3D cell culture Layout 1 - Drug Discovery World, Drug Discovery

Increasingly the term three-dimensional (3D) isbeing applied in relation to cell culture.Simplistically this involves growing cells in a

3D environment, matrix or on a scaffold with 3Darchitecture as opposed to the flat surface of a con-ventional two-dimensional (2D) culture vessel.However, what 3D cell culture encompasses is hardto define and varies widely depending upon theapplication. In most drug discovery areas and instem cell research we are mainly talking aboutanchorage-dependent cell culture on a 3D scaffold,which can in the least demanding cases be made ofthe same material as the 2D surface, although often

more complex biological gels or coatings arerequired. In tissue engineering and clinicalresearch, and in some aspects of safety assessmentand the delivery of stem cells, the focus is on actu-al organ and tissue development. In an attempt tobring some clarity to the subject, HTStec under-took a survey and report on 3D cell culture inFebruary 20101.

Main advantages of 3D cell cultureTo better understand what is driving the investiga-tion of 3D cell culture, the survey first sought toidentify what were perceived as the most important

By Dr John Comley

Drug Discovery World Summer 2010 25

Cell Culture

3D CELL CULTUREeasier said than done!

The transition from cell culture on the flat surface of a conventional two-dimensional (2D) culture vessel to a three-dimensional (3D) environment,matrix or scaffold with 3D architecture has begun, and is providing muchneeded support for emerging applications in tissue engineering and stem cellresearch. Biomimetic scaffolds (eg hydrogel or collagen) have shown potentialin culturing specific cell types and in investigating different aspects of the cell-matrix interaction in 3D. Structural scaffolds, made from the same material as2D plate surfaces (ie polystyrene), would seem to be compatible with manyroutine (easy) 2D assays. Microfluidic devices with moulded microchannelsincorporating biomimetic scaffolds are now available to support specific 3Dapplications, eg invasion assays and specific tumour cell models. Systemsdirectly supporting the automation of 3D cell culture and/or tissue creationare beginning to emerge and should facilitate the scale up of cell production,but also impact how 3D generated cells are used in drug screening assays andhow organs and tissues can be consistently produced. The prospect ofdeveloping more physiologically relevant 3D models systems for use in in vitrotoxicology is particularly compelling. However, the state-of-the-art is still someway off from providing fully validated or robust 3D culture solutions and toolsand the field remains open to major improvements at this point in time.

3D cell culture_Layout 1 01/07/2010 09:53 Page 25

advantages of 3D cell culture. This revealed thatbetter mirrors the environment experienced bynormal cells in the body was the most importantadvantage to survey respondents. In the rankingthis was closely followed by replicates complex tis-sue structures and in vivo-like morphology; andthen better reflects normal differentiation, polari-sation, cell behaviour and intercellular interac-tions. Ranked least important advantages weresimpler to automate and eliminate the need forharsh cell dissociation solutions (Figure 1).

Main application for 3Dscaffolds/formatsThe main application for the 3D scaffolds/formats(ie the substrates on which cells are cultured) underinvestigation by survey respondents are presentedin Figure 2. By application here we include boththe cell origin and the desired outcomes. Thisanalysis showed that the majority (64%) wasinvestigating stem cells, this was closely followedby primary cells (61% investigating) and humancell lines (57% investigating). Other importantapplications were investigated by fewer respon-dents, eg tissue engineering (38% investigating)and cancer cells (35% investigating).

Types of primary cells mostinvestigated for 3D cell cultureThe type of primary cells most investigated by sur-vey respondents for 3D cell culture was fibroblasts,this followed by endothelial cells, mesenchymalstem cells, and then hepatocytes (Figure 3).

3D scaffolds/formats that have shownmost promiseThe 3D scaffolds/formats that have shown mostpromise in 3D cell culture was gel/hydrogel, thiswas followed by ECM (extra-cellular matrix)sheet, aggregates/spheroids and then collagen tis-sue constructs (Figure 4).

Where 3D cell culture will impact the mostSurvey respondents ranked tissue/organ engineer-ing as the area where they expect 3D cell culture toimpact the most over the coming years. This wasfollowed by all aspects of basic research and thendrug discovery application areas (Figure 5).

Assay types most successfullydemonstrated with 3D culturesThe assay types survey respondents have most suc-cessfully demonstrated to date (2010) with 3D cellcultures were cell viability, closely followed by cellproliferation, then cell migration and cell signallingassays (Figure 6).

Transitioning from 2D to 3D culturesTwo-thirds of people surveyed plan to transitiontheir cell culture from 2D to 3D, with half of thesehaving already transitioned some part of theirwork to 3D. Greater biological relevance wasranked as the most important reason for transi-tioning from 2D to 3D cell culture. This was fol-lowed by enhanced cell viability/responsivenessand better quality of assay results (Figure 7).

26 Drug Discovery World Summer 2010

Cell Culture

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Simpler to automate

Less cell numbers required

Significant cost saving compared to alternative approaches

Shorter production times relative to similar to current monolayer cultures

of the target tissue

More realistic cell biology and function

Better reflects normal differentiation, polarisation, cell behaviour and intercellular interactions

Replicates complex tissue structures and in vivo-like morphology

Better mirrors the environment experienced by normal cells in the body

Eliminates the need for harshcell dissociation solutions

More mechanistically accurate modelling

and drug responsesMore predictive of disease states

MEAN RANKED ORDER 1 to 11, where 1 = least important and 11 = most important

Figure 1: Most important advantages of 3D cell culture

© HTStec 2010

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Figure 2: Main applications for 3D scaffolds/formatsinvestigated

© HTStec 2010


3D automationThe ability to automate 3D cell culture should facili-tate the production of large quantities of cells, butalso impact how 3D generated cells are used in drugscreening assays and how organs and tissues can beconsistently produced. When survey respondentswere asked about their use of automation in 3D cellculture today, most (86%) replied they were not yetusing it and/or don’t have a system, only 7% havealready implemented changes to existing 2D auto-mated cell culture systems to enable 3D, with a fur-ther 7% actively looking at or investigating 3Denhancements. In separate questions 43% of surveyrespondents indicated that the availability of auto-mated equipment would influence their future choiceof 3D scaffold/format; and 32% indicated that theyexpect to be able to use existing equipment to scaleup and automate their chosen 3D scaffold/format.

Latest developments in 3D cell CultureThe following vendor snapshots provide addition-al details and describe some of the latest develop-ments in 3D cell culture, tissue generation andrelated automation:

Inspired by the principle that tissue engineeringscaffolds can be used to create 3D in vitro tissueand organs, 3D Biotek (www.3dbiotek.com) hasdeveloped two 3D cell culture product lines, thebiodegradable scaffold, 3D Insert™–PCL, andnon-degradable scaffold, 3D Insert™-PS. With theproprietary 3D Precision MicrofabricationTechnology, 3D Biotek can produce 3D scaffoldswith well controlled and 100% connected porousstructure. The 3D Insert™-PCL scaffolds are madefrom biodegradable poly(γ-caprolactone) (PCL).These PCL scaffolds are 100% interconnected,have a very well controlled porosity, and are main-ly designed for applications in tissue engineeringand stem cell research. 3D Insert™-PCL has most


Cell Culture

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Figure 3:Primary cell types investigated using 3D cell culture

© HTStec 2010

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Bead/microsphere

Collagen tissue construct

Aggregates/ spheroids

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Gel/hydrogel

% Shown greatest promise

Figure 4: 3D scaffolds/formats that have shown most promise

© HTStec 2010Figure 8: 3D Biotek 3D Cell Culture Kit whichcontains 24 96-well 3D Insert™-PS scaffolds


recently been chosen by The National Institute ofStandard Technology (NIST) as the reference 3Dtissue engineering scaffold. The non-degradable3D Insert™-PS has been developed for conductingroutine 3D cell culture. Made from the same mate-rial as traditional tissue culture plates, 3D Biotekhas essentially engineered 2D polystyrene into a3D scaffold. Combining the precisely engineered3D structure with the inherent transparency ofpolystyrene material, creates an ideal 3D Insert™-PS scaffold for performing easy 3D cell culture.These PS scaffolds are compatible with most 2Dassays and allow researchers to monitor 3D cellgrowth by simply using an inverted light micro-scope. 3D Biotek has initiated a partnership withBioCellChallenge SAS, a drug delivery company inFrance, and developed the world’s first 3D celltransfection kit, which combines 3D Insert™-PSand Transfection Reagent into one kit. This is theworld’s first 3D in vitro transfection technology,which now allows researchers to achieve highdelivery efficiencies of plasmid DNA into 3D cul-tured cells (Figure 8).

BellBrook Labs (www.bellbrooklabs.com) hasdeveloped a device and methods to address theneed for automatable and high-content assays for


Cell Culture

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Drug discovery – DMPK

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Drug discovery – secondary screening/lead opt.

Drug discovery – target validation

Basic research – developmental biology

Basic research – cell biology

Basic research – cell culture methodology

Tissue/organ engineering

MEAN RATING SCALE 1 to 5, where 1 = no impact and 5 = major impact

Figure 5: Where 3D cell culture is expected to impact most

© HTStec 2010

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Simpler automation

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Enhanced cell viability/responsiveness

Greater biological relevance

MEAN RANKED ORDER, where 1 = least important and 7 = most important

Figure 7: Most important objectives of transitioningfrom 2D to 3D culture

© HTStec 2010

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Patch clamping

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Figure 6: Assay types successfully used/investigated in 3D cell culture

© HTStec 2010

Figure 9: A) Schematic of BellBrook Labs IUVOMC5250 Microchannel. B&C) BxPC3 cells suspended in3D Type I Collagen were incubated 18 hours and thenstained for EdU incorporation using Invitrogen ClickITEdU Imaging Kit. Hoescht staining of nuclei is shown inblue, and cells with incorporated EdU are shown in red.An average of 19% of untreated cells (B) haveincorporated EdU, compared to 0% for cells treated (C)with 10µM Cycloheximide. n = 4, p<0.001


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Cell Culture

pancreatic cancer cell models in 3D matrices. Thedevice consists of an array of microfluidic chan-nels specifically designed for the culture of cells in3D format. For the pancreatic tumour cell model,BxPC3 cells were added to the channel suspendedin neutralised Collagen Type I which traps thecells in 3D upon gelling. The channels are open ateach end and allow for a droplet placed at one endto flow through the porous fibrillar collagenmatrix to equilibrate the pressure across the chan-nel. As opposed to well-based 3D culture, this sim-ple addition and removal of droplets of growthmedia and assay reagents to the channels is donewithout disruption of the matrix and cells. Afteran overnight compound treatment, the cells werefixed, permeabilised and stained for EdU incorpo-ration during DNA replication using Invitrogen’sClickIT Alexa 594 EdU Imaging Kit. Compoundsthat inhibit cell proliferation or are overtly cyto-toxic have a lower incorporation of EdU. Thechannels are 140µm tall, which allows for imagingof the full depth of the 3D matrix with low powerobjectives. Here, (see Figure 9) we captured thechannel region in one shot for each label (Hoeschtand Alexa 594 EdU). Image analysis was per-formed with Metamorph’s Multi-wavelength CellScoring application in which cells are first seg-mented by their nuclear Hoescht stain and thenscored for EdU incorporation.

BioTek (www.biotek.com) is interested in theautomation of cell-based assays, particularly inGlobal Cell Solutions GEM 3-D matrix as it sim-plifies the work flow of cell-based high throughputscreening assays. Current cell culture techniquesare two-dimensional (2-D) where cells attach to themicroplate surface in a single monolayer.Trypsinisation is required to split cell cultures, andprepare them for downstream applications, whichbecomes increasingly difficult as scale-up isrequired for primary and secondary screens. 3-Dcell culture using GEM, where cells grow on micro-carriers suspended within the culture, eliminatesthe need for trypsinisation and enables the growthof high-density cultures in a small volume. Cells onthe optically-transparent microcarrier can be dis-pensed directly from culture or frozen in situ to bethawed when needed without further culturing.Figure 10 demonstrates the comparison ofHistamine 1 antagonist pharmacology usingGeneBLAzer® H1-NFAT-bla HEK 293T cellsfreshly cultured on GEM; frozen on GEM, thenthawed; and using standard 2-D cell culture proto-cols. Receptor pharmacology was found to beequivalent across the three formats.

CellASIC (www.cellasic.com/3D) has developed the3D:M microfluidic plate, enabling long term perfu-sion culture of cells in a 3D environment. Themicrofluidics are integrated with a standard 96-wellframe and do not require any external pumps, max-imising compatibility with existing assays. Eachperfusion unit consists of four well positions (inlet,culture chamber, cell/gel inlet, outlet) resulting in 24independent units per plate. The user loads theirselected cell/gel combination via capillary flow tofill the 150 nanolitre culture chamber. Differentprotocols allow for embedding cells in gel, overlayof gel on a layer of cells, or 2D culture without gel.The micro-chamber floor is a #1.5 thickness glasscover slide to facilitate high magnificationmicroscopy. The cells are fed by gravity driven per-fusion of medium, set to a rate of 40

Figure 11(a) The 24-unit CellASIC 3D:M microfluidic 3Dperfusion plate. (b) MCF-10Abreast epithelial cells culturedin Matrigel (BD Biosciences)for four days in the micro-chamber, and (c) actin staining highlights the acinarorganisation of cells in 3D culture

Figure 10: Comparison of Histamine 1 antagonist pharmacology using GeneBLAzer® H1-NFAT-bla HEK 293T cells. All assay steps were automated: cells and LiveBLAzer substrate weredispensed using the BioTek MicroFlo™ peristaltic pump dispenser; antagonist dose responseswere serially diluted using the Precision™ automated pipetting station; and GeneBLAzer FRETsignals measured using the BioTek Synergy™ 4 MultiMode Microplate Reader



Cell Culture

microlitres/day. An innovative microfabricated per-fusion barrier promotes uniform nutrient transportinto the gel chamber. They key advantages of the3D:M design are: 1) enabling 3D perfusion screen-ing; 2) high quality cell imaging in a 3D environ-ment; and 3) reduction of cell/gel usage by 10 times.CellASIC has also developed variations of the 3Dmicrofluidic culture concept to address applicationsrequiring: larger gel chambers, automated solutionexchange, maintaining long term spatial gradients,and in-chamber immunostaining (Figure 11).

CELLnTEC Advanced Cell Systems (www.cell-ntec.com) applies the latest developments in adultstem cell signalling to formulate speciality cell cul-ture media unique to the epithelial market.CELLnTEC began as a CRO developing 3D vaginaland bladder models. This led to a number of epithe-lial culture media, and primary cells isolated in thesemedia especially suitable to 3D cell culture.CELLnTEC has optimised a skin medium that sup-ports the conflicting requirements of 3D in vitromodelling. 3D models place very specific demandson the cell culture medium. Specifically, the mediummust encourage cells to reach terminal differentia-tion and stratify, while in parallel maintaining thepopulation of proliferative cells necessary in thebasal layer to establish a fully stratified model.These conflicting requirements are poorly addressedby conventional media, which were developed sole-

ly on the need for isolation efficiency and prolifera-tion, without consideration of these parallel require-ments of a 3D model. In combination with primaryhuman keratinocytes isolated in CELLnTEC’sProgenitor Cell Targeted media, this 3D medium hasbeen found to establish 3D epidermal models withaccurate representation of the in vivo structure,marker expression and lipid profile and to maintainthem for an extended period. CELLnTEC also has athriving CRO business testing compounds on theseskin models. Additionally, outside laboratories areusing its cells and media for 3D Airway models,corneal transplantation research and 3D co-cultureexperiments. CELLnTEC has recently invested innew facilities and staff to extend its offering of invitro 3D models both via CRO and packaged prod-ucts (Figure 12).

The ‘Cell Cycle’ is a partnership formed betweenthe European Collection of Cell Cultures (ECACC)(www.hpacultures.org.uk), Sigma Aldrich(www.sigmaaldrich.com), Corning (www.corn-ing.com) and XCellR8 (www.x-cellr8.com). Thispartnership fully recognises that proliferation anddifferentiation of cells in culture has becomeincreasingly important for basic research, drug dis-covery, tissue engineering and regenerative medi-cine. The way in which cells proliferate and differ-entiate is defined by the unique microenvironmentsrequired by the different cell types in a multicellu-lar organism. Several factors influence the cells’proliferative and differentiation status and thesefall into two broad categories: soluble cues (growthfactors, metabolites, dissolved gases) and insolu-ble, physical cues (the composition, architecture,

Figure 12: A fully differentiated 3D epithelial model, established from a population ofepidermal keratinocyte progenitor cells isolate in CELLnTEC Progenitor Cell Targetedmedium and differentiated in CELLnTEC optimised 3D medium

Figure 13: Human mesenchymal stem cells grown byECACC for five days on the surface of a HyStemhydrogel with non-covalently incorporated collagen I



Cell Culture

and elasticity of the extracellular matrix (ECM)and cell-cell interactions). The incorporation ofthese cues for in vitro cultivation of cells is crucialfor recreating a cell’s natural niche. For example, inmyogenic induction, it has been found that theMyoD1 muscle marker protein will only be fullyexpressed by Murine muscle cell cultures when thehydrogel matrix stiffness matches that of muscle(11kPa). Hydrogel systems such as GlycosanBiosystem’s HyStem™, allow researchers to designenvironments in which cells respond in a physio-logically relevant manner. HyStem™ is a customis-able synthetic hydrogel that can be used as an invivo animal-free cellular delivery vehicle which isbiocompatible, biodegradable and injectable.HyStem™ is optimal for culturing stem cells whosenatural environments are rich in hyaluronic acid.The HyStem™ hydrogel scaffold closely mimicsthe rich, natural extracellular matrix environment,complete with hyaluronic acid and collagen fibrils,into which appropriate growth factors, attachmentfactors, and proteins can be incorporated selective-ly by the researcher. The Cell Cycle partnership isworking to deliver complete cell culture solutionsfor researchers (Figure 13).

As the biomedical research community continuesto make extraordinary strides in the prevention,diagnosis, and treatment of debilitating diseases, ithas become more critical that reliable, efficient,affordable, and relevant techniques for the cultureof cells, particularly stem cells, become available.Global Cell Solutions (GCS) (www.globalcellsolu-tions.com) along with Hamilton Company(www.hamiltoncompany.com) have developed theideal cell culture system that allows for 3D stemcell cultures at the benchtop. This cell culture solu-tion is a magnetic, pipettable and application-friendly microcarrier called the GEM™. TheGEM™ consists of a magnetic core, an opticallyclear and non-autofluorescent alginate micros-phere, and a thin molecular layer of basement bio-mimetic coating. Cells are loaded on to the GEM™where the coating allows them to divide andexpand. GCS offers a variety of kits to researchers,including one with the pre-coated and ready-to-uselaminin and basement membrane GEM™ micro-carriers to promote the expansion and mainte-nance of most adherent stem cells feeder-free. Stemcells can now be magnetically manipulated open-ing the door for co-cultures and novel experiments.The cells can be sampled, transferred, monitoredfor pluripotency, assayed, imaged and cryopre-served directly on the microcarrier. Furthermore,they never have to come near trypsin during these

applications. The magnetic GEM™ can be gentlylevitated and dispersed in the growth media whilealso enabling full automation of the cell cultureprocess. The alginate core of the GEM™ sharesproperties with hyaluronic acid which has beenshown to affect differentiation. Thus, the GEM™makes 3D stem cell culture convenient, reliable andefficient (Figure 14).

Figure 14: Human neural stem cells begin directed differentiation on the Global CellSolutions GEM (image courtesy of Life & Brain)

Figure 15: Chicken embryonic cardiomyocytes (striations) and fibroblasts (no striations)cultured with Glycosan BioSystems HyStem hydrogel. Blue = Nuclei, Red = Actin and Green= Alpha-Actinin



Cell Culture

Glycosan BioSystems (www.glyconsan.com) man-ufactures and sells Extracel™ and HyStem™hyaluronic acid-based hydrogels for 3D and stemcell culture. As 3D cell culture increases in popu-larity, a key need for researchers is to gently andrapidly recover encapsulated cells for either nucle-ic acid or protein extraction. Glycosan has recent-ly launched Extracel-SS and HyStem-CSS hydro-gels which dissolve within two hours. The keyimprovement is the novel crosslinker, PEGSSDA,which has an internal disulfide bond which breaksupon exposure to reducing agents (Figure 15).

Hamilton Company (www.hamiltoncompany.com)and Global Cell Solutions (www.globalcellsolu-tions.com) have joined forces to develop theBioLevitator™, a unique 3D cell culture system.The instrument is a self-contained incubator andbioreactor with temperature and CO2 control. TheBioLevitator is used to culture adherent cell types

on a unique 3D magnetic substrate called theGlobal Eukaryotic Microcarrier or GEM™, devel-oped by Global Cell Solutions. The GEM is com-posed of an alginate core with covalently boundproteins, which allows the cells to grow on the 3Dcurved surface. Multiple protein coatings are avail-able to support established, cancer, primary, andstem cell lines. Cells cultured on the GEM, includ-ing primary and stem cells, are healthier and haveshown an improved in vivo phenotype. The GEM isnon-autofluorescent and optically clear makingcell-based assays much more user friendly. Cells canbe easily assayed via fluorescence, luminescence, orabsorbance detection methods. Additional down-stream applications, including electroporation andcryopreservation, have shown enhanced efficiencywhen the cells are attached to the GEM. The cellculture parameters of the BioLevitator are directlyscalable to the 3D-CellHOST. The 3D-CellHOSTintegrates up to four BioLevitators on Hamilton’sMICROLAB STAR automated liquid handlingplatform and has the ability to produce two billioncells per week. The automated platform eliminatesuser error and variation between cell lines andusers. The latest advancement in the BioLevitatortechnology is the addition of a pH reader, for onlinemonitoring of the quality of the cell culture mediumand automated medium change (Figure 16)

Microscale organotypic cell-culture technologiesare currently shifting from the academic environ-ment to an industry setting enabling better modelsystems to test for drug efficacy and toxicology invitro. Over the past few years, the hanging droptechnology has shown its versatility to recreateembryonic, tumour and primary tissues in vitro.However, the conventional hanging drop technol-ogy – placing drops of cell suspension on a surfacewhich is cultivated upside down – did not allowfor high-throughput production of microtissues.The GravityPlus technology from InSphero(www.insphero.com) allows for generating hang-ing drops as well as medium exchange from thetop enabled by a special well-design where theinlet and the culture compartment are connectedvia a vertical microchannel. The capillary andcohesion forces lead to highly stabilised hangingdrops which can be handled either manually or inan automated fashion. This system enablesInSphero to produce microtissues with a numberof substantial advantages: a) low cell numbersrequired for one tissue depending on the requiredsize (100-25,000); b) no adverse effects of artifi-cial materials on biochemical assays; c) standard-ised tissue size and one tissue per drop enables

Figure 16: Hamilton Company’s BioLevitator™ 3D cell culture system

Figure 17: Automated generation of hanging drops to produce microtissues using theInSphero GravityPlus technology



Cell Culture

precise microscopic analysis and excellent cellnumber-size correlation; and d) the possibility toincrease the complexity from homotypic to het-erotypic tissue models. These tissues are providedoff-the-shelf or custom-made in 96-well-compati-ble plates to allow for a straightforward upgradefrom 2D to 3D at the user lab using existinginstruments and assay procedures (Figure 17).

Kuraray (www.kuraray.co.jp) is developing a newcell culture plate called the Micro-Space CellCulture plate by utilising its micro-fabricationtechnology. The plate has a number of micrometersize compartments regularly arrayed on its surfacewhich provide cells ‘micro-space’ to form 3D struc-ture. The plate has several advantageous features:it conforms to the standard microplate footprintenabling simple handling; it has good observabili-ty; and there is uniformity in the size or shape ofthe microstructure. No special techniques, includ-ing gel formation, are required. The bottom of theplate is a thin film made of transparent material,polystyrene, so that it is suitable for microscopicobservation for fluorescent immunoassay. The sizeof the cell aggregates can be controlled by the sizeof the micro-pattern used. The shape of the micro-pattern can be designed as desired. It is believedthat an optimum micro-pattern varies dependingon cell type. For instance, primary hepatocytesform a spheroid-like structure in a square com-partment (eg surface which width x 100µ depth)(see Figure 18). Hepatocyte-specific functions, forexample albumin secretion and some ofcytochrome P450s activity, were well preserved onthe Micro-Space Cell Culture plate compared tothose on a conventional flat plate by several-fold asmeasured by mRNA expression. On the otherhand, cardiomyocytes form cell networking onanother pattern providing synchronous contrac-tion, and mammary epithelial cells present acinus-like structure on other pattern showing similarmorphology to that seen in vivo. The Micro-SpaceCell Culture plate is currently nearing commercial-isation. Kuraray has prepared 24-well and 96-wellsample plates for collaborators in Academia andPharma, but other formats 6-, 12- and 384-wellwill be made if Kuraray confirms demand. Kuraraybelieves the plate is applicable for HTS due to thesimple handling and good observable feature.

QGel (www.qgelbio.com) recently commercialisedQGel™ MT 3D Matrix (QGel™), a synthetichydrogel supported by more than 10 years of sci-entific experimentation and publications (Figure19). Among many of its applications, QGel™ can

be used as a matrix for 3D cell culture and is par-ticularly interesting for studies in regenerativemedicine, cancer research and drug screening. Thesynthetic biological and biochemical componentsused to bioengineer QGel™ extracellular matrix(ECM) are what make QGel™ unique comparedto currently available products. Protease-sensitivesites and cell-adhesion ligands, such as RGD (pep-tide containing the amino acid sequence Arginine-Glycine-Aspartic Acid), are key components ofQGel™ that mimic essential features of naturalECM (Figure 19). Unlike other synthetic products,QGel™ degrades like natural ECM, ie by cell-secreted and activated proteases, such as MMP(matrix metalloproteinase). QGel™ components

Figure 19: QGel™ issynthetic gel that consists of apolymer (a) structuralcomponent, (b) proteasesensitive sites, and (c) cell-adhesion ligands. These threecomponents are pre-mixed asa powder in a single vial. Aftersolving the powder withQGel™ Buffer (not shown) a(d) 3D hydrogel structureforms that mimics key featuresof the natural extracellularmatrix. Gelation occurs in 5-10 minutes, which allows youto encapsulate cells and shapethe gel as you wish, such as inthe form of a (e) 3D disc.QGel™ matrix products areavailable as (f) non-degradableand (g) degradable gels (withand without incorporatedRGD-peptide) delivered sterilein ready-to-use vials for 0.5mLof gelated matrix

Figure 18: Microscopic observation of primary hepatocytes on Kuraray Micro-Space CellCulture plate



Cell Culture

that define the biological and biochemical charac-teristics of its matrix are like building-blocks thatallow for QGel to be bioengineered on a molecularlevel. This building-block modularity gives QGel™advantages over natural ‘gold standard’ matrices,where ‘what you get’ depends on the origin of theorganism from which it was derived. The ability tosynthesise and bioengineer QGel™ offers scientistsan exclusive possibility to adapt these biomimetic3D cell culture models to specific cell types and toinvestigate different aspects of cell-matrix interac-

tion in 3D. QGel™ matrix products are availablein sterile, ready-to-use vials manufactured understrict GMP conditions.

RealBio™ Technology (www.realbiotechnology.com)has introduced the D4 Culture System, a revolution-ary tool for growing cells and tissues in a setting thatclosely mimics the in vivo environment. TheRealBio™ D4 Culture System represents an exponen-tial improvement over current 2D and 3D cell culturetechnologies because it supports mixed-cell popula-tions in varied microenvironments throughout anopen scaffold. As a result, tissues and cells cultured inthe D4 Culture System arrange as in vivo and demon-strate natural function. In addition, cells cultured inthe D4 system exhibit migration throughout and outof the tissue mass, just as cells naturally migrate invivo. The D4 Culture System also providesresearchers with unprecedented control of the cultureenvironment, including control of nutrient and gasgradients across the cultured tissue mass, by decou-pling the supply of nutrient media and metabolicgasses. The D4 Culture System was originally devel-oped with a $5 million grant from the US military andperfected within the RealBio™ Technology laborato-ry in Kalamazoo, Michigan. The patented D4 Systemis a valuable tool for studying various structured tis-sues (including polar tissues) and has demonstratedthe ability to support the growth and maintenance ofhuman bone marrow for longer than one year withdaily harvests of a mixed-cell population that includesearly progenitor stem cells. Mixed-cell populationsharvested from long-term cultures maintained in the D4 system exhibit similar CD marker profiles to the initial seed material thereby demonstrating that the RealBio™ D4 Culture System minimises the selective pressures observed in other culture systems(Figure 20).

RegeneMed (www.regenemed.com) provides 3Dhuman and animal tissue cultures in multiwellplates and specialised bioreactors to serve as morephysiologically relevant replacements to currentindustry-standard animal and cell-based tests thatare often not predictive of the human response totoxic compounds. Liver3, 3D liver co-culture, sus-tains tissue function for months, providing off-the-shelf available, reproducible, reusable, predictionof metabolism, toxicity and efficacy measures fornew drug candidates, chemical entities and con-sumer products. The long-term tissue-specific func-tion enables pre-characterised tissues from freshand cryopreserved hepatocytes for in vitro assess-ment of previously unattainable endpoints such asbioavailability, drug-drug interactions and chronic

Figure 20: Tissue Equivalent Culture in the RealBio™ D4 Culture System

Figure 21: RegeneMed Liver3 and RegeneTOX product line for safety and efficacyassessment of new chemical entities



Cell Culture

toxicity. The RegeneTOX product line provides tis-sues, customised assays and Contract TestingServices for safety and efficacy assessment of newchemical entities. Included are 2D hepatocytes and3D liver co-culture from the same liver donor andsibling animal dosing for in vitro 2D to 3D to invivo comparisons. Skin3 3D full- and partial-thick-ness skin models (based on the Skin2 product linepulled from the market in 1996 and expanded toinclude immune competent models to address sen-sitisation, inflammation, etc) will be re-launched toaddress the growing animal alternative market andregulations. Stem cell-derived 3D tissues, includingliver, cardiomyocyte and neuronal, for patient-spe-cific and genomic diversity research are in develop-ment. The mission of the company is to provide tis-sues and organs to address medical need regardlessof disease progression state; from miniaturised tis-sue for high throughput safety and efficacy assess-ment, to chemical/biological warfare biosensors,molecular diagnostics, personalised medicines,medical devices and tissue implants (Figure 21).

Reinnervate (www.reinnervate.com) hasresearched and developed Alvetex™, a uniquepolystyrene scaffold designed as a platform tech-nology to enable routine 3D cell culture. The com-pany is now focused heavily on preparing its com-mercial facilities to support quality controlled pro-cedures for the scaled manufacture and productionof Alvetex™, due for formal product launch in var-ious formats later this year. Its product quality test-ing levels exceed the various standards required fora consistently high performing cell culture product.This is essential for the global distribution ofAlvetex™ to the industrial sector. Its developmentwork includes the manufacture of various devicesdesigned to present the scaffold, to enable optimalfunction and versatility. For example, well inserts,well clips and large reservoir vessels are all impor-tant ancillary components to maximise the benefitsof 3D cell culture using Alvetex™. Reinnervate’stechnology is adaptable to scaled manufacture andit has overcome various challenges to deliver aquality 3D cell culture product. While its productis designed as a generic solution to 3D cell culture,scientists at Reinnervate are also developing specif-ic applications for Alvetex™, notably: an artificial3D skin construct for in vitro testing of cosmeticsand topical drugs; a liver toxicity assay using 3Dcultured hepatocytes; a 3D platform for enhancedcell differentiation by various stem cell types; anda cell invasion model to evaluate cell migration in3D to assess various anti-cancer compounds.Alvetex™-enabled 3D cell culture will represent

the core technology behind each of these applica-tions for which there is significant market demand(Figure 22).

Expansion of hematopoietic stem cells for bonemarrow transplantation using conventional 2Dculture systems with exogenous growth factors hasmet with little success. Recent research has shownthat the stem cell microenvironment (stem cellniche) plays a key role in the maintenance andexpansion of stem cells. Synthecon (www.synthe-con.com) has developed, with funding from theNational Institutes of Health, a 3D, bio-artificialstem cell niche in its Rotary Cell Culture System(RCCS™) to address this problem. The bio-artifi-cial niche consists of disk-shaped porous scaffolds,bone marrow stromal cells and umbilical cord

Figure 22: Reinnervate’s Alvetex™ is supplied as a 200 micron thick membrane that enablescultured cells to form a 3D structure. This sample has been fixed, embedded, transversesectioned, and cells stained with Toluidine Blue. Alvetex™ is seen in white

Figure 23: Bio-artificial niche assembled in the Rotary Cell Culture System. Bone marrowmesenchymal cells expressing GFP were grown on a porous 3D scaffold. Umbilical cordblood stem cells stained with DilC18(5)-DS were added and allowed to home to the scaffold



Cell Culture

blood stem cells. The bone marrow stromal cells(MSCs) are first seeded with the scaffolds in theRCCS and allowed to proliferate to confluence.The umbilical cord stem cells are subsequentlyinjected into the bioreactor and induced to home tothe scaffolds by chemoattractive signalling fromthe MSCs. Stem cells will be detached from thescaffolds by mobilising agents currently used invivo. Synthecon has designed a modification of its

standard RCCS that will allow harvesting of thestem cells without disturbing the bio-artificialniche. The goal of this project is to mimic the invivo production of hematopoietic stem cells in ascalable bioreactor system that can reliably expandthese cells for therapeutic applications (Figure 23).

RAFT (Real Architecture for 3D Tissue) is a new sys-tem for scientists to create consistent, well defined3D tissues in a convenient, simple-to-use format. Ithas been developed by The Automation Partnership(TAP) (www.automationpartnership.com) in collab-oration with leading tissue engineering academicsand uses a novel, patented technology for making 3Dcollagen tissue constructs rapidly (<1 hour), simplyand reproducibly. Cells and neutralised collagen aremixed, pipetted into a special 24-well plate and incu-bated. Gentle, controlled compression is applied tothe cell seeded hydrogels (by absorbent plungers)which removes some liquid, increasing the cell andcollagen concentration 100-fold. The construct sur-face can be embossed with micro scale topology, andcould be used for example, to mimic the in vivo stemcell microenvironment, and then culture a differentcell type on the surface. RAFT allows complex mul-tilayer tissues to be formed – with different cell typesin each layer – and cells to be co-cultured in a wellcontrolled way. The resulting biomimetic tissues,made from fibrillar collagen (the main component ofextracellular matrix), are strong, transparent and 50-100µm thick. TAP has developed a workstation andconsumables to automate and scale up this 3D tissueproduction process, enabling up to 24 tissues to bemade in parallel. Tissues are made either in the wellsof a 24-well plate; on permeable membrane insertsfor barrier assays or cultured at an air/liquid inter-face, for example to form stratified epithelia. The tis-sue remains in the same well from its creation untilthe end of the experiment and can be analysed usingstandard techniques. TAP will be unveiling RAFT atthe Tissue Engineering and Regenerative MedicineInternational Society (TERMIS) Conference, on June13-17, 2010 in Galway, Ireland (Figure 24).

The DermiGen™ Mechanical Bioreactor, manufac-tured by Tissue Growth Technologies (www.tissue-growth.com), imparts static and oscillatory strainto a skin-like sample in a unique air/media inter-face. The device may be used to stimulate thegrowth of tissue engineered skin or to act as a testbed for drug and cosmetic development. Featuringa linear motor driven mechanical stimulator, theDermiGen applies mechanical strain using opera-tor defined parameters. The bioreactor’s uniquechamber design provides nutrient media perfusion

Figure 24: The RAFT (Real Architecture for 3D Tissue) system from The AutomationPartnership forms tissues in less than an hour by controlling compression and incubation ofcollagen-based gels seeded with cells. The system, which uses a SBS standard 24-well plateformat can be filled manually and will fit into a standard laminar flow hood to preventcontamination of tissue constructs

Figure 25: Tissue Growth Technologies’ DermiGen™ Mechanical Stimulation BioreactorSystem



Cell Culture

along the underside of the specimen while simulta-neously exposing the topside to a gaseous environ-ment. The chamber accommodates sample dimen-sions of 70mm length, 20mm width and 0.1-2mmthickness. The company’s GrowthWorks Softwareand Controller runs on a Windows® laptop andcan operate up to four mechanical stimulators. Theentire system is designed to fit within a standardlaboratory incubator and the chambers can beautoclaved. Founded in 1990 with the charter ofbecoming a world-class provider of instruments forthe biomedical community, Tissue GrowthTechnologies has introduced bioreactors for ten-dons, ligaments, cartilage, bone, blood vessels,heart valves and other 3D tissue types (Figure 25).

DiscussionTable 1 attempts to summarise the main features ofthe 3D cell culture offerings that vendors submit-ted for inclusion in this review. Although this listdoes not represent all available offerings, it is fair-ly representative of what is currently commerciallyobtainable to support 3D cell culture.

The majority of these developments utilise somesort of biomimetic scaffold (eg hydrogel or colla-gen) (3D Biotek, BellBrook Labs, CellASIC,ECACC, Global Cell Solutions, Glycosan,Hamilton, QGel, The Automation Partnership). Itis generally accepted that biomimetic scaffoldsoffer a better prospect to culture specific cell typesand to investigate different aspects of the cell-matrix interaction in 3D. Several trends are evi-dent in this respect to biomimetic scaffolds: 1) themove to using synthetically derived materials tominimise the previously poor reproducibilitybetween batches and the resulting lack of consis-tency between cultures (especially primary cells)(QGel); 2) the ability to design scaffold environ-ments so cells respond in a physiologically rele-vant manner, eg stem cells are thought to do bet-ter in gels rich in hyaluronic acid (ECACC, GlobalCell Solutions, Glycosan); and 3) the developmentof biodegradable scaffolds, particularly to supportapplications in tissue engineering and stem cellresearch (3D Biotek, Glycosan). As 3D cell cultureemerges in popularity, a key need for researchersin these areas is to gently and rapidly recoverencapsulated cells from scaffolds for nucleic acidand protein extraction.

Apart from the biomimetic scaffolds there arewhat we have called in Table 1 3D structural scaf-folds. Most of these are made from the same mate-rial as 2D plate surfaces (ie polystyrene), but offeradditional 3D microstructure directly on the 2Dsurface, or are produced separately and mounted

on to a 2D surface or supplied as an insert to a 2Dculture vessel to create a 3D structurally matrix(3D Biotek, Kuraray, Reinnervate, Synthecon). Allprovide cells with a microspace to form 3D struc-ture and demonstrate enhanced functional activitycompared to cells grown under identical conditionson 2D culture plastic. Being for the most part inertpolystyrene structural scaffolds are non-biodegrad-able, but are compatible with most routine (easy)2D assays and have optically clarity allowingresearchers to monitor 3D cell growth by simplyusing an inverted light microscope. The uniformityof the 3D matrix does however vary; where theyare micro-fabricated directly on to the 2D surfacethey are usually highly uniform repetitive patterns.Where they are fabricated as an emulsion-templat-ed polystyrene scaffold which is subsequentlymounted on to a 2D surface the architecture isslightly less regular, but is highly porous and con-sists of voids linked to one another by pores.

We also report on several microfluidic devices(BellBrook Labs, CellAASIC) that have mouldedmicrochannels contained within a microplate for-mat for ease of handling and automation. Theseofferings incorporate a biomimetic scaffold (gel)and are structured to enable fluidic flow or longterm perfusion of cells and to facilitate high quali-ty cell imaging in a 3D environment. Currentlythey are optimised to quite specific 3D applications(eg invasion assay profiling) or to support specifictumour cell models.

Only three platforms that directly support theautomation of 3D cell culture and/or tissue cre-ation were reported in the vendor’s snapshots.Awareness of these platforms was not particular-ly high among HTStec’s survey participants,although since all are relatively new approaches,this is not surprising. The development of micro-carriers (magnetic, spherical, pipettable sub-strates) (Global Cell Solutions) and the associatedrelatively small scale automation of 3D cell cul-ture on these microcarriers (Hamilton Company)represents a major advance in manipulating, cul-turing and even cryopreservation of cells, howev-er the suitability of these microcarriers to a sig-nificant proportion of cell and tumour lines usedfor screening is in doubt1. Nevertheless the sim-plification to the work flow of cell-based screen-ing made possible by such a format has triggeredadditional investigations, particularly withrespect to undertaking bioassays directly onmicrocarriers (Biotek). An alternative approachthat has yielded some success in recreating 3Dembryonic, tumour and primary tissues in vitro ishanging drop scaffold-free culture. The advent of



Cell Culture

GravityPlus technology (InSphero) provides ameans for high-throughput production of micro-tissues and for the automation of media exchangefrom hanging drops. The RAFT (RealArchitecture for 3D Tissue) system (TAP) is a con-venient, simple-to-use semi-automated way ofbringing greater scale-up and consistency to gen-erating tissue constructs by controlling compres-sion and incubation of collagen-based gels seededwith cells. It remains to be seen what impact theseearly attempts at automation will have on 3D cellculture and tissue generation, and whether anywill be adopted to a large extent.

The need for more physiologically relevantreplacements to current industry-standard animaland cell-based tests of efficacy and in vitro toxicolo-

gy was highlighted by several vendors and 3D modelsystems of liver (hepatocytes), skin, cancer invasion,vaginal, bladder and airways are beginning to bedeveloped to meet this need (BellBrooks Labs,CellASIC, CELLnTEC, InSphero, RegeneMed,Reinnervate, TAP). We also report on a device thatmay be used to stimulate the growth of tissue engi-neered skin or to act as a test bed for drug and cos-metic development (Tissue Growth Technologies)and a culture system with a flow chamber that utilises a 3D matrix to support culture development and incorporation of normal tissue organisation(RealBio).

Stem cells are the prime application focus of 3Dcell culture. This is partly because the in vivomicroenvironment is believed to play a key role in

3D Vendor Company MicroplateBased

Device Type BiomimeticScaffolds

3D StructuralScaffolds

3D SpecificCulture Media

FacilitatesAutomation

TissueCreation

3D Biotek √ Well insert √ √ √

BellBrook Labs √ Microfluidic plate √ √

BioTek √

CellASIC √ Microfluidic plate √

CELLnTEC Advanced Cell Systems √ √

ECACC/HPA Culture Collection √ √ √

Global Cell Solutions √ √ √

Glycosan √ √

Hamilton √ √ √

InSphero √ Hanging drop √ √

Kuraray √ Wells √ √

QGel √ √

RealBio Technology Flow chamber √ √

RegeneMed √ Wells, well inserts √

Reinnervate Well inserts √ √

Synthecon √

The Automation Partnership √ Wells, well inserts √ √ √

Tissue Growth Technologies √

Table 1: Summary of the features of the 3D cell culture offerings discussed in this article



Cell Culture

the maintenance and expansion of stem cells and3D is perceived as a way of manipulating thismicroenvironment. Also conditioned mediaderived from mesenchymal stem cells grown in 3Dmatrixes has instructive effects on the differentia-tion of some stem cells. Quite a number vendorshighlighted in this review are developing and opti-mising their 3D offering to further stem cellresearch (ECACC, Global Cell Solutions,Glycosan, RegeneMed, Reinnervate, RealBio,Synthecon, The Automation Partnership).

Reading the vendor snapshots one might con-clude that 3D cell culture was a done deal and tis-sue generation is readily achievable, howeverHTStec’s survey uncovered many problems andunmet needs, for example:

l Poor reproducibility between batches of bio-mimetic scaffolds.

l 3D matrices have too many components andcreation of constructs is difficult and laborious.

l Limited ability to scale up or down a single 3Dformat.

l Post culturing processing/cell extraction frommatrix difficult to handle.

l Virtual lack of proven automated solutions.l All methods need higher throughput. l Lack of methodology directly applicable to

screening and bioprocessing.l Wider applicability needed, today everything

depends upon or is limited to the specificmodel.

l Lack of consistency between 3D cultures (espe-cially primary and stem cells).

l Greater flexibility to accommodate the manydifferent cell-lines and types.

l Better methods (readouts) for characterisingcells cultured in these geometries.

l Better visualisation, wider applicability to HCSand video imaging, better image analysis tools.

l Room for improvement with more physiologi-cal substrates.

l Limited stability in long term experiments.

In conclusion, the state-of-the-art seems to someway off from providing fully validated or robust3D culture solutions and the field is clearly open tomajor improvements at this point in time. DDW

Dr John Comley is Managing Director of HTStecLimited, an independent market research consul-tancy whose focus is on assisting clients deliveringnovel enabling platform technologies (liquid han-dling, laboratory automation, detection instrumen-tation and assay reagent technologies) to drug dis-covery and the life sciences. Since its formationseven years ago, HTStec has published more than50 market reports on drug discovery technologiesand Dr Comley has authored more than 30 reviewarticles in Drug Discovery World. Please [email protected] for more information.

Reference1 3D Cell Culture Trends 2010Report, published by HTStecLimited, Cambridge, UK,February 2010.

TissueCreation

3D ModelDevelopment

Stem CellFocus

Other Features/Applications:

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√ HCS imaging

Plate reading

√ HCS imaging

√ √ Services

√ √ Cell supply

√ √ Cell supply

√ √

√ Bioreactor, Cell supply

√ √ Cell supply

√ HCS imaging

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√ √ √ Prolonged culture,Cell harvesting

√ √ √ Services

√ √ √

√ √ Bioreactor

√ √ √

√ Bioreactor


Documents

3D cell culture Layout 1 - Drug Discovery World, Drug Discovery