HumanOrganandTissueEngineering:AdvancesandChallenges

inAddressingtheMedicalCrisisofthe21stcentury

MelanieP.Matheu,PhD

ErikBusby,PhD

JohanBorglin,PhD

ABSTRACT

Organfailureistheimminenthealth-carecrisisofthe21stcentury.IntheUnitedStates,theestimated

costof lung,kidney,and liverdiseasecombined isupwardsof$256Bannually.1,2,3One insevenadult

Americansissufferingfromsomeformofprogressivekidneydisease,andcurrently,660,000adultsinthe

USaresuffering fromend-stagerenal failure.4,5,6Lungdisease is the3rd leadingcauseofdeath,7 liver

diseaseisthe12thleadingcauseofdeath,4andheartdisease,thenumberonecauseofdeathintheUS,

canbecaused,orexacerbatedby,lossoforganfunction.7,8Endocrinediseasescanalsobethoughtofas

afailureinanorgansystem;anotableexampleisDiabetes.Globally,Type2Diabetesaffectsover400

millionpeople,9whileType1Diabetesaffects fewerpatients, thenumbersare in themillions.Type1

Diabetesisautoimmune-drivenandhasanannualglobalincreaseinincidenceof10percentyearover

year.10,11BothType1andType2DiabetescanbenefitfromtransplantationoftissueknownastheIslets

of Langerhans,anessentially curativeprocedure that leads independence from insulin injections.12,13

Transplantationistheonlyknowncurativeoptiontoaddressorganfailure.Developmentofvascularized

organsandtissuesfortransplantationisnotfeasiblewithcurrenttechnology.Despitethedevelopment

of numerousmethods, none have broachedwhatwill ultimately become amulti-trillion-dollar global

market that can address hundreds of millions of patient’s needs: living human organ and tissue

replacement.Here,wereviewcurrentapproachesintheracetoproducefunctionalhumanorgansfor

transplantation, discuss roadblocks, and present novel technology that will pave the way to building

humanorgansinthelaboratorysetting.

AuthorAffiliations:

Founder,CEOPrellisBiologics,Inc.(MPM),PrincipleOpticalEngineer(EB),PrincipleOpticalEngineer(JB)

INTRODUCTION

Organ and tissue transplantation can restore physiologic homeostasis, reduce stress on other organ

systems,andimprovethelivesoftherecipients.Donor-derivedtransplantation,however,issignificantly

limitedbysupply,withonly34,771organtransplantsbeingperformedintheUSin2017,andwellovera

million potential recipients turned away from a waitlist, or waiting for an immunologically matched

donor.14Effortsintheregenerativemedicinespacehaveadvancedtreatmentoptions,andincludework

with patient-derived stem cells to promote tissue repair, as well as development of improved

immunosuppression options. However, medical options to treat end-stage organ failure have not

advancedfornearly50years,andstillrequirehuman-to-humandonation.Thisleadstoalimitednumber

ofhealthyorgansavailable for transplantationandonly selectcandidatesareadded to thewaitlist to

receivealife-savingorgan.Tremendousadvancesinregenerativemedicinehavebeenreportedinthelast

decade,butintermsofhumanorganengineering,thefieldremainsstalledatthinsheetsofcellsthatlack

capillarybloodflow,andthereforearenotsuitabletransplantation.

IntheUnitedStates,anestimated150millionpeoplearesufferingfromkidney,lung,orliverdisease,the

3rd,9th,and12thleadingcausesofdeath,respectively.15,16,17Co-morbiditiesincludingheartfailureprecede

palliativecare,oftenfordecades,takingasignificanttollonpatients,theirfamilies,andthehealthcare

system.1, 8, 18, 19 Compounding the toll on our healthcare system, it has recently been reported that

extended periods of time with low organ function are associated with progressive loss of cognitive

function and physical abilities.20, 21 Even in a reasonablywell-managed disease such as Diabetes, it is

reportedthatautonomicandcentralnervoussystemdeclineisaccelerated,leadingtoincreasedincidence

ofdementia,cognitivedecline,aswellasanincreasedriskofheartattackandrenalfailure.22,23,24Aswe

age, decline in organ systems may contribute to and precipitate neurological disorders, or induce

significantstressonotherorgansystemsincludingtheheart.21,25,26,27

Organtransplantationallowspatientstoreclaimnormalcy,returntowork,andinmanycases,extends

lifebydecades.However, lifesavingtransplantationis limitedbytoofewviableorgans.Donationfrom

16,468deceasedindividualsledtoarecord-breaking34,771transplantsperformedin2017(morethan

oneorgancanbeharvestedperdonor).14The increase inhealthyorgans for transplantation iswidely

attributedtothecurrentopiateaddictioncrisis,28anddoesnotrepresentasustainedincreaseindonation

which,until2015,remainedbelow30,000transplantsurgeriesperyearintheUS.29Typically,fewerthan

16,000organdonorsgivethegiftoflifeeachyear,farfewerthanthemillionsofpatientswhocouldbenefit

fromanorgantransplant.

Althoughsignificanteffortshavebeenmadetobridgethechasmbetweenorganavailabilityandneed,

increasingorgandonornumbersandimprovingorganprocurementprocedureswillnotbridgethegap.

Advancesinstemcellbiologyandnovelengineeringapproacheshavebroughtscientistscloserthanever

beforeinmedicalhistorytoproducingafunctionalreplacementhumanorgan.Theracebetweenthree

technologies in the fieldwill likelybring thecapability toproduce fully functional replacementhuman

organsintomedicalreality:recellularizationoftissuestructures,xenografttransplantation(fromanimals),

and3Dprintingtomanufactureorgans.Here,wefocusonthefieldoforganandtissuedevelopmentin

the laboratorysettingusing3Dprinting,andprovideabriefoverviewofother technologies, including

recellularization,andtheeffortstodevelopporcinebreed-stockforxenographtransplantation.

MANUFACTURING3DPRINTEDHUMANORGANSANDTISSUES

Thereare four requirements for scalable3Dprintingof functionalhumanorgans: Resolution, Speed,

Complexity,andBiocompatibility.

RESOLUTION

High resolutionprinting is critical for building single-cellwalled capillaries andmicrocellular niches,

necessarycomponentsoffunctionalorgansandtissues.

Alllivingtissuesandorgansareperfusedby5-10micron(indiameter)bloodvesselsthatareknownas

capillaries.Capillariesarethesmallestbloodvesselsinthebodyandserveastheprimaryexchangepoint

foroxygen,nutrients,andcellularwastes.Afternutrientandwasteexchangecapillaries recombine to

formtheveinsthatferrybloodbacktotheheartandthenlungs.Thesesmallbloodvesselsarethecritical

functional unit in all tissues. Without a network of capillaries, tissues and organs starve for oxygen

(hypoxia),andcellsbegintodieoffinamatterofminutesleadingtotissuenecrosis(death).Therefore,

theresolutionofagivenprintingprocessmustbeaboutornearthatofasinglecell.

Organsneedoxygenforcellularrespiration,butalsorequirethin-walledcapillariestofunctioneffectively.

Withinmanyorgans,capillariesplayadditionalfunctionalroles.Kidneys,forexample,requiresingle-cell

walledcapillariesforbloodfiltration,lungsrequiresingle-cellwalledcapillariesforgasexchange,andthe

liverrequiressingle-cellwalledcapillariesforwasteprocessing.Thefunctionalnetworksofcapillariesin

anorganareextensive.Ahumankidney contains12milesof glomerular capillaries that areprecisely

placedtofilterbloodandreabsorbsaltsandnutrients.30Thehumanbrain,whichuses20%ofourinhaled

oxygen,boastsover400milesofcapillaries,whereeachneuronhasadedicatedcapillary.31,32

Living tissuedependenceoncapillariesmakesbioprintingatahigh resolutiona criticalelement in3D

printing and manufacturing of organs. Although large structures can be created with current 3D

bioprinting technologies, the smallest blood vessels created to date are on the order of 50 to 1,500

microns in diameter, too large to act as functional capillary networks.33, 34, 35, 36, 37 The average inner

diameterofacapillaryis5to10microns,aboutoneone-thousandthofacentimeter.Thisinnerdiameter

is so small, red blood cells pass through in single file. In organs and tissues, capillaries are spaced a

maximumof 250-300microns apart, the limit of diffusion for oxygen (O2). Capillary spacing can vary

significantly,andintissueswithhighmetabolicrequirements,suchascardiactissues,capillariesaremuch

closer together, an average distance of 20microns apart.38, 39 Therefore, a printing technologymust

achievebothfineenoughresolutiontocreateathin-walledcapillary,whileallowingforcellstobeplaced

betweencapillaries.Withouttheabilitytobuildafunctionalvascularsystem,humantissueengineering

willnotprogressbeyondthinsheetsofcells.40Thelowestreportedresolutiontodatewasachievedwith

two-photonraster-scanningofalaser.41Spray-baseddepositionprintingcanprintthelowestresolution

of thenon-lightbasedsystemsandallows for50micron resolution, still 5 to10 fold to large tobuild

functionaltissues.42

Alongwithbuildingvasculature,theabilitytoplacesinglecellsallowsforthecreationofmicrocellular

niches,animportantaspectoftissueengineering.Microcellularnichesaresmallgroupingsofdiversecell

typesthatcreateaself-sustaining,highlyspecializedmicro-environmentthroughchemicalcross-talkand

cell-cellinteractions.Often,aswiththecryptsofthesmallintestine,thesenichescontainstemcellsto

replenish local cell populationsduring tissue remodeling, repair, andduring thenatural courseof cell

death.43Invitrocellscanbeinducedtoself-organizetocreatetheseniches,howeveroftenfailtoachieve

afullyorganizedsystem,thismaybeduetogeometriesandintracellularsignalingfactors.44,45,46However,

thecapacityforcellstoorganizeanddifferentiatedonceplacedinalargertissuewithcirculationremains

tobedetermined.Itislikelythatcellswillhavetohavespecific3Dorganizationtorecapitulatefunctional

microcellularniches.Therefore,theabilitytoplacegroupsofcellswithhighresolutionislikelytobean

importantcomponentoftissueandorganengineering.

Insum,thephysiologicrequirementsfororganstructureandfunctionrequiresinglecellplacementto

build capillaries and microcellular niches. Therefore, the resolution for creating tissues, organs, and

extracellularmatrixshouldfallwithintherangeofasinglecell,between1to10microns.

SPEED

Unlikeastandardmanufacturingprocess,humantissueengineeringisconstrainedbythefinitelifetime

andhealthofthecellsbeingusedtocreatethetissue.

Complexorganscontaincellsatvariousstagesofdifferentiation(ordevelopment).Extendedperiodsof

time ina sub-parenvironmentmay induceundesirable celldifferentiation.Whilemanycell types can

surviveincarefully-controlledcultureconditionsforuptotwotothreemonths,highpressure,speeds,

sheerforce,andheatcreatedbyextrusionordroplet-basedprintingarenotwelltoleratedbycells.47,48,

49,50

Ideally,printinganextracellularmatrixthatcontainscellswouldtakelessthan12hoursbeforecellsare

returnedtophysiologicconditions;generally37°C,and5%CO2inanutrientsufficientenvironment.Cell

deathduringthetissueprintingprocessisanimportantconsiderationasitcanresultina‘dead’tissueor

significantlyaltertheenvironmentfortheremainingcells.51Thelimitforthepercentageofcellsthatcan

be lost during a printing process is unknown, and different cell types have different tolerances for

environmentalchanges.Extrusionandelectrospinbasedbio-printingcausessignificantcelldeath,while

ink jetbasedbioprintingandothermethodshave reportedhigher cell viability.52,53 42Otherpotential

methodsforimprovingcellviabilityincludecoolingofmaterials,includingcryogenicprintingwhichoffers

aninterestingmethodformaintaincells.However,theresolutionofcryogenicprintingistoolowtocreate

vasculature, and defects such as air-bubbles in the range of hundreds of microns in diameter are

reported.54

Tocalculatethree-dimensionalprintingtimesatagivenresolution,thetypeoftechnologybeingusedto

printmustbeconsidered.Threedimensional(3D)lightbasedprintingatitsfastestdescribed,todate,is

performedthroughlayer-by-layeradditivemanufacturingthroughthedepositionofasingle lineor2D

planarillumination,oftenviaUV-lightbasedexcitation.55,56UVlightisknowntobemutagenic,andthus,

despitethedemonstrationofprintingwithbioinksthatarebothcellcompatibleandUVreactive,repeated

UV light exposure limits the safety of these engineered tissues for use in transplantation. Extrusion

printingof5-10micrometerlayershasyettobedescribedandthespeedofdepositionattheseresolutions

isnot rapidenough for thecreationofmicrovasculature for transplantable tissues.Therefore,wewill

focusprimarilyonthelight-baseddepositionofstructuresorphotolithography,forassessingspeedsof

tissueengineering.

Laserorlight-basedpolymerizationof3Dprintingmaterialscanprintpolymerizebiologicalproteinsand

softnon-toxictransplantablematerials.Thepolymerizationoccursmuchlikethecuringprocessofan

epoxyresinwithlight.Lightcanbeusedoutsideofthevisiblerangeofwavelengthstoinducephoto-

crosslinking(Figure1).

Figure1.Photo-polymerizationofaliquidpolymer

Thisprocessisbothfastandcanreachtheresolutionnecessaryforsinglecelllayerdeposition.Current

laser-based printing approaches utilize a one-dimensional line scan of a pinpoint laser, or 2D planar

depositiontoprint3Dstructuresinlayers.Scalingoftheprintvolumein3Dleadstosignificantgainsin

speedwithout compromising resolution. Toestimate time required toproducea structurewith light-

basedpolymerizationofamaterial,wemustaccountforboththetimerequiredtoscanthelaserthrough

the sample and the time that must be spent to polymerize each voxel. A is the voxel cross section

orthogonaltothescandirection,visthescanvelocity,fisthefillfactor,𝑛"#$%&'isthenumberofvoxels

intheprintvolume,t isthedwelltimenecessaryformaterialpolymerization,andRistheholographic

exposurerate(voxels/s).57,58

Equations1-3

𝑡𝑖𝑚𝑒,-&"# =𝑉𝐴𝜐

+ 𝑓 ∗ 𝑛"#$%& ∗ 𝑡

𝑡𝑖𝑚𝑒6#&7,#8 = 𝑛"#$%& ∗ 𝑡

𝑡𝑖𝑚𝑒9#&#,:-697 = 𝑓 ∗ 𝑛"#$%& ∗ 𝑅

Usingtheseequations,wepresentcalculationscomparingtheprintingtimesofacentimetercubedwith

onemicronresolution,a100μsdwelltime,andtwodifferentfill factors(Table1). Notethatpolygon

scanners are rate-limited by the dwell time, which leads to fill factor independence. 3D holographic

printingcanbescaledbyscalingupopticalcomponents.Thefirsttwonumbersarerepresentativeofthe

fastesttechnologybuiltbyPrellisBiologicspriortointroducingcustomopticalcomponentsfollowedby

theestimatesforthenextgenerationsystemwhichwillprintatarateof12millionvoxelspersecond.

Table1.Estimatedfastesttimetoprint1cm3with1micronresolution 1%fillfactor 15%fillfactorScanspeeds(high/low) Galvanometer(Scanner)

10mm/s 3.2years 3.6years10m/s 12.7days 175days PolygonScanner 10mm/s(limitedbydwelltime) 3.2years 3.2years

3DMulti-PhotonHolography 36,000voxels/s 3.2days 48days240,000voxels/s 11.6hrs 7days12Mvoxels/s 14minutes 3.4hours

Evenat10mm/s,theresolutionisrelativelylow(10sofmicrons),andasmuchas30-50%whatwouldbe

structurally necessary to print is skipped in each illumination pass, severing limiting the cohesive

structures thatareconstructedat this speed.59 60,613DMulti-PhotonHolographydevelopedbyPrellis

Biologics, Inc. rapidlyprints3Dstructuresand isdescribed indetail later.Holography iseffectively fill-

factorindependentifsufficientlaserpowerexists.Therefore,inholographicbasedlaserprintingspeed

notlimitedbyresolutionwhereinspeedisvolumedependent,andtheprintvolumeisdictatedbystatic

opticalcomponents.Holographyisachievedwithmulti-photonexcitationthroughacombinationofbeam

expansionwave-frontshapingwhichcanbethoughtofasbeam-steeringthatallowsforcompleteornear-

completestructuredeposition(Figure2).

Figure2.Singlephotonabsorptionascomparedtotwophotonabsorptionandholographiclaserprinting.

Figure 2.Graphics demonstrating laser printing output based on optics of single photon andmultiphoton printing processes and the expected structural outcomes. (A) Graphic depictingsinglephotonlaserprojectionintoaprintmediacontainingbathwithoutmaskingorisolationoftheintendedplaneoffocus,whichwouldbeexpectedtoleaveaprintedstructurebehindintheshapeof the entire coneof lightalong the focal length. (B)Graphic depictingamulti-photonabsorptionprocesswherethephotondensityishighenoughonlyatthepointoffocus,leavingonlyapinpointstructurebehindinaphotosensitiveprintmediabath.(C)Representativegraphicofwavefrontshapingtoproduceaholograminwhichthemultiphotonabsorptionprocessoccursatmultiplepointsoffocusinthex,y,andzplanes.Inthisprocess,rapidswitchingbetween3Dprojectedportions of a complete structuremaybeused to build the complete structure. (D)Representative graphic showing a complete image projection in multiple planes allowing forholographicprintingofacomplexstructure.

COMPLEXITY

Biological complexity in 3D organ printing can be defined as the close placement of structural

componentsthatworkinconcerttocarryoutthefunctionalpurposeoftheorgan.

Forexample,theprimaryfunctionalpurposeofthelungisgasexchangebetweenthecirculatorysystem

andtheenvironment.Themajority,90%,ofgasexchangeoccursacrosstwocellsthinlystretchedcellsin

closeassociation,onethatcomprisesthecapillarywall,onethatmakesupthealveolarcellwall;theend-

pointforallbranchesofthelung.62Alveolarspacesarestructurallysimilartoabunchofgrapes,where

eachalveolusor‘grape’isthesmallestsubunitoflung,measuringamere200micronsacross.Toferryair

andbloodtothismembrane,airwaysdivideanaverageof23timesbetweenthetracheaandthealveoli

wheregasexchangeoccurs.Branchedairwaysandadualcirculatorysystemcreate thestructuraland

physiologicalsupportsystemofthe lungwhich iscomprisedof1,500milesofairwaysand700million

alveolar spaces.63 The complexity of the lung is not unique: kidney, liver, and even skin each have

structuresofsimilarcomplexitythatarerequiredforphysiologicalfunction.Anengineeredtissuemust

be able to match the complexity of these functional subunits, structural requirements, and nuances

thereintobeconsideredviableforhumandonororganreplacement.

Layer-by-layer extrusion printing can achieve some measure of complexity but cannot match the

complexitynecessaryforcreationofhumantissue.Shortwavelengthlight,muchlikethatusedinresin-

basedpolymerization, canachievequiteabitof complexity if controlled frommultiplepoint sources;

however,itstilllacksbiocompatibilityandtheabilitytobuildtruecomplexity.64Thisisreviewedfurther

inbiocompatibilityofprintingprocesses.

Multi-photonexcitationofafluorophore,anditsuseinphotolithography,hasinherentadvantagesinthat

ityieldshighcomplexity.3Dcontrolofstructuralpolymerizationandtheabilitytoprintbehindandinside

ofalready-printedstructuresallowsforunprecedentedcomplexity.Multi-photonexcitationallowsfora

3Dhologramtobegeneratedwithultra-fine,one-micronresolution,inamask-lesslithographyprocess,

whileusing2Plongwavelengthlightthatiscanpassthroughalreadypolymerizedstructuresorcellsand

befocusedontheothersidetocontinueprinting.Thisisachievablebecausefocalplaneorvoxelsinthe

caseofaholographicprojectionaretheonlyplacewherepolymerizationoftheprintmaterialsisinduced.

Therefore, complexity is easily createdbyprintingbehind andevenwithin alreadyexisting structures

usingmulti-photonpolymerization.

BIOCOMPATIBILITY

Tobuildanorganortissue,biocompatibility,isofparamountimportance.

First,theprintingsubstrateormaterialsandprocessusedtocreatethetissuemustbecompatiblewith

livingcells.Second,thetissuemustbetoleratedbythehost’simmunesystem.Third,thematerialsmust

bestructurallysound;matchingthepropertiesoftissuessuchthatitcanoperatewithinthephysiologic

requirementsofanorgan.

Printing materials that are highly biocompatible range from extracellular matrix proteins such as

hyaluronicacidandthemostabundant,collagen(60-80%ofagiventissue),tobiologicallyinertmaterials

suchaspolyethyleneglycol(PEG).65,66,67Collagenishighlyevolutionarilyconservedwhereinhumanand

animal collagen is often indistinguishable at the biochemical level, leading to cross-species

compatability.68 Furthermore, collagen is generally immunologically inert with only 2-4% of patients

reacting to cosmetic injections of bovine collagen, a response that can be tested for well ahead of

transplantation.69Furthermore,recombinantsourcesofpurecollagenhavebeendevelopedtobypassany

complicationswithanimal-basedproductionofmaterialsmakingthisanidealmaterialforthescaffoldof

transplantabletissues.70Mildcellulartoxicitycanoccurinsomeprintingformulationsthatincludehigh

amountsofphotoinitiators;however,withprotectiveformulationsandlowexposuretime,theseissues

arenotexpectedtohindertissueandorganprintingvialight-inducedpolymerization.71,72,73

The printing process that creates a cell-containing extracellular matrix must also be biocompatible.

Extrusionandspray-basedprintingofcell-containingbioinkscreatessignificantsheerforce,evenatlow

resolution, and therefore is not scalable to a higher-speed lower resolution that could maintain cell

viabilityatthespeedsnecessarytobuildwholeorgans.Light-basedpolymerizationprocessesdonotexert

sheer force on the cells and can print at high resolutions; however, photo-damage and heat-induced

damagecanbesignificantissues.Short-wavelengthlightinthevisiblerange,projectedatahighintensity

isphoto-toxictocells,andUVlightsourcescausesingleanddoublestrandedDNAbreaksinasmanyas

10%ofthecellswithpotentialtoresultinoncogenictransformation.73,74Transplantationofapotentially

cancerouscellpopulationwouldbeofprohibitiveconcern,makingUVorshort-wavelengthlight-based

lithographyforlivingbioinksahigh-riskproposition.

PRELLISBIOLOGICS3DPRINTINGTECHNOLOGY

Manufacturingmethodshavemadesignificantprogresstowardsmeetingeachofthefourrequirements

forbuildinghumanorgansfromscratch;Resolution,Speed,Complexity,andBiocompatibility.However,

to date, no single technology has been reported to meet all four. Here we present our laser-based

holographicprintingprocessthatprovidesasingleplatformtechnologythatmeetstheresolution,speed,

complexity,andbiocompatibility,requirementsnecessarytobioprinthumantissuesandorgans.

Byusingholographic3Dprintingwithafar-redlaser,weeffectivelydecouplespeedfromresolution,while

introducingpreviously impossiblecomplexity.Thishasallowed, for the first time, thecreationof sub-

cellularresolutionstructuresusingcell-ladenbioink.Theapplicationsofthistechnologyarenumerous,

rangingfromultra-fastsinglecellencapsulationtoprintingofreplacementhumanorgansandtissues.

SubcellularTunableResolutionandPrintAreas

Inthelithographyfield,oftenusedforcomputerchipmanufacturing,theresolutionobtainedbyprinting

withlightcanbesub-wavelength(ontheorderoftensofnanometers).Althoughtheextracellularmatrix

thatholdstissuesandorganstogetherissub-cellularinresolution,ontheorderofone-hundredthofa

humanhair, these structuresare far larger than thecomponentsofa siliconwafer, ranging from200

nanometerstoseveralmicronsindiameter.

AtPrellisBiologics,wehavebuiltaphoto-lithography-basedsystemthatprintswithinagiven3Dfieldof

viewwithsimultaneousmulti-voxelprojection,muchliketheprojectionofahologram(holography)with

voxels(3Dpixels)thatmaintainaprintresolutionof0.5x0.5x3micronsinthex,y,andzdimensions.

Theresolutionofthesystemisdependentupontheopticalcomponentsusedtoguideandfocusthelight

source.

Customopticalcomponentscanbeintroducedtodecreaseresolutionorincreasethespeedofprinting.

Subcellularopticalresolution(lessthanabout1micron),however,maynotberequiredordesiredfor

printingvasculatureembeddedtissues.Becausetheholographyprocessutilizesoptics,ratherthanaprint

nozzleformaterialdeposition,thewidthoftheprojectedlaserlightcanbecanbealteredontheorderof

milliseconds to increase or decrease structural dimensions of the printed materials. Optics-based

holographicprintingthereforeoffersbothhighresolutionandnear-immediatechangesinresolutionthat

canbeappliedtocreateultrafinestructures.

PrintingSpeedsCompatiblewithOrganandTissueGeneration

Thepolymerizationprocessiscontrolledbythelocalizedabsorptionoflight,lendingflexibilityonparwith

thatofprojectingaseriesofimagesonamoviescreen.Indeed,thehologramsutilizedtorecreatethe

structure are projected as a series of images at speeds well over video rate speeds, up to 250 Hz.

Manufacturingof3D-printedcomponentsisdependentuponthespeedoflayerdeposition.Inthiscase,

thelayersarethree-dimensionalanddepositedbyalight-inducedchemicalreaction(seeFigure1)that

occursontheorderof5orfewermilliseconds.Atthesespeeds,printingtimeshiftstobemoredependent

upon the chemistry of the printing formulation than the physical properties of laser projection. By

removingthemechanicalencumbrancesoflaserlightscanninginthecaseofpinpointlaserprinting,orz-

step requirements of planar printing, while scaling the manufacturing technique from 1D or 2D, a

significant reduction inprint timesoccurs.Thisallowsbiologicallycomplexstructures tobecreatedat

unprecedented speeds. Increasedprint speedsdecreasemanufacturing timesof complex tissues such

thatprintingofhumanorgansandorganstructuresisnowintherangeofpossible.

3DLayeringAllowsforOrganSystemsLevelComplexity

Beyondmeeting requirements of resolution and speed, Prellis Biologics canutilize far-redbased light

polymerizationtointroducepreviouslyundescribedcomplexitiesintothe3Dprintingprocess.Printinga

fully formed structure inside of another already formed structure is possible using multi-photon

polymerization,where the structure is optically clear to the far-red excitation source. This process is

demonstratedbelowinFigure3,whereasinglesphereisdepositedinsideofaprintedtube.

Figure3.Demonstrationofprecisionmultiphotonprintinginsideanalreadyformedstructure.

Todate,theabilitytoprintbehindorwithinanotheralreadydepositedlayerhasnotbeendescribedwith

anyother3Dprintingmethod.Thisfeatureisusefulforaddingadditionaltissuelayerswithinorbehind

already deposited layers in amulti-layered cell printing process. This capability is critical for building

complextissues,suchasthekidney,especiallytheglomerulustheprimarypointofbloodfiltration.The

glomerulusiscomprisedofafinenetworksofrenalvasculaturesitwithinthecapsulelikeaballandsocket

joint,orbaseballinsideofabaseballglove.Therefore,3Dlayeringwithlivingcellsallowsforcreationof

structuresaroundfinely layeredmicrovasculatureandthusthedevelopmentoffullyfunctionaltissues

andinthefuture,organs.

BiocompatibilityofMulti-PhotonHolographicPrinting

3Dprintinginkisofparamountimportanceforbuildingcompletetissues.Traditionalcell-ladenbioinks

canbeformulatedformultiphoton-basedprintingthroughtheadditionofaphoto-initiator.Thephoto-

initiator undergoes a photoreaction on the absorption of far-red (infrared) light, catalyzing the

polymerization of the bioink. There are a variety of bioink/photo-initiator combinations that are

compatible with multiphoton printing, all of which have demonstrated high biocompatibility when

subjectedtocellviabilitystudies.

Relative to UV and single wavelength polymerization, far-red multi-photon excitation, provided by a

femtosecond(10x10-15second)pulsedlasersource,ensuresthathighenergyisonlyabsorbedatthe

focal plane, therefore the rest of thebioinkonly experiencesbrief pulses of far-red lowenergy light.

Figure 3. A representative graphic ofprinting holographically printing a sphereinside of an already formed three-dimensionaltube(toprow).Ahollowtubestructure printed with multiphotonholography allows for the projection of asphericalhologram into the centerof thetubedepositingthecompletesphereinsideof the tube without disrupting thestructure. The sphere is printed in 5 orfewermilliseconds(bottomrow).

Studies using far-red light at various wavelengths have demonstrated far-red laser light to be non-

oncogenicandprotectivefromoxidativestressincellculture.75Accumulatedcelldamageispulse-length,

power,anddwell-timedependent;thereforerapidprintingandshortlaserexposureminimizestheriskof

celldeath. Furthermore, laser irradiationat lowpowershasnomeasurableeffectsoncelldivision,at

infraredwavelengthsroughlyabove1nm.76,77

Summaryof3D-BioprintingTechnology

PrellisBiologics ispoised tomove into thehumanorganand tissuemarket rapidlyandwithaunique

approachthatcanbescaledtogeneratewholeorgansintimeframesthatwillallowfortransplantationin

high-need patient groups. 3D printing approaches that canmeet all four requirements of resolution,

speed,complexityandbiocompatibilitywillenableamyriadoffunctionalapplicationsoftissueandorgan

engineering.ExamplesofapplicationsthatPrellisBiologics’technologyisuniquelysuitedforincluderapid

single cell encapsulation, organoid printing for disease models, and tissue and organ printing for

transplant.

PrellisBiologicshasdemonstratedbiocompatiblesinglecellencapsulationcapabilitiesatestimatedrates

ofupto20,000cellspersecond.Usingadeterministic,ortargetedapproach,PrellisBiologics’holographic

projection of encapsulation spheres is being developed with computer vision capabilities to enable

selection and rapid encapsulation of specific cells based on label or morphology, while preventing

accidentalencapsulationofmultiplecellsatonce.Singlecellencapsulationenablessinglecellprecision

duringtheengineeringofcomplextissues,andwillfacilitatedeliveryofcellsinvivofortherapeuticuses.

Thesinglecellencapsulationtotaladdressablemarketisestimatedat$1.67Bin2017.

Vascularized organoids for detailed study of human disease models has numerous advantages over

studying cells in a dish and animalmodel use. The discrepancy between in vitro efficacy and clinical

outcomescanbeattributedto limitationsof2Dcellculturemodels.Withprintresolutions inthesub-

micronrange,PrellisBiologicswillhavethecapacitytorecreatecomplex3Dtumormicroenvironments

that combine tumor cells with extracellular matrix. This will allow for improved screening of drug

responsesandthestudyoftumor-stromamolecularinteractions.Thetotaladdressablemarketfor3Dcell

culturesystemswasestimatedat$559Min2017.

ScalableHumanTissueandOrganEngineering

Morethan2millionpeopledieprematurelyeveryyearforlackofaccesstotreatmentforkidneyfailure.92

Numerouschronicillnessesaremanagedbytherapeuticinterventionsthatarebothcostlywithnumerous

repeat interventions required and lead to high patientmorbidity andmortality. Replacement human

organsandtissueswillbothhelptoalleviatesky-rocketingmedicalcostsandallowforpatientstoregain

functional independence from dialysis, oxygen tanks, multiple daily insulin injections, and other life-

alteringtherapeuticinterventions.Scalablehumanandorganengineering,usingapatient’sowncellsto

maximizecompatibilityisamedicalinterventionwiththetruehallmarksofadisruptivetechnologyasit

canbeappliedtonearlyeverymajordiseasestate.Insolvingthefinalhurdleinorganmanufacturing,the

abilitytorapidlyproduceavascularsystem,holographic3Dprintingcansupportforthefirst-timelarge-

scaletissuemanufacturingthatwillallowforsignificantadvancesandpavethewaytofunctionalorgan

replacements.Thesizeofthehumanorganandtissuemarketwhenestimatedbythenumberofpatients

inneed,multipliedbythestandardprocurementcostofadonatedorgan,totalswellover$3Tworld-wide.

Other approaches to solving the human organ shortage and the impending medical crisis including

recellularizationofexistingtissuestructuresanddevelopmentofanimalsforxenograftsthatwouldallow

foreasytransplantationduetoremovalofimmune-systemtriggeringelements.Bothtechnologiesoffer

promiseinspecificareas,howeverbothfacesignificanttechnicalhurdlesthatmaybecostandenergy

prohibitiveindevelopmentoffullhumanorgantransplantation.

Recellularization of tissue scaffolds involves dissolving cells with detergents, washing the remaining

extracellular matrix, then re-introducing the desired cells by bathing the tissue structure in a cell-

containingmedia.Recellularizationisoneapproachthathasbeendevelopedwithmoderatesuccessin

thelaboratorysetting.Indeed,visuallystunningexamplesofcellularbehaviorindecellularizedstructures

such as cardiomyocyte repopulation of a spinach leaf 78 have inspired the field in the direction of

replacementtissueengineering.To-dateoneanimal transplantwitha rat lung lobehasdemonstrated

partial success,wherein the lung retainedat leastpartial function for 6hoursbut the tissue function

ultimatelyfailed,andtheratsrequiredintubation.79Furthermore,thisprocesshasyettoproduceworking

bloodvesselsandmicrovasculature,howevernumerouspromisingstudiesespeciallyintheareaoflung

recellularization are underway. In some skin transplant studies, blood vessels begin to grow into the

transplantedtissues,howeverthickerportionsoftissuerapidlybecomenecroticduetolackofoxygen.

Another hurdle faced by recellularization is that it does not solve the critical problem of single cell

resolutionorplacementofcellswithinfinitefunctionalcellniches.Forexample,airwayendothelialcells

andvascularendothelialcells,alongwithalveolarmacrophagesmusthavethecorrectrelativeplacement

for the lung tissue todifferentiateand functionproperly. Inonere-cellularized lungstudies, scientists

observedincompletedifferentiationoftheengraftedcells,notingalackofmatureciliatedorsecretory

cellsintheconductingairways.79Recellularizationiseffectiveinpaintinglayersofcellsonatissuescaffold

which works for thin tissues. Ultimately sourcing of the organ scaffold may become a significant

bottleneckthatcanbealleviatedbyeffective3Dprintingofidenticalcellscaffolds.

XenographTransplantation

Xenograftmodelsareparticularlyattractivesourceoforgansfortransplant.Heartvalvesandligaments

fromporcine,bovine,andovinesourceshavebeenusedsuccessfullyfordecades.Theselargerstructural

elementsarechemicallytreatedpriortoremovecellularcomponentspriortotransplantationtoremove

cellular components and there are few reported cases of rejection. Chemical pretreatment is not

compatiblewiththetransplantationofmorecomplexfunctionorgans,however,leavingtwosignificant

hurdlespreventingthewidespreadadaptationofxenotransplantation:transmissionofzoonoticdiseases

andimmunologicalrejection.

Recent advances in gene editing approaches have catalyzed efforts to breed and develop genetically

modifiedpigsthatwillproduceorganssuitableforhumantransplantation.Muchlikehumans,mammals

carry diseases and viruses that could be transmitted to xenograft recipients. Of concern are porcine

endogenousretroviruses(PERVs),whichareinsertedintothegenomeofeverypigandcaninfecthuman

cells.Recently,scientistshaveusedthegenomeeditingapproach,CRISPR,toreportedlyeliminateall62

known PERVS from their geneticallymodified porcine population.80While significant additionalwork,

particularlyinimmunologicalcompatibilitywillberequiredtooptimizethisapproachinpigsdeveloped

fororgantransplant,thisrepresentsapositivestepinthedevelopmentoforgansfortransplantation.

Thesecondsignificantbarrier isrejectiondueto immuneresponse,orgraftv.hostdiseasewherethe

organgraftbringsenoughofitsownimmunecellsalongtomountanattackagainstthehost.Ourimmune

systemsarehighlyevolvedtorejectanyforeignmaterial;organsfromaporcinesourcebreedstockthat

exists today could provoke a significant immune response. Scientists have begun efforts to use gene

editingtomodifyporcinespecific-genestodeveloppigswithorgansthathaveimprovedbio-compatibility.

However,thiswillbeadifficultapproach.First,tomanipulatetheporcinegenometobeacceptedbythe

human immune system numerous genes must be altered. Second, there are thousands of protein

differencesbetweenhumansandporcinespecies,andmanymoreare likely tobecomerecognizedas

immunogenic.Third,andperhapsthemostprohibitive,manyof thegenes inthe immunesystemand

similar proteins play a significant role in the developmental process of the animal during gestation,

mutationsandalterationstothesemaybeanon-starterindevelopingabreedingpopulation.Finally,even

ifmostofthefactorsthatcauserejectionwereeliminated,apatientwould likelystill require life-long

immunosuppression.Immunosuppressiondrugsaverage$20,000peryearandincreasesthelikelihoodof

deathfromaggressivecancerbythree-foldacrossallagegroups.81

Analternativeapproachwouldbetogrowhumanorgansinhostanimalsusinghuman-pigchimeras.In

recentstudies,scientistsusedgeneeditingtoremovethegenesresponsibleforpancreaticdevelopment

inapigembryo.Withtheintroductionofhumanstemcellsduringthegestationalperiod,scientistsare

determiningifafunctionalhumanpancreaswilldevelopinsideoftheporcinehost.Whilethisapproach

wassuccessfulinmouse-ratchimeras,82recentattemptstocreatehuman-pigchimerasshowedextremely

lowratesofhumancellengraftment.83

The xenograft approach is beneficial in that tissue will not be as supply limited as re-cellularization

approaches using human organs and combining the two approaches may prove fruitful for the

developmentofmanytissuesfortransplantation.

SUMMARY

Fewerthan20,000organtransplantsarecarriedouteachyearintheUnitedStates.Everyyearlessthan

one-sixthofthepeopleontheorgantransplantwaitlistreceiveanorgan.Anestimated330peopledie

everydayintheUnitedStatesduetoorganfailure.Requirementsforbeingonatransplantationlistare

strict;theover120,000peoplewaitingareonlyafractionofover90millionpeopleintheUnitedStates

whoaresufferingfromsomeformofprogressiveorganfailureandcouldbenefitfromanorgantransplant.

Progressiveorganfailure,specificallykidneyfailure,isasilentdiseasethatwillaffectoneinsevenadults

intheUnitedStates,andisacontributortoheartdisease,8,84,85dementia,86,87,88lossofbonedensityand

catastrophicfractures,86,89andParkinson’sdisease.90,91

Advances in immunotherapeutics, vaccines, and treatments for communicable disease have

demonstratedsignificantimpactinhealthandlife-span;however,fewadvancementshaveoccurredin

thefieldoforganreplacement.Asthesecausesofdeathareminimized,anewchallengeisonthehorizon:

howdoesourhealthcaresystemcopewiththetremendouscostsassociatedwithorganfailure?

Building human organs with 3D printing not only has tremendous potential for improvement of

therapeuticsdevelopmentbutindevelopinganinterventionalprotocolthatwillminimizetheprogression

ofthemajorpointsofhealthfailureandcost.

Significantissuesexistintechnologicalapproachestoorganengineering,suchasre-cellularization,and

thedevelopmentofanimalsforxenotransplantation.Standard3Dprintingmethodsarehinderedbythe

trade-offbetweenspeedandresolutioninmanufacturing.Theslowspeedatnecessarilyhighresolutions

doesnotallowforcell-containingnativetissuestructures,completewithbloodvessels,tobeprintedfast

enoughtokeepcells,orapatient,alive.

AtPrellisBiologicswehavedevelopedalaser-basedtechnologythatdecouplesspeedfromresolution,in

a biocompatible printing process with capacity to build highly complex structures. Our mission is to

developthisplatformtechnologytobuildfullyfunctionalhumanorgansandtissuesondemand.These

organswillrepresentadisease-free,fully-human,cell-basedalternativetodonororgantransplantation.

In addition, this technology can create perfectly matched organs that will eliminate the need for

immunosuppressionandofferthepossibilityofremovingdiseasecausinggeneticmutations.Thevision

ofPrellisBiologics istogivethemillionsofpatientssufferingfromprogressiveorganfailuretheir lives

back.

Acknowledgements:TheauthorwouldliketothankRichardHutton,RisaPatterson,BarbaraKrause,and

NoelleMullin,PhDforeditingandreviewofthemanuscriptcontent.

PrellisBiologics,Inc.wasfoundedinSanFrancisco,CAinOctoberof2016,hasfiled2patentapplications

andhasraisedatotalof3.1Minpre-seed,seedfundingfromleadinvestorsIndieBioandTrueVentures.

Forfurtherinformationpleasecontactinfo@prellisbio.com

FormediainquiriespleasecontactBarbaraKrauseofKrause-Taylor:barbara@krause-taylor.com

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