptacle 31 composed of PyrexR

glass with an inner diameter of 1 mm is filled with an

electrode solution 32 with KCl and AgCl dissolved to sat-urated concentrations. The KCl concentration was 3.3M/L and AgCl was added to approximately 1.1 mM/L. ForKCl, the saturated concentration is approximately 3.3 M/Lat ordinary temperature. For the AgCl/Ag electrode 33housed in the electrode receptacle 31, AgCl is coatedonto the surface of a silver wire. Such an AgCl/Ag elec-trode 33 can be formed by coating AgCl powder onto thesurface of a silver wire, or by dipping a silver wire into ableaching agent or the like containing sodium hypochlo-rite. Alternatively, it can be formed by electroplating in aKCl solution.[0152] The tip section of the electrode receptacle 31is plugged with an inorganic porous material 34 such asporous glass or porous ceramic. The inorganic porousmaterial 34 actually used was Vycor glass (Corning, Inc.).The tip of the inorganic porous material 34 composing apart of the receptacle wall of the electrode receptacle 31is dipped in a conducting liquid (cell culture solution orbuffering solution). The KCl concentration in the conduct-ing liquid is a few millimoles, but since mixing betweenthe electrode solution 32 and the conducting liquid out-side the receptacle is small enough to be ignored whilethe inside and outside of the electrode receptacle 31 arein a state of electrical conduction, due to the effect of theinorganic porous material 34, a large KCl concentrationdifference is maintained between the inside and outsideof the receptacle, thereby maintaining a fixed AgCl/Agelectrode 33 interface potential and liquid/liquid interfacepotential. The base section of the electrode receptacle31 is sealed with a sealant, and the electrode pin 35 pro-trudes from it.[0153] When the channel current is controlled usingcells expressing ion channels where the channels openin response to light, placement of the electrode sections28, 29 on the first surface side and the second surfaceside causes a liquid pool section on the first surface sideto be formed by an optically opaque spacer 16 or plate20, and therefore light irradiated onto the main pool 22is not irradiated onto the AgCl/Ag electrodes of the elec-trode sections 28, 29 on the first surface side and thesecond surface side. In addition, since a neuron 11 isplaced in the main pool 22 and the potassium ion con-centration of the conducting liquid on the exterior of thecell is as small as about several mM, it is preferred tominimize the effect of KCl leaking from the electrode sec-tion even if it is a trace amount, and for this purpose asecondary pool 25 is formed in addition to the main pool22 in the liquid pool section on the first surface side, themain pool 22 and secondary pool 25 being connected bya narrow liquid flow channel 24 with a width of no greaterthan 1 mm.

[Second example]

[0154] A second example is shown in Fig. 6. The sec-ond example and the following third example further dem-onstrate in detail the essential points of the first example.

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The part numbers in these examples differ from those ofthe first example, but the same part names have essen-tially the same structures.[0155] A negative photoresist SU8 is coated onto thesurface of a Si plate 1 to a thickness of 8 to 10 mm usinga spinner, and a previously prepared photomask is usedfor development by a common process, to form a cellplating section 13 composed of a plurality of palisadedprotrusions 12, examples of which are shown in Fig. 6(a)and Fig. 6(b).[0156] The protrusions in this case are square columnswith bottoms of 10 mm 3 10 mm and heights of 8 to 10mm, the mutual gaps between the protrusions being 8 to10 mm. The diameters of mouse or rat cerebral corticalor hippocampal neuronal cell bodies are generally about10 mm when seeded and 15 to 20 mm when plated, andtherefore the neuron 3 situated in the cell plating section2 formed by the plurality of protrusions does not migrateout of the cell plating section 2. However, the cell culturemedium migrates into the cell plating section 2 so thatculturing of the cell takes place satisfactorily in the cellplating section 2. The shapes of the protrusions may becircular columnar or elliptic cylindrical, and may even besolid spherical.[0157] Placement of the neurons 3 by the cell platingsection 13 composed of the plurality of protrusions 12 isuseful for formation of a neuron network. As shown con-ceptually in Fig. 6(c), placing a neuron 3 on the inside ofthe cell plating section 13 results in formation of a networkwith the neurons 3 on the outside of the cell plating section13. In such network formation, the neuron 3 undergoeselongation of the axon to bind with adjacent neurons,while neurotransmitters are released from the tip of theaxon, while the neurons 3 at the receiving end receivethe neurotransmitters and extend their dendrites, formingsynaptic junctions.[0158] It is a feature of the invention that the neuron 3inside the cell plating section 13 and the neurons 3 out-side the cell plating section 13 are present on the sameflat surface of the plate 1, so that communication betweenthe neurons is accomplished without interference, stableculturing is continued, and a stable network is formed. Inthe case of this example, the neuron 3 situated insidethe cell plating section 2 could be cultured for a monthor longer.[0159] It is useful in many respects to form a networkby setting the locations of the neurons in this manner.The effect is particularly notable when applying a planarpatch-clamp device for culturing, such as shown in Fig. 5.[0160] The inner diameter of the cell plating section 13can be easily changed as shown in Fig. 6(a) and Fig.6(b). The inner diameter may be the inner diameter al-lowing numerous (comparatively numerous) neurons 3to be placed for stable culturing of the neuron networkfor a prolonged period, or an inner diameter allowing oneto a small number of neurons 3 to be placed for easyfunctional analysis of a network, determined based onthe optimal number of neurons (cluster size) for the pur-

pose. For example, a cluster of 1 to 4 is satisfactory forrelatively hardy rat cerebral cortical neurons, but with rel-atively fragile iPS cells or neurospheres differentiatedand induced from them, clusters comprising more nu-merous cell bodies are preferred for stable culturing.

[Third example]

[0161] A third example will now be described based onFig. 7. In this diagram, the circled section labeled "c" atthe bottom right of Fig. 7 (A) is a magnified section of thecross-section near the fine through-hole 4 at the centersection of the cell plating section 2. The culturing planarpatch-clamp has a construction with a fine through-hole4 with a diameter of 1 to several mm formed in a plate 1such as Si, or plastic, ceramic or glass, a neuron 3 beingplaced over the fine through-hole 4, and a prescribedbuffering solution filling both above and below the plate1, and with an upper electrode 7 and lower electrode 8installed. The lower electrode 8 is connected to a currentamplifier 5.[0162] Fine pores are opened in the cell membrane ofthe neuron 3 contacting with the fine through-hole 4, form-ing a whole-cell state with electrical conduction betweenthe neuron 3 and the buffering solution pool below theplate 1. The method of opening fine pores in the neuron3 is a method of applying negative pressure to the lowerliquid pool to breach the cell membrane, as explained forthe first example. Another method involves causing abuffering solution dissolving antibiotics such as nystatinand amphotericin to flow into the lower liquid pool, andimplanting the antibiotics into the cell membrane to pro-duce a state of electrical conduction between the cellinterior and the lower liquid pool.[0163] In this case, coating an extracellular matrix-forming substance 9 onto the surface of the plate 1 sur-rounding the fine through-hole 4 is effective for prolong-ing the lives of the neurons 3. While many extracellularmatrix-forming substances 9 are known, poly-L-lysineand laminin are among those more well known. For seed-ing of the neurons 3 into the system, it is particularly ef-fective to utilize a micropipette 6 such as shown in thediagram, since it is necessary to reliably seed a singlecell or a plurality of cells inside the cell plating section 2.[0164] Furthermore, in this case, as shown by the cir-cled section labeled "c" in the partial magnified view atthe bottom right of Fig. 7 (A), the suspension of a neuron3 is injected onto the cell plating section 2 at a prescribedrate from the micropipette 6 while a prescribed negativepressure is applied to the lower liquid pool, so that theneuron 3 can be efficiently placed on the fine through-hole 4 as shown in Fig. 7 (B). If the negative pressure istoo great, however, the neurons 3 will die, and thereforeappropriate pressure must be set for each type of cell.In Fig. 7 (B), in a suction experiment using HEK293 cells,it was confirmed that neurons 3 do not suffer damage ifthe negative pressure is 2 kPa. It was also confirmed that80% of the neurons 3 do not suffer damage if the negative

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pressure is 5 kPa. However, it was confirmed that if thesuction pressure begins to be removed immediately afterthe neurons 3 have been placed on the fine through-holes4 by suction, the extent of damage is minimal.[0165] In this example, a probe molecule for Ca imag-ing was introduced into the neurons 3 beforehand. Also,the neurons 3 also expressed photoreceptor ion chan-nels such as channel rhodopsin by gene transfer, allow-ing stimulation by light such as laser light. In this case, itis important for the excitation wavelength of the channelrhodopsin and the excitation wavelength of the Ca probemolecule to be sufficiently separate so that they do notinterfere. The excitation wavelength of the channel rho-dopsin utilized in this example was 470 to 480 nm, whilethe Ca probe used was Oregon Green BAPTA-1 havingan excitation wavelength of 494 nm and a luminous wave-length of 523 nm. The operational modes implementedin this third example were the following four types.

(First operational mode)

[0166] A prescribed membrane potential (normallyfrom -80 to +80 mV) is applied to the neuron 3 on the finethrough-hole 4 by an upper electrode 7 and a lower elec-trode 8, and the ion channel current flowing in the neuron3 by spontaneous firing is observed in whole-cell mode(arrow symbol "a" in Fig. 7(A)). In this case, synaptic cur-rent of Na+, K+, Cl- and the like is observed with the inflowof Ca ion by spontaneous firing of the surrounding neu-rons 3 and the reception of neurotransmitters (K.S. Wil-cox et al., Synapse 18 (1994) 128-151), and this can yieldinformation regarding the state of the axons and the stateof the neurons 3.

(Second operational mode)

[0167] A prescribed current is injected into the neuron3 on the fine through-hole 4 by the upper electrode 7 andthe lower electrode 8, or voltage is applied (the arrowsymbol "b" in Fig. 7 (A)) to stimulate the neuron 3 andgenerate an action potential. This causes influx of Caions into the stimulated neuron 3. Thus, the fluorescenceof the Ca probe is observed (the arrow symbol "d1" inFig. 7 (A)), while the generated action potential is prop-agated to the surrounding neurons 3, producing lumines-cence of the Ca probe in the surrounding neurons 3 (thearrow symbol "d2" in Fig. 7 (A)). Observation of this lu-minescence allows propagation of the signal to be con-firmed. That is, information can be obtained regardingthe signal propagation properties of the neuron network.

(Third operational mode)

[0168] Laser light of 470 to 480 nm is focused and ir-radiated on a single neuron 3 present near the neuron 3on the fine through-hole 4 (in the cell plating section 2)and expressing channel rhodopsin (the arrow symbol "e"in Fig. 7 (A)), generating an action potential in the single

neuron 3. Thus, the action potential signal is propagatedto the neuron 3 on the fine through-hole through the net-work, and the Ca channels are opened inducing influx ofCa ions. As a result, it is possible to observe ion channelcurrent by the upper electrode 7 and the lower electrode8 that are applying a membrane potential to the neuron3 on the fine through-hole, previously set to whole-cellmode. According to the third operational mode, the signalpropagation properties of the neuron network can bemeasured on the single cell level and analyzed in detail.

(Fourth operational mode)

[0169] In the first to third operational modes, the struc-ture shown in Fig. 7(A) which is a combination of the cellplating section 2 and the fine through-hole 4, operatesas an element so long as at least one location is present.Also, if a plurality of such structures are formed on theplate 1, it will operate as a high throughput screeningdevice. In contrast, in the fourth operational mode de-scribed below, an element is constructed having struc-tures as shown in Fig. 7(A), i.e. one each correspondingto the trigger cell and the follower cell.[0170] A system that is a combination of a fine through-hole 4 and cell plating section 2 as shown in Fig. 7(A) isformed at multiple locations on the plate 1. An actionpotential is generated in several of the neurons 3 on thefine through-hole 4 (trigger cells) by current injection orvoltage application. Also, propagation of the action po-tential to the other neurons 3 on the fine through-hole 4(follower cells) can potentially be analyzed by recordingthe ion channel current of the follower cells in whole-cellmode. In these devices it is very important for the neurons3 to be placed at specified locations, and it is obviousthat the invention is highly useful for formation of a stableneuron network with specified locations.[0171] Furthermore, in the first, third and fourth oper-ational modes, Ca imaging is observed simultaneouslynot only with the ion channel current at the fine through-hole 4 but also at the top side of the plate 1, therebyallowing functional analysis of the network to be accom-plished in a more precise manner, and this is thereforehighly effective.

[Fourth example]

[0172] The fourth example is illustrated in Fig. 8(a) and(b). In Fig. 8(a), which is a flat photograph, a cell platingsection 2 comprising circular columns 12 with diametersof about 10 mm and heights of about 8 mm, appearing asa small circle, has been formed on the surface of a Siplate, neurons 3 harvested from 17-day-old rat embry-onic cerebral cortex have been seeded, and formationof a neuron network after 14 days of culturing has beenconfirmed with a fluorescent microscope. After the me-dium was exchanged with a prescribed buffering solution,Oregon Green BAPTA-1 as a Ca probe was mixed withthe buffering solution, and after approximately another 2

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hours elapsed, the buffering solution was exchanged witha solution containing no Ca probe and the neurons 3were observed with a fluorescent microscope.[0173] It can be seen that several neurons 3 are stablysituated in the cell plating section 2, and that neuronsinside the cell plating section 2 and outside the cell platingsection 2 have formed a network. Moreover, Fig. 8(b)shows the results of observing the time-dependentchange in fluorescence intensity of the neurons 3 insidethe cell plating section 2, where there can be seen re-peated active spontaneous firing of the neurons 3 andfluctuation in the Ca concentration inside the neurons 3.In this example, only one the cell plating section 2 groupis set, but if numerous such groups are set it is possibleto observe the state of signal propagation from a specificcell plating section 2 to another cell plating section 2, andto accurately and stably carry out functional analysis ofa network.

[Fifth example]

[0174] A fifth example is shown in Fig. 9. A cell platingsection composed of a plurality of protrusions surround-ing a fine through-hole in a culturing planar patch-clampelement constructed using a Si plate, was formed by thesame method as the second example to the fourth ex-ample, using a negative photoresist. Neurons harvestedfrom rat embryonic cerebral cortex were seeded by thesame method as the fourth example, and after 14 daysof culturing, it was confirmed that the neurons in the cellplating section were situated on the fine through-hole andthat the neurons had formed a network with the surround-ing neurons.[0175] Next, the media on the top side of the plate andthe bottom side were each exchanged with prescribedbuffering solutions, a 500 mg/ml concentration of nystatinwas mixed with the buffering solution on the bottom side,and after standing for approximately 10 minutes, the cur-rent flowing into an electrode set on the bottom side ofthe plate 1 in a whole-cell mode configuration was de-tected with a current amplifier (Axopatch200B).[0176] The results are shown in Fig. 9(A). Fig. 9(A)shows the membrane potential dependency of the cur-rent. Also, 40 mV-TTX is the current observed with amembrane potential of 40 mV, when the Na channelblocker tetrodotoxin (TTX) had been mixed with the buff-ering solution on the top side. When the membrane po-tential changes from minus to plus the current waveformorientation switches from down (-) to up (+), and there isexhibited the feature of signal propagation from the spon-taneously firing neurons and synaptic current from spon-taneous release of neurotransmitters. These waveformsreflect the nature of the network, and the waveform isaltered by chemical agents such as antagonists and ag-onists. Because the area occupied by the cell plating sec-tion and fine through-hole is very small it is easy to ac-complish multipoint measurement at about 100 points,easily allowing the multipoint measurement necessary

for high throughput screening.[0177] Fig. 9(B) and Fig. 9(C) show in summary theresults of measuring the time-dependent change in flu-orescence intensity by Ca imaging of each of the neu-rons, from above the plate. The abscissa represents time,and the ordinate represents the numbers assigned toeach of the neurons. The sizes of the circles representthe strengths of Ca fluorescence intensity. In Fig. 9(B),the density of the neurons was low at only 2 3 105 per35 mm dish, and the luminescence from the neurons wascompletely random.[0178] In contrast, in Fig. 9(C), the density of the neu-rons was high at 2 3 106 per 35 mm dish, thus demon-strating that luminescence by Ca imaging occurs in syn-chronization. These results mean that it is possible toaccomplish more precise measurement and analysis bymeasuring ion channel current on the bottom side of theplate while simultaneously conducting Ca imaging on thetop side.

[Sixth example]

[0179] The sixth example corresponds to a modifiedexample of the cell plating section 13. As shown in Fig.10, a cell plating section 13 is formed surrounded by aplurality of protrusions 12 in the same manner as the firstexample, on the plate surface above the opening on thefirst surface side of the fine through-hole, with the neu-rons 11 placed in the cell plating section 13. An outer cellplating section 36 is also formed on the outside of theplurality of protrusions 12 forming the cell plating section13 in a ring shape, and further surrounded by numerousprotrusions 12. That is, according to the sixth example,the rings that are formed by the protrusions 12 are in adouble ring structure with the cell plating section 13 andthe outside cell plating section 36.[0180] Fine through-holes are present in the cell platingsection 13 which is the region surrounded by the innerring, and in this region there are plated 1 to several neu-rons 11, the neurons 11 being definitely situated on thefine through-hole. Numerous neurons 11 are simultane-ously seeded in the outer cell plating section 36, whichis the region between the inner ring and the outer ring.Thus, a neuron network is formed not only between theneurons 11 inside the cell plating sections 13 but alsobetween the neurons 11 of the cell plating sections 13and the neurons 11 of the outer cell plating sections 36.[0181] Such a structure comprising cell plating sec-tions 13 and outer cell plating sections 36 is advanta-geous in that neurons that are unstable as single cellsand cannot be stably cultured without aggregation of alarge number of cells, such as iPS cells for example, canbe reliably plated as single neurons each on a finethrough-hole, while being cultured for prolonged periodsin a stable manner.[0182] A seventh example will now be briefly describedas an example of a method of seeding neurons 11 sep-arately in a cell plating section 13 and an outer cell plating

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section 36 according to the sixth example.

[Seventh example]

[0183] The seventh example corresponds to a neuronseeding device according to the invention. The devicebody of the neuron seeding device is a device for seedingof neurons in multiple cell plating sections surroundedby a plurality of protrusions on an apparatus plate, seton the apparatus plate of the culturing device for forma-tion of a neuron network or the planar patch-clamp deviceaccording to the example described above.[0184] As shown by the perspective view in Fig. 11(a),the device body 40 of the neuron seeding device accord-ing to this example is a flat board having a thickness ofabout 4 to 6 mm and a square planar shape of about 23 2 cm, and it comprises an upper board 41 and a lowerboard 42. The upper board 41 and lower board 42 areeach composed of a transparent plastic such as PDMS(polydimethylsiloxane), PMMA (polymethyl methacr-ylate) or the like or silicon rubber, that has been surface-cleaned by plasma treatment, for example, and they areheat-bonded together in a closely joined state to form atwo-layer structure.[0185] At one side of the center section on the top sideof the upper board 41 there is opened a suspension sup-ply port 43 forming a first suspension flow channel sys-tem, for external supply of a neuron suspension compris-ing neurons suspended at a fixed density. Also, at theother side of the center section on the top side there isopened a second suspension supply port 43a forming asecond suspension flow channel system. The first sus-pension flow channel system will be explained first. Thesecond suspension flow channel system will be ex-plained below.

(First suspension flow channel system)

[0186] The first suspension flow channel system com-prises a suspension supply port, a suspension flow chan-nel and a suspension injection port according to the ninthinvention to twelfth invention.[0187] First, a cross-sectional view (partially simplified)along line X-X in Fig. 11(a) is shown in Fig. 11(b), andas shown in Fig. 11(b), fine grooves with inner diametersand depths of both about 50 to 500 mm extend in abranched fashion from the suspension supply port 43 onthe bottom side of the upper board 41 (the bonding sur-face with the lower board 42). Thus, when the upperboard 41 and the lower board 42 are joined in a closelybonded state, a plurality of fine suspension flow channels44 for flow of the neuron suspension are formed by thesegrooves and the top side of the lower board 42.[0188] Fig. 12(a) shows a lower view (bottom sideview) of the upper board 41. While Fig. 11(b) shows onlya single suspension flow channel 44, in actuality a plu-rality of suspension flow channels 44 extend in abranched fashion from the suspension supply port 43

shown at the lower end of the drawing, as seen in Fig.12(a). By providing extra by-pass routes at appropriatelocations in these suspension flow channels 44, the flowchannel lengths can be adjusted to be nearly equivalent.[0189] In Fig. 12(a), the suspension flow channels 44are represented simply by dark solid lines for conven-ience. The second suspension flow channel 44a, shownhere and described below, is also represented simply bya dark solid line.[0190] Fig. 12(b) conceptually shows a top view (planview) of the plate 1 of a culturing device for formation ofa neuron network or a planar patch-clamp device, illus-trating five designated plating areas each with a pluralityof cell plating sections 2 surrounded by a plurality of pro-trusions on the top side of the plate 1 (indicated by singledots for simplicity) set in a collective manner. Fig. 12(b)also shows second suspension injection ports 45a, whichare described below, although these are in actualityformed by the lower board 42 and not formed on the plate1, but merely shown in order to highlight the positionalrelationship with the aforementioned five designatedplating areas.[0191] As further explanation based on Fig. 12(a) withthe relationship shown in Fig. 12(b), assuming the statewhere the device body 40 of the neuron seeding deviceis set on the plate 1, the plurality of suspension flow chan-nels 44 extending in a branched fashion from the sus-pension supply port 43 each arrive at a point right abovethe five designated plating areas in which the plurality ofcell plating sections 2 on the plate 1 are set in a collectivemanner, and at those locations they form five injectionhole sections 46 communicating with the plurality of sus-pension injection ports 45 formed on the lower board 42.[0192] Fig. 13 shows a magnified view of the injectionhole sections 46. At each of the injection hole sections46, the suspension flow channel 44 branches into 5 lon-gitudinal flow channels, and the five branched flow chan-nels are each connected to one of a plurality of suspen-sion injection ports 45 provided in the lower board 42.That is, as shown in Fig. 12(b), five rows of cell platingsections 2 are formed in the longitudinal direction in thefive designated plating areas on the plate 1, with five cellplating sections 2 included in each row, and five rows offlow channels are situated running in the longitudinal ori-entation of the suspension flow channels 44, completelycorresponding to all of the cell plating sections 2, whilesuspension injection ports 45 are also formed in the lowerboard 42, completely corresponding to all of the cell plat-ing sections 2.[0193] The inner diameters of the suspension injectionports 45 are essentially the same as or slightly largerthan the sizes of the interior regions of the cell platingsections 2, but smaller than the outer shapes of the cellplating sections 2 including the protrusions 12. Thus, theconstruction is such that the protrusions 12 do not enterinto the suspension injection ports 45.[0194] Consequently, when the neuron suspension issupplied in a pressurized state, for example, to the sus-

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pension supply ports 43 composing the first suspensionflow channel system, the neuron suspension is injectedinto all of the total of 250 cell plating sections 2 providedin the five designated plating areas on the plate 1 throughthe suspension flow channels 44 and the suspension in-jection ports 45, in a very short period of time and inessentially equal amounts to all. As a result, neurons areseeded in the cell plating sections 2, according to thepreferred mode described above under "Effect of the In-vention".

(Second suspension flow channel system)

[0195] The second suspension flow channel system isa suspension flow channel system for injection of a neu-ron suspension into the regions other than the cell platingsections of the apparatus plate, according to the thir-teenth invention, comprising the second suspension sup-ply port 43a shown in Fig. 11(a), a plurality of secondsuspension flow channels 44a extending in a branchedfashion from the second suspension supply port 43a asshown in Fig. 12(a), and second suspension injectionports 45a formed on the lower board 42 at each of theends of the second suspension flow channels 44a. Eachof the second suspension injection ports 45a are shownas broken lines in Fig. 12(a), and as solid lines in Fig.12(b).[0196] The structural relationship between the secondsuspension supply ports 43a, second suspension flowchannels 44a and second suspension injection ports 45a,is the same as for the suspension supply port 43, sus-pension flow channel 44 and suspension injection port45 shown in Fig. 11(b), for the first suspension flow chan-nel system. In the second suspension flow channel sys-tem, however, the flow channel lengths of the plurality ofsecond suspension flow channels 44a may be different,or the second suspension injection ports 45a may openinto regions other than the cell plating sections on theplate 1, as shown in Fig. 12(b).[0197] Therefore, when the neuron suspension hasbeen supplied in a pressurized state, for example, intothe second suspension supply ports 43a forming the sec-ond suspension flow channel system, neurons are seed-ed into regions other than the cell plating sections on theplate 1, through the second suspension flow channels44a and the second suspension injection ports 45a.[0198] When cell plating sections 13 and outer cell plat-ing sections 36 are formed as according to the sixth ex-ample, third suspension flow channels (not shown) forseeding of neurons into the outer cell plating sections 36may also be formed in the device body 40 in addition tothe suspension flow channels 44 and second suspensionflow channels 44a, into seed neurons in the outer cellplating sections 36 in the device body 40. Alternatively,neurons may be seeded in the outer cell plating sections36 utilizing the suspension flow channels 44 after thelocation of the device body 40 on the plate 1 has beenslightly shifted.

[Eighth example]

[0199] As explained above, the first suspension flowchannel system formed in the device body 40 of the neu-ron seeding device is utilized to inject a prescribed con-centration of neuron suspension into each of the cell plat-ing sections 2. Also, since the mutual gaps between theplurality of protrusions 12 of the cell plating sections 2are smaller than the dimensions of the neuronal cell bod-ies during seeding, with sufficient room allowing easyflow of the medium solution of the neuron suspension(for example, the neuron culture solution), introductionof the neuron suspension from above the cell plating sec-tions 2, with flow of the medium solution, causes the neu-rons to pool inside the cell plating sections 2 and be seed-ed therein.[0200] It is thereby possible to seed cells without dam-age in the multiple cell plating sections 2 in a short periodof time, and in an essentially simultaneous manner. Asa result, it is possible to stably and easily form a neuronnetwork comprising multiple cell plating sections (multi-ple channel current measuring points of the planar patch-clamp device).[0201] When the gaps between the protrusions 12 arethus utilized for seeding, it is important to more specifi-cally examine the relationship between the dimensionsof the gaps and the cell bodies. Specifically, neuronalcell bodies generally differ in their sizes during seedingand during culturing. Also, the cell body shapes are notperfectly round but rather elongated ellipsoid.[0202] Thus, it is necessary for the mutual gaps be-tween protrusions 12 to be smaller than the smaller valueof the minimum dimension of the cell body during seeding(the dimension in the short axis direction) and the mini-mum dimension of the cell body during culturing, but it isalso necessary for them to be formed as large as possibleso as to allow the medium solution of the neuron sus-pension to easily flow out and so that the neuronal axonsor dendrites can easily move into and out of the cell plat-ing sections 2.[0203] As an example, the results for an experimentwith rat hippocampal neurons will now be described withreference to Fig. 14 and Fig. 15. In Fig. 14, the distributionof the maximum dimension (the dimension in the longaxis direction) during seeding of numerous rat hippoc-ampal neurons is shown in the graph at the left, and thedistribution of the minimum dimension (the dimension inthe short axis direction) is shown in the graph at the right).As shown in Fig. 14, the minimum dimension of the cellbodies during seeding was approximately 7.5 mm.[0204] In Fig. 15, the distribution of the maximum di-mension (the dimension in the long axis direction) duringculturing of the same cell bodies is shown in the graphat the left, and the distribution of the minimum dimension(the dimension in the short axis direction) is shown in thegraph at the right). As shown in Fig. 15, the cell bodieswere long and narrow during culturing, with the minimumdimensions of the cell bodies being approximately 8 mm.

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[0205] A different experiment with rat hippocampalneurons will now also be described with reference to Fig.16. Fig. 16 is an optical microscope photograph of thestate of neurons on the 4th day of culturing, the four largecircles in the photograph being four groups of protrusions12 forming cell plating sections 2, and the outlines indi-cated by white solid lines representing neurons. As seenin Fig. 16, a neuron is attempting to infiltrate the cell plat-ing section 2 through a gap between the protrusions 12,from the outside. This neuron in fact did not infiltrate butregressed after some elapse of time. The gaps betweenthe protrusions 12 were 11 mm, and the dimensions ofthe neuronal cell bodies in the short axis direction were8.5 mm. In the case shown in Fig. 16, therefore, this showsthat it is safer for the gaps between the protrusions 12 tobe somewhat smaller.

[Ninth example]

[0206] The ninth example corresponds to a method ofpreparing a rat neuron suspension to be used for seeding.The suspension was prepared in the following manner.Specifically, cerebral cortexes or hippocampi were har-vested from 17- to 18-day-old Wistar Rat fetal brains andthe tissue was dispersed with enzyme treatment (37°C,20 minutes) using a 0.25% Trypsin solution. Next, a cellsuspension was prepared at 1.0 3 107 cells/ml usingserum-containing medium with Minimum Essential Me-dium (MEM) as the basal medium. The cell suspensionwas introduced and seeded in the cell plating sectionsusing a microflow device or microinjector.

[Tenth example]

[0207] The tenth example corresponds to preparationof iPS cells to be seeded. Specifically, the human inducedpluripotent stem cell (iPS cell) strain 201B7 was obtainedfrom CELL BANK by the independent administrative in-stitution RIKEN (Japan), and STO cell-derived cells(SNL), rendered proliferation impotent by inactivationwith mitomycin C, were cultured as feeder cells. Feedercells are other cells that play a role in aiding in auto-replication of iPS cells.[0208] The culture solution used was mammalian cell-culturing medium (DMEM/F12 medium) containing KSRas serum replacement, L-glutamine, non-essential ami-no acids and 2-mercaptoethanol, and it was added im-mediately before using recombinant human basic fibrob-last growth factor (bFGF). Seeding was in a 6 cm dishcoated appropriately for feeder cells, with a feeder cellconcentration of 3 3 104 cells/cm2, and the iPS cells wereseeded on the feeder cells after one day. SatisfactoryiPS cells have distinct colony outlines and high inner celldensities. Subcultures of iPS cells are usually at a fre-quency of once every 3 to 4 days. After subculturing of3 to 4 generations, in order to induce differentiation tomotor neurons, the cells were transferred from culturingon the feeder to a 6 cm dish surface-coated with gelatin

or Matrigel, for feederless culturing.[0209] After subculturing for 3 to 4 generations by feed-erless culturing, differentiation to motor neurons beganto be induced. The feederless cultured iPS cells wereinduced to differentiate to neural stem cells by suspen-sion culture in the presence of a growth factor. The dif-ferentiation-inducing medium was DMEM/F12 mediumcontaining added glucose, glutamine, insulin, transferrin,progesterone, putrescine and selenium chloride. Thesuspension culture step for inducing differentiation wassuspension culturing for 2 days at a density of 5 3 104

cells/ml. The medium was then exchanged with differen-tiation-inducing medium containing added retinoic acid(10-8 M), and suspension culturing was carried out for 4days. It was again further exchanged with differentiation-inducing medium containing added FGF2 (20 ng/ml) andSHH-N (30 nM), and culturing was carried out for 7 days.As a result of this procedure, the cell forms developedinto neural stem cells.[0210] The neural stem cells were dispersed and ad-hesion culturing was carried out on a culturing dish coat-ed with poly-L-lysine, and differentiation to mature motorneurons occurred by 5 weeks after the start of adhesionculturing. When the neuron network was to be formed ona sensor plate, the plate surface was coated with poly-L-lysine and the dispersed neural stem cells were seededon it and adhesion cultured for 5 weeks, after which anetwork containing motor neurons formed. For formingthe neuron network on a plate, the plate surface wascoated with poly-L-lysine and the dispersed neural stemcells were seeded on it and adhesion cultured for 5weeks, upon which a network containing motor neuronsformed.

Industrial Applicability

[0211] According to the invention there is provided aculturing device for formation of a neuron network, allow-ing a satisfactory neuron network to be constructed withneurons in a live state in cell culture medium while re-stricting their movement, as well as a means of utilizingthe same.

Explanation of Symbols

[0212]

1 Plate2 Cell plating section3 Neuron4 Fine through-hole5 Current amplifier6 Micropipette7 Upper electrode8 Lower electrode9 Extracellular matrix-forming substance11 Neuron12 Protrusion

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13 Cell plating section14 Plate15 Fine through-hole16 Spacer17 Spacer18 Culturing space19 Notched section20 Plate21 Plate22 Main pool23 Liquid pool section24 Liquid flow channel25 Secondary pool26 Liquid introduction flow channel27 Liquid discharge flow channel28 Electrode section29 Electrode section30 Extracellular matrix-forming substance31 Electrode receptacle32 Electrode solution33 AgCl/Ag Electrode34 Inorganic porous material35 Electrode pin36 Outer cell plating section40 Device body41 Upper board42 Lower board43 Suspension supply port43a Second suspension supply port44 Suspension flow channel44a Second suspension flow channel45 Suspension injection port45a Second suspension injection port46 Injection hole section

Claims

1. A culturing device for formation of a neuron network,wherein cell plating sections surrounded by a plural-ity of protrusions are formed on a flat plate that canbe filled with a cell culture medium, characterizedin that the cell plating section satisfying the followingconditions (1) to (3).

(1) gaps are set between the plurality of protru-sions which defines the cell plating sections,wherein the gaps are wide but do not allow aneuronal cell body to pass through;(2) the inner diameter of the cell plating sectionsdefined by the plurality of protrusions is of sizescapable of accommodating one to several neu-ronal cell bodies;(3) the plate surface forming the bottom of eachcell plating section comprises at least one con-stituent of the following (a) and (b):

(a) the plate surface is coated with an ex-

tracellular matrix-forming substance.(b) fine through-holes for suction of mediumby an aspirator provided below the plate sur-face are provided, wherein the hole diame-ters is such that the neurons cannot passthrough.

2. The culturing device for formation of a neuron net-work according to claim 1, characterized in that theculturing device corresponds to any one of the fol-lowing (1) to (3):

(1) cell plating sections are formed as selectedregions on the plate, and one to several neuronsare placed in the cell plating sections as the se-lected cells, while other neurons are simplyseeded on the plate;(2) multiple cell plating sections are formed onthe plate with appropriate gaps between them,and one to several neurons are placed in thecell plating sections, while one cell plating sec-tion is used as a selected region in which theneuron is placed;(3) a culturing device for high-throughput anal-ysis of a neuron network, wherein the cell platingsections are formed dispersedly at appropriatelocations so that several or multiple units of neu-ron networks according to (1) or (2) can beformed on the plate.

3. The culturing device for formation of a neuron net-work according to claim 1 or claim 2, wherein theculturing device for formation of a neuron network isa planar patch-clamp device designed for a neuronnetwork, characterized in that:

(1) the plate is an electrical insulating plate,wherein fine through-holes are formed so as topass through both sides of the plate surface,which composes the bottom of the cell platingsection of the electrical insulating plate;(2) liquid pool sections and electrode sectionsare provided on the neuron network-formedside, as the first surface side of the fine through-holes, and on the second surface side which isthe opposite side, respectively, wherein the liq-uid pool sections hold the conducting liquid asthe cell culture medium, and the electrode sec-tions are disposed to be electrically conductiveto the conducting liquid of the liquid pool section;and(3) the liquid pool sections on the first surfaceside are liquid pool sections for neurons platedin the cell plating sections.

4. The culturing device for formation of a neuron net-work according to claim 3, characterized in that theelectrode sections on the first surface side and sec-

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ond surface side disposed in the planar patch-clampdevice comprise the following constituents (a) to (c):

(a) an electrode receptacle, wherein at least aportion of the receptacle wall that is in contactwith the conducting liquid, when the conductingliquid is introduced into the liquid pool sections,is composed of an inorganic porous material.(b) an electrode having a precious metal chloride(NmCl) layer formed on a surface layer sectionof the precious metal (Nm), and housed in theelectrode receptacle.(c) an electrode solution filled into the electrodereceptacle, wherein the precious metal chloride(NmCl) and an alkali metal chloride are dis-solved at saturated concentration.

5. The culturing device for formation of a neuron net-work according to any one of claims 1 to 4, charac-terized in that the culturing device for formation ofa neuron network is used for a purpose according toany one of the following (A) to (C):

(A) for measurement and analysis of neuronalion channel current in a neuron network;(B) for imaging analysis, including at least Caimaging analysis, imaging analysis with synap-tophysin or synapsin labeling as synaptic sitemarkers, imaging analysis with MAP2 as a den-drite marker, and imaging analysis with FM1-43or FM4-64 which labels endosomes or exo-somes;(C) for high throughput screening systems forneuron networks.

6. The culturing device for formation of a neuron net-work according to claim 5 characterized in thatwhen the culturing device for formation of a neuronnetwork is used for imaging analysis according to(B), the culturing device comprises one or more ofthe following constituents (D) to (F).

(D) a photodetector for detecting light emittedby neurons is set above the plate;(E) an irradiating device that irradiates light ontothe neurons or plate surface is set above theplate;(F) the irradiating device of (E) is equipped withan optical focusing system for irradiation of lightonly on a prescribed single neuron.

7. A method of forming a neuron network under cultur-ing for any research purpose by means of a culturingdevice for formation of a neuron network accordingto claims 1 to 6, the method comprising:

(1) seeding neurons on said flat plate that is filledwith cell culture medium,

(2) placing and/or plating one or several neuronsin each cell plating section by an extracellularmatrix-forming substance in the cell plating sec-tion, and/or by suctioning liquid medium fromthe fine through-holes at the bottom of the cellplating section, and(3) forming synaptic junctions between neuronsby axons or dendrites, wherein while movementof the neurons plated in the cell plating sectionis restricted by the plurality of protrusions, thepresence of adjacent neurons is mutually rec-ognized through the gap sections of the pluralityof protrusions.

8. The method of forming a neuron network accordingto claim 7, characterized in that during seeding ofthe neurons in step (1), glial cells are also seededat sections other than the cell plating sections.

9. A neuron seeding device for seeding neurons in mul-tiple cell plating sections surrounded by a pluralityof protrusions on a flat apparatus plate, wherein theneuron seeding device is set on the flat apparatusplate that can be filled with cell culture medium in aculturing device for formation of a neuron network orin a planar patch-clamp device utilizing such a cul-turing device, characterized in that:

a board-shaped device body that can be set onthe apparatus plate has a size covering the mul-tiple cell plating sections and the flat bottom ofthe device body is in contact with the apexes ofthe plurality of protrusions at the multiple cellplating sections in the set situation,the device body is provided with (1) a suspen-sion supply port for externally supplying a neu-ron suspension having neurons suspended at afixed density, (2) a plurality of fine suspensionflow channels extending in a branched mannerfrom the suspension supply port, inside the de-vice body, and (3) a suspension injection portfor injecting the neuron suspension into eachcell plating section, opened at the bottom of thedevice body at the end of each suspension flowchannel.

10. The neuron seeding device according to claim 9,characterized in that the plurality of suspensionflow channels are set to have substantially the samelengths, and the design is such that when the neuronseeding device is placed on the apparatus plate ofthe culturing device for formation of a neuron networkor the planar patch-clamp device, the individual sus-pension injection ports are located to precisely cor-respond with the individual cell plating sections.

11. A neuron seeding device according to claim 9 orclaim 10, characterized in that the board-shaped

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device body comprises an upper board provided withthe suspension supply ports and a lower board pro-vided with the suspension injection ports, joined ina closely bonded state, and grooves composing thesuspension flow channels are formed on at least oneof the bonding surfaces of the upper board and lowerboard.

12. A neuron seeding device according to any one ofclaims 9 to 11, wherein the culturing device for for-mation of a neuron network is a culturing device forformation of a neuron network according to claim 1,and/or the planar patch-clamp device is a planarpatch-clamp device according to claim 3.

13. A neuron seeding device according to any one ofclaims 9 to 12, wherein the device body further com-prises a second suspension flow channel system forinjection of the neuron suspension into regions otherthan the cell plating sections of the apparatus plateof the culturing device for formation of a neuron net-work or the planar patch-clamp device.

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the Europeanpatent document. Even though great care has been taken in compiling the references, errors or omissions cannot beexcluded and the EPO disclaims all liability in this regard.

Non-patent literature cited in the description

• G. ZECK et al. PNAS, 2001, vol. 98, 10457-10462[0008]

• J. ERICKSON et al. J. Neurosci. Methods, 2008, vol.175, 1-16 [0008]

• F.W. PFRIEGER et al. Science, 1997, vol. 277,1684-1687 [0059]

• K.S. WILCOX et al. Synapse, 1994, vol. 18, 128-151[0166]