Pleasecitethisarticleinpressas:E.Garreta,etal.,Mater.Today(2017),http://dx.doi.org/10.1016/j.mattod.2016.12.005
MaterialsTodayVolume00,Number00January2017 RESEARCH
Tissue
engineering
by
decellularization
and
3D
bioprinting
Elena
Garreta
1,
Roger
Oria
2,
Carolina
Tarantino
1,
Mateu
Pla-Roca
3,
Patricia
Prado
1,
Francisco
Ferna´ndez-Avile´s
4,
Josep
Maria
Campistol
5,
Josep
Samitier
6,7,8and
Nuria
Montserrat
1,8,*
1
PluripotentStemCellsandActivationofEndogenousTissueProgramsforOrganRegeneration,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac 15-21,08028Barcelona,Spain
2
InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain;UniversityofBarcelona,Spain
3
NanotechnologyPlatform,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain
4
DepartmentofCardiology,HospitalGeneralUniversitarioGregorioMaran˜o´n,InstitutodeInvestigacio´nSanitariaGregorioMaran˜o´n,Universidad Complutense-FacultaddeMedicina,c/DrEsquerdo46,28007Madrid,Spain
5
HospitalClinic,UniversityofBarcelona,IDIBAPS,08036Barcelona,Spain
6NanobioengineeringGroup,InstituteforBioengineeringofCatalonia(IBEC),c/BaldiriReixac15-21,08028Barcelona,Spain 7DepartmentofEngineering:Electronics,UniversityofBarcelona,08028Barcelona,Spain
8NetworkingBiomedicalResearchCenterinBioengineering,BiomaterialsandNanomedicine(CIBER-BBN),Madrid,Spain
Discarded
human
donor
organs
have
been
shown
to
provide
decellularized
extracellular
matrix
(dECM)
scaffolds
suitable
for
organ
engineering.
The
quest
for
appropriate
cell
sources
to
satisfy
the
need
of
multiple
cells
types
in
order
to
fully
repopulate
human
organ-derived
dECM
scaffolds
has
opened
new
venues
for
the
use
of
human
pluripotent
stem
cells
(hPSCs)
for
recellularization.
In
addition,
three-dimensional
(3D)
bioprinting
techniques
are
advancing
towards
the
fabrication
of
biomimetic
cell-laden
biomaterial
constructs.
Here,
we
review
recent
progress
in
decellularization/recellularization
and
3D
bioprinting
technologies,
aiming
to
fabricate
autologous
tissue
grafts
and
organs
with
an
impact
in
regenerative
medicine.
Introduction
Regenerativemedicineholdsthepromisetoreplaceorregenerate humancells,tissueororgansinordertorestoreorestablishthe normalfunctionlostduetodiseaseordamage[1].Bythe combi-nationofnovelbiomaterialswithcells,oneoftheaimsof regen-erativemedicine is to create autologous tissue graftsfor future replacement therapies [2,3]. In the last three years, discarded human donororgans, such askidney [4,5], lung[6], heart[7], andliver[8],havebeenusedtoobtaindecellularizedextracellular matrix (dECM) scaffolds,proving their potential applicationin
tissue engineering. Despite the translational value of these
advances,wearestillfartogeneraterelevanttissuesforimmediate clinicalapplications.
Theuseoforgan-deriveddECMscaffoldsfor bioengineering
of human-scale patient-specific organs using hPSCs is
envi-sioned as a major platform for therapeutic applications
(reviewed in [9]). Interestingly, theconcept of organprinting
has lately takencenter stage due to recent three-dimensional
(3D)bioprinting advancements(reviewed in [10–13]). Current
3D bioprinting techniques have shown to simultaneously
de-positcombinations of differentcell typesencapsulatedwithin
biomimetichydrogelsviaalayer-by-layerprocess,leadingtothe
generation of 3D bioinspired tissue constructs (reviewed in
[10,12,14,15]) [16–20]. Such approach could offer new
venues when translating hPSCs-related technologies to a
high-throughput 3D setup (e.g., patient induced pluripotent
stem cells (iPSCs)-derived organoid screening platforms)
(reviewed in[21]).
RESEARC
H:
Review
*Correspondingauthor:.Montserrat,N.([email protected])
1369-7021/ß 2016 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).http://dx.doi.org/10.1016/
Sofar,differentlaboratories haveshownthatitispossibleto build up tissue- and organ-like structures either by the use of
organ-derived dECM scaffolds(reviewed in [22–27]) or 3D
bio-printingtechniques(reviewedin[10,12,14,15])[16–20].However,
thebioengineeringofvascularizedhuman-scaleorgananalogues
with optimal functionalactivitystillrequires much effortfrom multidisciplinaryresearchgroupsbeforethiscanbecomeareality. Here,wereviewthelatestadvancementsintheapplicationof decellularization/recellularization technologyforthegeneration
of autologous tissue graftstaking advantage of hPSCs. Wealso
examine how 3D bioprinting technologies may benefit from
hPSCsderivativestofabricatehumanorgananalogues.
The
advent
of
decellularization
technology
Tissue-specific
extracellular
matrix
(ECM)
through
decellularization
of
tissues
and
organs
Pioneerfindings onthe production oftissue-specificECM were
firstreportedinthe1970sand1980s[28,29].Despitethesemajor steps,itwasnotuntil10yearslater thatBadylakand coworkers generatedintactacellularsmallintestinalsubmucosamatricesby
mechanically removing allmesenteric tissues whileleaving the
trilaminateconnectivetissuelayersintact[30].Indeed,such ma-tricesdemonstratedhealingcapacityinadogmodelforAchille’s tendonrepair[30].Soon,differentworksondecellularizationof othersimpletissuessuchasskin[31],vasculartissue[32],heart
valves [33] and bladder [34] showed promising results on the
generation of biological scaffolds for biomedical applications, representingarealisticalternativetotheuseofsynthetic bioma-terialscaffolds.Fromthatmoment,furtherworksbegantoapply decellularizationmethodologiesforthefabricationofdECMslices fromcomplexorganssuchasliver[35].
In2008,theseminalworkbyOttandcoworkers,who
success-fullygenerated wholeratacellular heartsby meansofperfusion decellularization,representedabreakthroughinthefieldoftissue
engineeringand thebeginningoftheeraofwholeorgan
decel-lularizationtechnology[36].Overthelastyears,differentresearch groupsfollowingsimilarapproacheshavereportedthepossibility toderivefull-scaledECMscaffoldsfromdifferentorgansincluding liver, heart, lungs, and kidneys;and multiple species including mouse,rat,pig,rhesusmonkey,andhuman(reviewedin[22,23])
[37]. The main milestones on decellularization technology are
summarizedinFig.1.
InvivothecompositionandultrastructureofECMisinconstant remodelingbytheresidentcellsdependingonthemetabolicand
mechanical demands of the tissue, a concept called ‘dynamic
reciprocity’[38].Suchdynamicremodelingmaybealteredduring injuryordisease,leadingtomodificationsinthecompositionand biophysicalpropertiesoftheECM,andultimately,compromising
organ function. Accordingly, it hasbeen suggested that ‘organ
specificity’,maydeterminewhycellsbelongingtoaspecificorgan exhibitaninnatepreferencetowardsdECMscaffoldsderivedfrom the organ oforigin[39,40]. Alongthis line, Nakayamaand
co-workersshowedthathumanembryonicstemcells(hESCs)
differ-entiated intorenal-likecells whenseeded onto rhesusmonkey
kidneydECM,butthiswasnotthecasewhenlungdECMwasused
instead[40].Themainhypothesissupportingsuchfindingsrelies ontheideathatspecificECMproteinsaredistinctivelydistributed alongthedifferentcompartmentsoftheorganasfootprintsfrom
resident cells. Interestingly, site-specific ECM micromechanical
properties have been described to delimitate different tissue
regions[41].
Overall,dECMscaffoldsprovideacomplexsite-specific
combi-nation of biochemical and mechanical cues, which have been
hypothesizedtoguidecelladhesion,proliferationand
differentia-tion during recellularization and further tissue formation
(reviewed in [42]) [39–41,43–45]. The main advantages using
dECMscaffoldsforthedenovobiofabricationoftissuesandorgans aresummarizedinBox1.
Methods
of
decellularization
Decellularizationtechniquesaim toremove allthe cellsfroma tissueororganwhilepreservingthenativeECMcompositionand architectureintegrity.Assuch,tissueandorgandecellularization, canleadtotheproductionof3DdECMscaffoldsretainingtheir biologicalactivityandmechanicalproperties.Ifeffective,dECM scaffoldsshouldnotelicit immune-mediatedrejectionafter
im-FIGURE1
Timelineofkeyeventsleadingtowholeorgandecellularization methodologiesandmajormilestonesusinghPSC-derivedcellsto repopulateorgan-deriveddECMscaffolds(*).
BOX1
PotentialofdECMscaffoldstorecapitulatetissuesandorgans
PreservationofdECMultrastructureandcompositioninducefavorable tissueorganizationandremodeling(reviewedin[23,24,36]).
dECMcanmodulatecellbehavior:attachment,migrationand differentiation(reviewedin[23,24,36])[39,40,43,44].
dECMdonotelicitimmune-mediatedrejection,sinceECMcomponents arelargelyandhighlyconservedacrossspecies[46,47].
dECMexhibitsuitablemechanicalperformance,similartothatoftheir nativecounterparts(reviewedin[23,24,36])[41].
dECMpromoteconstructiveremodelingresponse,asshownforavariety oftissuesinbothpre-clinicalandclinicalstudies(reviewedin[42])[45]. ThepotentialadvantageofdECMorganspecificityensuresthe maintenanceofselectedcellfunctionsandphenotypes(reviewedin[38])
[39].
2
RESEARCH:
plantation [46,47]. Extensive research has shown the mode of actionand peculiaritiesofeach kindofdecellularizationagents employed,aswidelydiscussedelsewhere(reviewedin[48–50]).In general, the optimal decellularization protocol will generate a
DNA-freedECMscaffoldbyfindingtherightcompromisebetween
thedurationandcomplexityofthetreatmentandthe
conserva-tion of the desired compositional, structural and mechanical
propertiesofthegenerateddECMconstruct.
Withrespecttothetechniquesusedtoinfusethe decellulariza-tionagentswithinallregionsoftheorgan,perfusionthroughthe
vasculature and immersion/agitation are nowadays the most
employed, though others techniques have been also described
(e.g.,pressuregradient[51,52],supercriticalfluid[53]).Perfusion decellularizationtakesadvantageoftheinnatevasculatureofthe organ to deliver the decellularization agents across the entire
organ.Suchapproachisoftenperformedinorgansinwhichits
mainarterycanbecannulatedforperfusionwithdecellularization solutionsunderphysiologicperfusionpressures.Intheirseminal work,Ottandcoworkersdevelopedaprotocolforwholeratheart
decellularization using a homemade bioreactor to perfuse 1%
sodiumdodecylsulfate(SDS)throughthecoronaryvasculature,
showingthat12hoursperfusionata physiologicalpressure suf-ficedtoyieldafullydecellularizedratheart[36].Thesameresearch
group subsequentlyapplied a similar approach to decellularize
wholeratlungs[54]andkidneys[55].Forlungdecellularization,
boththeairwayandvascularcompartmentswereinvestigatedto
deliverthedecellularizationsolutions,renderinginallcases
acel-lular organ scaffolds with preserved ECM composition,
micro-structure and 3D architecture [54,56–59]. Overall, perfusion
decellularizationisthepreferredtechniquetodecellularizewhole organs,especiallyinlargeanimalsorhumans[4–8,58,60–63].
Onanotherhand,inimmersion-baseddecellularization proto-cols,thetissueororganofinterestissubmergedintothe decel-lularizationsolutionswhilebeingsubjectedtoagitation.Inthis way,decellularizationagentsenterthroughthetissuebydiffusion.
Thedurationoftheprotocolwillthendependontheinitialtissue thicknessandcelldensity.Suchapproachismainlyusedontissue samplesthatdonothaveeasyaccesstothevascularnetwork(e.g., skeletalmuscle,skin)[64–66],aswellasonorganslicestypically
obtained from a segmental resection. Box 2 summarizes the
advantagesanddisadvantagesofthedifferentmethodsof decel-lularizationdiscussedinthisreview.
Due to the large diversity of decellularizationprotocols and tissue sources reported so far, there has been the necessity to
establish common criteria to evaluate the effectiveness of any
decellularizationprocess.Decellularizationrequirementsand
as-sociatedmethodologiesaresummarizedinBox3.
Although successful decellularizationwas achieved formany
organs,stillmuch effortshouldbedirectedonthe definitionof standardized decellularization protocols with the final goal to advanceinthecreationofbiocompatibleandpersonalizedorgan scaffoldsforclinicalapplications.Forthat,issuesincluding
bio-degradation,cytocompatibility,pathogenicityand
immunogenic-ityshouldalsobefurtherstudied(reviewedin[67]).
Recellularization
of
whole
organ
dECM
scaffolds
Oneofthemajorissuesinthefieldoforganbioengineeringisthe precisepositioningofspecificcelltypesinsidetheircorresponding
specificorgancompartment.Methodsofcellseedingintowhole
organ-derived dECM scaffoldswilllargelydependonthe organ
itself and usually will require the use of bioreactors. Complex organsincludingkidney,heart,lung,andliverentailthe develop-mentofchallengingcellseedingandculturemethodstopromote
tissue formation and maturation. Oxygen diffusion across an
engineered tissue is limitedto a maximumtissue thickness of
200micrometers (reviewedin[68]),meaningthat highertissue thicknessesmustrequirefunctionalvasculaturetosupplythecells
with oxygen and nutrients as well as facilitate the removal of
metabolicwasteproducts.Inthisregard,bioreactortechnologyfor
whole organ engineering stillneeds to overcome many issues:
MaterialsTodayVolume00,Number00January2017 RESEARCH
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Pleasecitethisarticleinpressas:E.Garreta,etal.,Mater.Today(2017),http://dx.doi.org/10.1016/j.mattod.2016.12.005
BOX2
Methodsofdecellularization
Perfusion Immersion/agitation
Modeofaction Infusionofthedecellularizationagentsthroughtheorgan vasculature
Immersionofthetissueororganintothedecellularization solutionswhileshaking
Preferredwhendecellularizinglargeanimalorhuman organs
Usedwhentheaccesstothevasculatureisdifficultor absent
Advantages Facilitateshomogeneousexposuretothedecellularization reagentsandremovalofcellularcontent
Mechanicalagitationfacilitatescellularcontentremoval Thepossibilitytoapplyphysiologicalperfusionpressures
wouldfavorpreservationoftissueECMcompositionand architecture
Easyandfastprocedurewhendecellularizingsmallanimal organsortissues,andhumantissuesobtainedfrom segmentalresections
Controlledperfusionconditionsbytheuseofbioreactors enhancestherobustnessandefficiencyoftheprocess
Doesnotneedoverlyspecificbioreactorequipment
Disadvantages UnappropriateperfusionpressurescandisruptECMand impactontheviscoelasticbehaviorofthedECMscaffold. Optimizationisrequiredforeachtissue/organ
Decellularizationconditions(e.g.,agitation,reagents’ exposuretime)havetobeoptimizeddependingonthe tissuethickness.ExcessiveagitationcandisruptECM Needscannulationofthemainorganartery Itisanunreliablemethodwhendecellularizinglarge
animalorhumanwholeorgans Thismethodusuallyneedstheuseofspecificperfusion
bioreactors
Thismethodusuallyneedsincreasingtimesofexposureto thedecellularizationagentswhencomparedtoperfusion References [4–8,54,56–63] [39,64–66]
3
RESEARC
H:
BOX3
Establishedcriteriaforeffectivedecellularization
Requirement Methodology Outcomeforeffectivedecellularization References Removalofcells
andDNAcontent
Decellularizedtissuesshouldaccomplish Avoidadversecellandhostresponseaswell asnegativetissueremodelingand inflammationresponsesafterimplantation
[34](reviewedin[48])
(i)<50ngofdouble-strandedDNA(dsDNA) permgofdryweightdECM,asquantifiedby commerciallyavailabledsDNAintercalators suchasPicoGreenandgelelectrophoresis (ii)<200bpDNAfragmentlength,analyzed bygelelectrophoresis
Histologicalstainings(Hematoxylinand Eosin,Masson’sTrichome,Movat’s Pentachrome,orSafrinO)or
immunohistochemistryanalysisindECM shoulddenotethelackofvisiblenuclei(as stainedDAPIorHoechst).Thesecanbeused toqualitativelydetectifnucleicontent, cytoplasmicproteinsorsomeextracellular componentsarestillpresentafter decellularization
Quantificationof residualdetergents
QuantificationofremnantSDScanbe determinedusingStains-Allreagent
Avoidtoxiceffectsformthese decellularizationagents,ensuringcell viabilityduringdECMscaffold recellularization.
[67]
SimilarlyresidualTritonX-100canbe quantifiedbyusingderivative spectrophotometry.
Undetectableamountsofsuchcomponents aredesired
Preservationof ECMcomponents
Qualitativeevaluationby
immunohistochemistryanalysisofthemain ECMproteinsincludingcollagens,laminin, fibronectinandelastin
Retentionofthemainbasementmembrane componentsandstructuralECMproteinsas comparedtotheirnativecounterparts
[8,43,56,84]
Quantitativecolorimetricassaysare employedtodeterminetheamountof collagen,elastinandglycosaminoglycans usingcommerciallyavailableSircol,Fastin andBlyscankits,respectively
Massspectrometry-basedproteomics allowspreciseidentificationofmatrisome proteinsandothertissue-specificproteins Maintenanceof
3Darchitecture andvascular integrity
Micro-andnano-architectureofrelevant parenchymalstructuresofeachorgancanbe assessedbyscanningelectronmicroscopy (SEM)
Conservationofthemainstructuralfeatures specificofeachorgan,whichwillfacilitate tissueorganizationandmaintenanceof specificcellphenotypes
[4,5](reviewedin[22,36])
Conservationofthehierarchicalvascular bedoftheorganafterdecellularizationcan beevaluatedbyMicroCT,dyeormicrobeads perfusionassays,angiographyorcorrosion casting
Conservationofthehierarchicalvascular bedoftheorgan,whichisessentialtofurther achieveaneffectiverecellularization outcome
Biomechanical performance
Traditionalmaterialscienceand
engineeringtechniquesincludinguni-or bi-axialmechanicaltestingandatomicforce microscopy(AFM)havebeenmainlyusedfor mechanicaltestingonproduced
decellularizedscaffolds
OptimaldECMscaffoldmechanical properties(mechanicalstrengthand viscoelasticbehavior),thatfollowing recellularizationshouldbesimilartothoseof theirnativecounterparts
[4,41,56,63]
Lungmechanicshasbeenassessedusing pressure-volumecurvesandmeasuringforce tensionrelationshipsinlinearstripsof decellularizedlungs
4
RESEARCH:
fromthedefinitionofoptimalcellseedingmethodologiestothe possibilitytomonitorbiochemicalandbiophysicalmarkers indic-ativeoforganmaturationandfunctioninanon-invasivemanner. Otherissuesarerelatedtotheapplicationofphysiologicalrelevant stimulithatcanenhancetissueformationandfunctionality.The
principle bioreactor requirements for whole organ culture are
listedin Box4.Extensivediscussiononrecentbioreactor devel-opmentsisreportedelsewhere(reviewedin[69])[70–77].
Vascularandnon-vascularrouteshavebeenemployedto deliv-ercellsintowholeorgan-deriveddECMscaffolds,mostly
follow-ing dynamic seeding procedures, which consist of introducing
cellsataspecificconcentrationintothevascularperfusionline. Followingthismethodology, researchershaverecellularizedthe
vasculature and parenchyma of liver, heart, lung and kidney
(reviewed in [22–27]). Cells introduced into the vascular flow
maytraversethe vascularliningthroughporesproducedduring
decellularization, thenreaching the organ parenchyma. In this
regard,ithasbeen proposedthattheproperadjustmentofflow
rates may reduce shear stress on cells, while minimizing the
potential damage on the dECM scaffold. Moreover, multiple
inoculationsof cellsarepreferablethana uniqueone withthe sametotalcellnumber.Performingmultiplecellinfusionsdirectly intothelivervascularcircuitinastep-wisemannerledtomore than85%cellengraftment[77–79],alsoshowingamoreefficient distributionofcellsacrossallregionsoftheorgan-deriveddECM scaffold.Alternatively,directinjectionofcellswithasmallgauge
needle by performing multiple injections throughout different
areas of organ parenchyma has shown lesssuccess [80]. Other
non-vascularroutescommonlyusedtoreintroducecellsarethe
tracheainlungs[54,56–59]ortheureterinkidney[55,81–83]. Sofar,thelargebodyofworkregardingrecellularizationofwhole
organdECMscaffoldshasbeenperformedinsmallanimalmodels
usingdifferentbioreactor settings, celltypesand seeding condi-tions(reviewedin[22–27]).Themainproblemsencountered dur-ingrecellularizationweretheuncompletere-endothelizationofthe organvasculature,andtheinsufficientrepopulationoftheorgan parenchyma[55,56,60].Notably,recently thisyear,Guyetteand coworkershavepartiallyrepopulatedwholedecellularizedhuman
heartsusingacustomhumanheartbioreactorcapableofproviding coronaryperfusionandleftventriclewallmechanicalstimulation,
showing metabolically active repopulated myocardial segments
after14days oforgan culture[84].Also Nicholsand coworkers havereported,forthefirsttime,thedevelopmentofabioreactor systemtosupportrecellularizationofwholehumanpaediatriclung dECMscaffolds,identifyingthemainconditionsandcell require-mentsnecessaryforbioengineeringwholehumanlungs[85].
Moreover, many different cell types and sources have been
reported for recellularization strategies of different organs
(reviewedin[22–27]).Initialworksmadeuseofneonatalorfetal cellsderivedfromtheorganofinterest,showingretentionoftheir
tissue-specific phenotype after seeding into the organ-derived
dECMscaffoldtogetherwithrelevantorgan-specificfunctionality. Overall,theseworksservedasaproof-of-conceptofthefabrication
of whole organs de novo by decellularization/recellularization
techniques.However,thosefindingsalsohighlightedthe necessi-tytofindmoreamenablecellsourcesthatcouldbeeasily expand-edanddifferentiatedintofunctionalandmultiplecelllineages.In
this regard,hPSCshave beenproposed aspromising candidates
duetotheirself-renewalcapacityandthepotentialtogiveriseto anycelltypeinthebody[86–88].TheuseofhPSCsasacellsource
for the development of bioengineered organs based on dECM
scaffoldsisfurtherdiscussedinthefollowingsection.
Pluripotent
stem
cells:
a
long-standing
cell
source
for
regenerative
medicine
Pluripotent
stem
cells
Pluripotencyisdefinedastheabilityofasinglecelltodivideand
produce differentiated cells from the three germ layers of the
embryo[86–88].Theideatogeneratefunctionaltissuesandorgans frompluripotentstemcells(PSCs)hasbeenalong-standinggoalin stemcellbiology,representinganunprecedentedopportunityto
study development and evento heal degenerative diseases and
aging-related disorders.Inthis regard,thepossibilitytocapture
and culture indefinitely hESCs from the pluripotent inner cell
mass(ICM)oftheblastocysthasbeenamajorbreakthroughinthe areaofregenerativemedicine[86].
BeforehESCswerefirstderived,seminalstudiesalreadytriedto
answerhowshapeandpatternemergefromthesimplebeginnings
ofanembryo,andevenhowspecializedcellsdifferentiateduring
embryodevelopmentbecomingorganizedintoa3Darchitectural
context (reviewedin [89]) [90].Pursuingthe idea toreprogram differentiatedcellstoan‘embryonic’state,Takahashiand Yama-nakain2006discoveredthatthepluripotentstatefoundinhESCs derivedfromtheICMcouldbeartificiallyinducedinasomaticcell
through the overexpression of just four transcription factors
(OCT4, SOX2, cMYC,and KLF4-OSKM) [87](reviewed in [91]).
Theproducedcells,socallediPSCs,exhibitedallthemolecularand functionalfeaturesofESCs.Importantly,inthelastyearshuman
iPSCs(hiPSCs)haveshowntobecomeinstrumentalplatformsfor
thestudyofhumandevelopmentanddiseasewiththe identifica-tion,insomecases,ofmolecularandcellularmechanisms respon-siblefordiseasegestationandprogression(reviewedin[92]).
Overall, one major limitation in the field of hiPSC disease
modeling is the lack of a systemic context and disease-related
environmental cues [e.g., disorganized ECM, insufficient
bio-chemical signals from the niche, among others], opening new
MaterialsTodayVolume00,Number00January2017 RESEARCH
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BOX4
BioreactorrequirementsforrecellulatizationofwholeorgandECM scaffolds
Includeindependentaccesslinesforcellseedingthroughvascularand non-vascularroutes
Integratepressuretransducerstocontrolflow/volume-basedpressure Allowcontinuousorpulsatileperfusionthroughthevasculatureto providenutrientswhileremovingmetabolicwasteproducts Monitoringandmaintenanceofphysiologicalvascularpressure Monitoringandmaintenanceofpropergasessupply
Monitoringorganmechanicsduringrepopulation
Canincludeorgan-specificbiophysicalstimuli(e.g.providemechanical ventilationinlung,providemechanicalstretchandelectricalstimulationin heart)
Allownon-invasiveorminimallyinvasivemonitoringofrelevant biochemicalandbiophysicalmarkersindicativeoforganmaturationand function
Allowautomationofcriticalparameters
Maintainsterility(disposableoreasilysterilizedcomponents)
Goodmanufacturingpractices(GMP)amenable/Clinicalgradebioreactors
5
RESEARC
H:
challengeswhenintegratingbiomaterialsmimickingdisease pa-thology.Inthisregard,ithasbeenrecentlyshownthattheproper fine-tuningof3Dscaffoldsrecapitulatedtheabnormal
contractili-tyinhiPSC-derivedcardiomyocytesfrompatients withlongQT
syndrometype3,andnotwhenhealthycounterpartswereused.
Suchplatformalsoallowedforthescreeningofcardiotoxic com-poundsindifferent3Dsettings,highlightingthepotential appli-cationoftheseapproachesforhumandiseasemodeling[93].
Differentiation
strategies:
from
pluripotent
colonies
to
organoids/organogenesis
in
a
dish
OneofthemajorareasofresearchinthefieldofhPSCshasbeen thedevelopmentofprotocolsforthegenerationoffunctionalcell typessuitablefordiseasemodelingandcellreplacementtherapies. Until recently,most protocolsof differentiationrelayedon the generationofsinglecellpopulationsratherthancompletetissues. However,inthelastthreeyearspivotalstudieshavedemonstrated
thatit ispossible to generate 3Dcultures ofdevelopingtissues namedorganoids(reviewedin[94,95]).
Afterthe firstderivation ofhESCs [86], differentlaboratories worldwideexploredhESCscapacitytoundergocontrolled differ-entiationeitherinmonolayer,byseedingcellsinthepresenceof differentECMproteincoatings(majormatricesusedforthe cul-tureofhPSCsaredescribedinBox5) (reviewedin[94–96])[97– 101],orasspheroid-likestructuresnamedembryoidbodies(EBs) (Fig. 2). Although EBs can recapitulate several aspects of early
development(reviewed in [94]), asanyother methodology, EB
formationstillhampers the translationofthis approach intoa clinicalsetting(e.g.,lowreproducibilityand scalability). Never-theless,alltheseadvanceshavebeenfundamentalfortheproper instructionofhPSCs to formself-organized tissue-specific orga-noidsincludingtheopticcup,brain,intestine,liverandkidney (reviewedin[94]).
Organoids are similar to in vitro derived EBs, but they can
recapitulatea largenumberofbiological processesrelated with spatialandtemporalorganizationofheterogeneoustissue-specific cellswithinthe3Dstructures(Fig.2).Eveninsomecases, orga-noidshaveprovedtoexhibitphysiologicalfunctionsbeingclose totheinvivosetting.Inthisregard,kidneyorganoidsderivedfrom
hiPSCs havebeen recentlyshown tocontainmultiplenephron
segments surrounded by nascent blood vessels, being able to
respondinfrontofnephrotoxiccompounds[102].Despitethese
findings,most hPSC-derived organoid modelsonly represented
single or partial components of a tissue, hindering the proper
control of cell-cell interactions, cell-matrix interplay, and cell
organization.Moreover,commonhurdlesinorganoid
technolo-giesarebeingrelatedtoincompletematurationofhPSC-derived celltypesandthelackofvascularization.
Bioengineering
approaches
for
hPSCs
differentiation:
dECMs
as
biomimetic
platforms
for
generating
tissues
on-demand
Bioengineeringapproachescanbeusedtoovercomemajorissues
associatedwithhPSCsdifferentiationasmaturationand function-ality.Inthisregard,matrigelhasbeena fundamentalmatrixfor organoidmethodologies,includingcerebral,opticcupand intesti-nalorganoidsfromhPSCs(reviewedin[94,95]).However,matrigel
BOX5
ExtracellularmatrixcomponentsusedinhPSCscultureanddifferentiation
ECMcomponents References
CollagensarethemostabundantECMmacromoleculesfoundinourbody.Amongthe28typesofcollagenthatexist,collagen IVandIhavebeenwidelyusedinhPSCsdifferentiationstudiesdemonstratingtobeimplicatedinmesodermaldifferentiation.In addition,collagen’sabilitytoself-aggregateandcrosslinkmakesitanattractivemacromoleculeforbiomaterialscience
[97]
Lamininisatrimericproteinfoundinthebasementmembrane,whichhasbeenlargelyinvestigatedforitsinfluenceinhPSCs differentiationtowardsectodermaltissues
[98]
FibronectinisanECMproteinhighlyexpressedduringtheearlystagesofembryonicdevelopment,beingessentialforproper developmentofthemesodermandtheneuraltube.Generally,itiswidelyusedascelladhesionproteinduetothepresenceof thepeptidesequencearginine-serine-asparticacid(RGD)initsstructure,whichisimplicatedinintegrin-mediatedcelladhesion
[99,100]
MatrigelisgeneratedfromthebasementmembraneofmousesarcomacellstherebycontainingavarietyofECMmolecules andgrowthfactors.LamininisthemajorconstituentalthoughcollagenIVandproteoglycansalsotakepartofitscomposition. Sinceitisderivedformbasementmembraneitprovidesarichenvironmentthathasbeenlargelyusedforthemaintenanceof hPSCsaswellasdifferentiationtomanylineagesincludingcellsfromthethreegermlayers,mesoderm,endodermandectoderm
Reviewedin[94–96]
Cell-depositedECMcanbeextractedfromdifferentcelltypesbyfirstallowingthecellstoproducetheirownECMandthen removingthemwithoutdisturbingthenewlysynthesizedECMstructureandbiochemicalcomposition.Suchcell-depositedECM hasbeenthenusedasacoatingforstemcellstemnessanddifferentiationstudies
[101]
FIGURE2
ThemainstrategiesusedforhPSCsdifferentiationincludeguided differentiationin2Dmonolayercultures,theformationofembryoidbodies andtheinductionof3Dself-organizationgivingrisetoorganoids.The generationoforganoidsneedsahighercellculturecomplexitythanthe othertwoapproaches.
6
RESEARCH:
Materials Toda y V olume 00, Number 00 Januar y 2017 RESEARCH MA TTOD-849; No of Pages 13 Please cite this article in press as: E. Garreta, et al., Mater . T oday (2017), http://dx.do i.org/10.1016/j.mattod.2016.12.0 05 BOX 6 3D bioprinting techniques Bioprinting techniques
Additive unit Actuation method Cell
viability
Commercial bioprinter
Bioink viscosity
Disadvantages Advantages References
Inkjet printing Drop Piezoelectric pulse
Thermal induced pulse
>85% Yes Low Microvalves for inkjet are
fragile
Requires fast material gelation/stabilization Height of 3D constructs limited due to low bioink viscosity
High control on the amount of material deposited (1 pL to 0.1 nL per drop) Reviewed in[12] Laser assisted bioprinting
Drop Laser induced pulse >95% No Medium-high High cost of future
commercial bioprinters Long fabrication time Long preparation time of material ribbons
Low diversity of bioink for ribbon preparation
High printing resolution Single cell deposition
Reviewed
in[12]
Micro extrusion Material strain Pneumatic pressure 40–95% Yes Medium-high Pneumatic pressure
Cells may suffer important shear stress
Medium printing accuracy Low printing resolution
Pneumatic pressure Abrasive materials can be used
Disposable cartridge that avoids cross-contamination Non mechanical parts Used by most of commercial bioprinters due to its robustness and simplicity Allows printing cell high density
Reviewed in[10,12]
Mechanical pressure Mechanical pressure
Cells may suffer important shear stress
Medium printing accuracy Low printing resolution Mechanical parts make this system more fragile Screw based system require cleaning of mechanical parts
Mechanical pressure Abrasive materials can be used
Piston based systems uses disposable syringes Allows printing cell high density
Stereolithography (SLA)
Cured bioink voxel
Laser based curing UV and visible light projection curing
>85% Yes Medium Only can be used with light
crosslinkable bioinks Multicellular structures are challenging
Printer are not specifically designed for biofabrication
Affordable
High printing velocity
Reviewed in
[10,123,127,128]
7
compositionisnotwelldefinedandbatch-to-batchdifferencesmay leadtoimportantdifferencesinexperimentaloutcomes[103].
Alongthisline,biomaterialscanbeusedtocreatestem-cell-like nichesprovidingkeyelementstocontroltheregulationofstem cellfateandfunction.Indeed,materialpropertieshavebeenoften designedtomimicphysiologicallyrelevantECMstiffness, topog-raphy,andadhesion-ligandtype,densityandaffinity.These
fea-tures,whencombined withhPSCshaveledtothe derivationof
protocols for hPSCs differentiation building personalized tissue
constructs using human organ-derived dECM scaffolds
[43,84,104,105],andeven forpartiallyor totally reconstructing
mouse[106]andhumanwholeorgans[84,105].
Ottandcoworkersrecentlyreportedforthefirsttimethewhole repopulationofdecellularizedhumanheartswithhiPSCs-derived cardiomyocytes[84].Inthesameline,ourgroupdevelopedarapid protocolforthegenerationofhumanheartgraftsbyco-culturing
hPSC-derived cardiomyocytes on top of 400micrometers-thick
slicesofhumanventriculardECMscaffolds[43].
Importantly,rat and humanlung dECM scaffoldshave been
alsorecentlyshowntoberepopulatedwithendothelialand peri-vascular cellsdifferentiated fromhiPSCs [105].Concerning kid-ney,onlytwoworkshaveinvestigatedtheroleofrhesusmonkey kidneydECMscaffoldsonhPSCsrenaldifferentiation[40,107].
Alternatively, 3D bioprinting technology have opened new
venuesforthebottom-upgenerationoftissueandorgananalogues bythedepositioninanadditivelayer-to-layerapproachof differ-entiatedhPSCs and biomaterials,specifically arrangedto
repro-duce native 3D architectures (reviewed in [11,12,95]).
Nevertheless,attemptstogeneratehPSCsderivedbioprinted con-structsarestillinitsinfancy,withonlyonereportonthe genera-tionofminiliversfromhPSCs[16].
Tissue
engineering
of
human
organ
analogues
by
3D
bioprinting
3D
bioprinting
techniques
Bioprintingtechniquesaimtoperformsimultaneousdepositionof
single or multiple combinations of living cells together with
supportivematricescontainingbiochemicalandbiophysicalcues (altogether termed asbioink). In this manner, organs or tissue analoguesareconstructedfollowingapredefinedarchitecturein 3D(reviewedin[12–14]).Thesemethodologiesaregenerally clas-sifiedbasedonthetechnologyusedtogeneratethestructuresof thecell-ladenmaterialasfollows:
Inkjet bioprinting, also referred as ‘drop-on-demand printers’ appeared early in 2003 [108]. Firstly developed inkjet printers
modifiedcommerciallyavailabletwo-dimensional(2D)ink-based
printers by replacing the ink in the cartridge by a biological
material, and the paper, by an electric-controlled elevator that
moves on the z direction providing three-dimensionality
(reviewed in[12])[109].Nowadays,inkjet printersmake useof nozzles thatgenerate isolateddroplets ofcell-ladenmaterialby meansofpiezoelectric[110]orthermal(reviewedin[111]) actu-ationsystems.Inthismanner,bymeansofeitheracousticwavesor thermalforces,respectively,liquiddropsareejectedontoa sub-strate. In the last years, inkjet bioprinting has allowed for the efficientintroductionofgradientsofcellsorgrowthfactorsalong the3Dconstructsbythemodificationofdropdensitiesandsize
[17,112,113].Despitethismajoradvances,onecommondrawback
ininkjetprintingistheneedtoworkwithbiologicalmaterialsin liquidforms,whichinmostofthecasesdemandafast polymeri-zation procedure post-printing (e.g., by eitherchemical, pHor ultravioletmechanisms,amongothers).Alltheseprocedures di-rectlyaffectthebioprintingprocess,compromisingthechemical andmechanicalpropertiesofECM-derivedmaterials.Asreviewed elsewhere,otherdisadvantagesarerelatedtotheimpedimentof building3Dcell-ladenconstructswithgeneraltissuesize,nozzle cloggingwhenusingsolutionswithhighcell densities,andcell viabilityconstraints(relatedtotheuseofcross-linkers)(reviewed in [12]). Nevertheless, inkjet-based bioprinters have become a massiveusedtechnologyreducingcostsand facilitatingtheuse offreedesignsandsoftwaresworldwide.Currentresearchisnow focusedinthedevelopmentofnoveltechnologiesallowingtheuse ofmultiplecelltypesandmaterials.
Microextrusionsystemsappearedasamodificationofinkjet prin-ters.Heretheextrusionofthematerialtakesplacethrough micro-metricapertures(usuallyaneedlewithinnerdiametersthatrange
from few to hundred micrometers). By applying a continuous
force, this technique allows to print uninterrupted cylindrical
lines. Commonly, these systems robotically extrude biological
materialsby pneumaticormechanical dispensing systemsonto
asubstrate.Ithasbeenreportedthatalmostallkindofhydrogels withvaryingviscosities,aswellasaggregateswithhighcelldensity canbeprintedwiththisapproach(reviewedin[10,12]).Compared toinkjetprinting,microextrusionprintingenablesthedeposition oflargeamountofcells,allowingthegenerationofconstructswith generaltissuesize.Sofar,highconcentrationsofhydrogelssuchas alginate,fibrinandPluronicF-127,amongothers,haveprovedto beeffectivewhenproducingstable3Dcell-ladenstructures[114– 120].Importantly,microextrusionsystemshavebeenparticularly effectivewhenprintingmulticellulartissuespheroidsthatfurther self-assemble into the desired 3D structure (reviewed in [15]).
Although all the advantages described here, one of the major
limitationsofthistechniqueisthedecreasedcellviabilityresulting fromthe shearstresswhencellsareinviscousfluidsduringthe extrusionprocess[121].Thus,oneofthemainchallengesconsists
in the retention of cell viability and printing speed without
decreasingpressureor reducingnozzlesize. Severaltissueshave
been fabricatedusing this system, including branched vascular
trees,aorticvalves,andinvitrotumormodels(reviewedin[12]).
Recently, Atala and coworkers have lead the first work on the
fabricationofbioprintedtissuefunctionalconstructsinvitroandin vivoformandiblebone,ear-shapedcartilageandorganizedskeletal muscleathuman-scale[18].
Inlaser-assistedbioprinting(LABP)dropsofcell-ladenbiomaterials aregeneratedafterlaserpulses.Thefallingbioinkdropletisfurther collectedonthesubstrateandcrosslinked,avoidingshearstressand resultinginhighcellviabilities,evenwhenusinghighlyviscous materials.Thisrecentmethodologyreliesontheuseofalaserpulse
that createsa high-pressure bubbleon a ribbon containing the
materialtobeprinted,therebygeneratingabioinkdroplet.LABPis nozzle-free,thusminimizingclogging-relatedissues.Moreover,the achievedresolutionallowsthedeliveryofsingle-cellsoneachdrop. Sincethissystemgeneratesscaffold-free3Dcellconstructsthrough
a layer-by-layer manner, lately LABP is becoming used for the
deposition of different living cells and biomaterials in a well-defined3Dstructure.Besidestheseadvantages,stillpossibleside
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RESEARCH:
effectsoflaserexposuretocellsremainelusive.Otherlimitations arerelatedtothepreparationofcell-ladenribbons(specificforeach celltypeandhydrogel,hinderingscaling-upprocedures)andthe depositionofmetallicresiduesinthe finalbioprintedconstruct. Similarly,targetingandpositioningcellsbecomesdifficultbecause ofthenatureoftheribboncellcoating(reviewedin[12]).Todate, fewworkstookadvantageofthissysteminregenerativemedicine
[19,122],andthehighcostofLABPprecludestheiruseformany researchersworldwide.
Stereolithography(SLA)wasinitiallyusedtocreatecell-free scaf-foldsbuttheincreasingdevelopmentintheformulationsofnew cross-linkablematerialsallowedtheuseofSLAfor3Dbioprinting (reviewedin[123])[124].InSLA,thelaserfocusingpointmoveson theX/Yaxesalongtheuncuredbioink,whilethestagewherethe materialis polymerized lowers allowing polymerization. Lately, directlightprojection(DLP)hasemergedasanaffordableand versa-tilevariant ofSLA.InDLP,thelightfromadigitalmicromirror deviceorprojectorisusedforcuringphotocrosslinkablehydrogels in a layer-by-layer approach. By curing the structures
plane-by-plane, DLP offers enormous advantages in front of SLA (e.g.,
printingtimeisnotdependingonthedesigncomplexityofeach plane).DLPhasbeenrecentlyusedforgeneratingbiocompatible
scaffolds[125–127],andmorerecentlyWangandcoworkers dem-onstrated that DLP enabled forthe fabricationof3D cell-laden structureswithresolutionsof50micrometers,andreaching85% cellviabilitywhenencapsulatinghumanfibroblasts[128].DLPand SLAoffernewalternativesforthefabricationof3Dbioconstructs with precisemicro- and nano-architecture, beingaffordable sys-temsintermsofcosts.
Asummary ofthe aforementioned bioprintingtechniques is
presentedinBox6.
3D
bioprinting
of
cell-laden
hydrogels
Since 2000, when rapid prototyping technologies were first
adapted forthe depositionof cell-ladenhydrogel3Dstructures in cell-compatible printing conditions, researchers all over the worldhavetriedtoproducebiologicaltissue-likeconstructsusing
different cell types and hydrogel formulations. The basic 3D
bioprintingequipmentneedswhenaimingtofabricatecell-laden 3DstructuresaredescribedinBox7.
Current effortsaredevoted to developnovel biomaterial
for-mulations thatcanmimic the complexityofthenativeECM–a
conceptcalledbiomimicry–withanimpactforbioprinting appli-cations.ECMcompositionandrigidityhaveprovedtodictatecell
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BOX7
3Dbioprintingofcell-ladentissueconstructs
3Dbioprinterneeds Allowcombinationofmicro-extrusion,fuseddepositionandinkjetprintingatthesametimeduringa printingprocedure
Haveatleasttwohydrogel-compatibleprintingheads,whichfacilitatesdepositionofdifferentcelltypes inthesameprintedconstruct
Possesstemperaturecontrolsystemsforprintingheadsandsubstrate Allowopticalmonitoringoftheprintingprocess
Allowtheinterchangeofmaterialcartridgesduringtheprintingprocedure Precisecontrolofthepressureappliedduringtheprintingprocess
Allowtheuseofdifferentsubstratestoprintthedesired3Dtissueconstruct(e.g.petridish,multiwall plates)
Possesanaccuratecalibrationsystemforneedletippositioning Possesasourceoflight(UV,bluelight)forlight-crosslinkinghydrogels Workundersterileconditions
Keymaterialpropertiesfor3Dbioprinting Printabilitydefinesthesuitabilityofamaterialforaspecificprintingprocessandlargelydependsonthe materialphysicochemicalproperties(viscosity,shearthinning,yieldstress,hydrogelcrosslinking mechanism)undertheconditionsprovidedbythebioprintinginstrument
Biocompatibilityreferstotheabilitytoperformasamaterialthatwillsupporttheappropriatecellular activity,includingthefacilitationofmolecularandmechanicalsignalingsystems,inordertooptimizetissue regeneration,withoutelicitinganyundesirablehostresponses
Biodegradabilitydescribesthebiologicalprocessesinsidethebodythatcauseagradualbreakdownofa material.Degradationkineticsshouldbematchedtothenovotissueformationandthebyproducts generatedshouldbenontoxic
Biomechanicalpropertiesofthematerialshouldprovidesufficientstructuralintegritytotheprinted constructandmatchtissuespecificbiomechanicrequirements
Biomimicryreferstotheabilityofreproducetissue-specificendogenousmaterialcompositions 3Dbioprintingprocessingparameters Thefourmainvariablesthatneedtobebalancedtooptimizetheprintingprocedureandensurethe
designfidelityare:theneedle/tipsize,thedistancefromthetiptothesurface,thematerialflowrateand thelinearwritespeed
Thesevariablesarebalancedwhentheleadingedgeoftheprintedbioinkiscontinualwiththeneedle, beingpossibletodispenseauniformstrand
Theseaforementionedvariablesneedtobeoptimizedforeverydifferentmaterialused
Changesonenvironmentalconditionssuchashumidityandtemperaturemayinfluenceprintingfidelity Asprintedstructuresincreaseincomplexity,theincorporationofsupportstructureswithinthefabricated patternisnecessary
Theprintingtimeshouldbeconsidered,sinceextendedperiodsoftimeatnon-optimaltemperature, humidityormaterialbiocompatibilitywillaffectbothmaterialpropertiesandcellviability
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fateandfunction(reviewedin[129]).Inthehumanbody,tissue rigidityrangesfrom0.2–5kPainsofttissuesasbrain,to15,000kPa
in bone, beingan important parameterto beconsidered when
aimingtodesign3Dtissueandorgananalogues.
Besides the need to be biocompatible and biodegradable, a
biomaterial formulation for bioprinting must possess suitable
physicochemical properties in order to fabricate 3D constructs
withhighresolutionandprintingfidelity–acharacteristicnamed printability(Box7)(reviewedin[130–132]).Inaddition,itmust alsobeoptimizedinordertominimizestress-induceddamageto
the cells and biological components, which occur during the
deposition process (reviewed in [130–132]). The ideal hydrogel
formulationshouldreachacompromisebetweenpreservingcell
viabilityandmatchingoptimalprintability.
Viscosity,shearthinningandyieldstressofadefinedbioinkwill directlyaffectprintingfidelityduringthebiofabricationprocess. Similarly,thespecificprocessingparameterswilldefinetheshear stress that cells willsuffer during the deposition and the time requiredforthefabricationofagiven3Dconstruct(e.g.,tipsize, flowrate,temperature;Box7).Anotherimportantoutcomeisthe maintenanceofsufficientstructuralintegritybythebioprinted3D construct,whichwillbemainlydependentonthegelationofthe
hydrogelformulation.Naturalandsynthetichydrogelsappliedto
3Dbioprintingandtheircorrespondentgelationmechanismare
furtherreviewedelsewhere(reviewedin[130–133]).
Elegantworkshavedemonstratedthefeasibilityof3D bioprint-ingforthegenerationofseveraltissues,includingbone[18],skin
[19],vasculargrafts[118,120,134],trachealsupports[135],heart
[20,117,136]and cartilage[18,20]tissuestructures,usingseveral cellsourcessuchashumanumbilicalveinendothelialcells,
mes-enchymal stem cells, human meniscus cells and fibroblasts
(reviewedin[10,12]).However,thefabricationofhumansizeorgan analogueswithcomplexarchitecturesrequiresamoreelaborate3D bioprintingstrategy,usuallyinvolvingtheco-depositionofother materialcomponents(suchassupportiveandsacrificialmaterials), togetherwith cell-ladenhydrogels,ensuringsufficient structural integritytothe printedconstructwhile maintaininganinternal porosity (Fig. 3). In this regard, the recent work by Atala and coworkersprovidesa3Dbioprintingplatformfortheproduction oftissuesforhumanapplications,pavingthewayforfuture build-ingofsolidorgans[137].
Acellular
organ-specific
dECM
hydrogels
for
3D
bioprinting
As decellularization protocols emerged, hydrogels made from
decellularized tissues including urinary bladder [138], heart
[139],liver[140],dermis[141],adiposetissue[142],bone[143], and lung[144],among others,weredevelopedand reportedto supportgrowthandfunctionofdifferentcelltypes.However,itis notuntilveryrecentlythattissue-specificdECMhydrogelshave
beenenvisionedasa newclassofhydrogelsfor3D bioprinting
[20,145–147].Nowadays, one of the main hurdles when using
dECM hydrogelsasbioinks relieson their lowviscosity, which
inevitablycompromise shapefidelityofthebioprinted3D
con-struct,worseningprintingresolution.
To date,only fewstudies have used dECM hydrogels for 3D
bioprinting,applyingdifferent strategies toimprove their print-ability[20,145–147].Patiandcoworkerswerethefirstto
success-fully apply dECM hydrogels for 3D bioprinting. Their strategy
consisted on the co-deposition of an open porous structure of
polycaprolactone (PCL) as a supportive material, together with
thecell-ladendECMhydrogelmadefromcartilage,heartoradipose porcinetissues[20].Recently,thesamegroupusedskeletal muscle-deriveddECMhydrogelsfor3Dprintingofmuscleconstructs[147]. Followingadifferentstrategy,Skardalandcoworkerselegantly
developedmodularhyaluronicacidandgelatin-basedhydrogels
supplemented with porcine liver, cardiac and skeletal muscle
dECM solutions. Following a two-step crosslinking procedure,
the authors achieved printable bioinks with different stiffness rangingfrom100Pa to20kPa, thus allowingthe possibilityto mimicthe mechanical characteristics ofdifferent tissuesin the body[145].OtherrecentworkbyJangandcoworkerstook advan-tageofpigheartdECMtopreparecardiac-specifichydrogelsthatin
combinationwithhumancardiac progenitorcellswereusedto
fabricate3Dbioprintedcardiacconstructs.Thegelationofcardiac
dECMhydrogelswasbasedonthermalandchemicalcrosslinking
usingvitaminB2viaUVAactivation[146].
Future
outlook
Amajorlimitationwhengeneratingartificialorgansondemand
standsinthedevelopmentoftechniquestoproperlyreintroduce
FIGURE3
Schematicrepresentationofa3Dbioprintingsystemconsistingofa computeraided3-axisstagecontrollerandadepositionmoduleincluding threedifferentprintheadsconnectedtoapressurecontroller(a).Computer aideddesignandcomputeraidedmanufacturing(CAD/CAM)processfor 3Dbioprintingofahumansizekidney.A3DCADmodelgeneratedfrom medicalimagingdata(CT:computedtomography;MRI:magneticresonance imaging)producesavisualizedmotionprogramwhichdictatestheXYZ stagemovementstogeneratethe3Dbioprintedkidneyprototype(b).
10
RESEARCH:
cellsintotheorgan-specificdECMscaffolds,assessingboth
com-plete organ re-endothelization and functional activity. Due to
theirintrinsiccharacteristics,hPSCshavebeenenvisionedasan optimalcellsourceforthegenerationofcomplextissuestructures liketheorganparenchymaandthevascularsystem,offeringmajor
advantageswhencomparedwithadultsomaticor stemcellsfor
thesamepurposes.Moreover,targetedgenomeediting,asCRISPR
platform, is a powerful tool to manipulate and correct disease
relatedgenesinpatient-derivedhiPSCs,allowingforthe genera-tionof autologous-correctedcellssuitable fordisease modeling anddrugscreening[148].Webelievethattargetedgenomeediting
approachescombined with recentprogressin the formation of
patient-specific hiPSCs-derived organoids could provide an
un-precedentedsource of organ-specific cell types suitablefor cell
replacement therapies. In this regard, common efforts on the
definitionofchemicallydefined conditions toculture
hPSC-de-rived organ-specific cells has led to the examination of novel
approachesguidinghPSCsmaturation.Followingthesequestions, differentworkshaverelayedontheuseofhumandECMscaffolds
together with hPSCs in order to generate human tissue grafts
[43,84,104],and even toreconstruct wholeorgans [84,105], re-vealingtheimpactoforgan-deriveddECMsontheproper instruc-tionofhPSCsfateandfunction[40,43,84].
Alternatively,3Dbioprintingrepresentsaformidable technol-ogyforartificialorgangeneration.Besidesthedifferentlimitations of this nascenttechnology (e.g., printing resolution and time, combinationofdifferentbioinkssimultaneously,amongothers),
the possibility to print human-scale tissues has been recently
demonstrated [18].Inthis regard,seminal studies havealready provedthe feasibilityto print3Dtissue constructsusing
organ-specific dECM hydrogels as biomimetic bioinks [20,145–147],
openingthedoortothefabricationofnovelbioinkformulations
matching cytocompatibility and mechanical strength
require-mentsfor3Dbioprinting.
Overall,webelieve that recentadvancementsinthe fieldsof hPSCsdifferentiationtogetherwithorgan-deriveddECMscaffolds
ornoveldECM-basedhydrogelsaimedfor3Dbioprinting
repre-senta stepforward in the fabrication ofautologous functional tissueson-demand(Fig.4andBox8).Tothisend, multidisciplin-aryresearchinthefieldofengineering,biomaterialsscience,stem
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FIGURE4
3Dbioprintingapproachforthedevelopmentofpatient-specificorgananaloguessuchaskidney.Patient-specificiPSCs(a)aredifferentiatedintorenal progenitorcellsin2Dmonolayercultures(representativeimageforSALL1andWT1doublepositiverenalprogenitorsbyimmunofluorescence)(b)thatare furtherinducedtodevelopintokidneyorganoids(representativeimageforPAX8andE-cadherinpositive3Drenal-likestructuresbyimmunofluorescence) (c).ThecombinationofkidneydECM-basedhydrogels(d)withpatientiPSC-derivedrenalprogenitorcellscouldbeusedforthebiofabricationofspecialized 3Drenalconstructs(representativeimageforPAX8renalstructuresdevelopedintoprintedkidneydECM-basedhydrogels)(e).Thisapproachrenderstothe generationoftissueandorgananaloguessuitableforregenerativemedicineapplications(e.g.,diseasemodeling,drugscreening,cellreplacementtherapies, amongothers).
11
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cellbiologyandmedicinewillbeessentialtofurthersucceedinthe biofabricationofautologousorgansforfutureclinicalreplacement strategies.
Acknowledgements
WethankSOLIDCAMSTUDIOfor3Dmodelling,printingand
rendering.E.GwassupportedbyStG-2014-640525_REGMAMKID.
R.OwasfoundedbyaFIfellowship(Secretariad’Universitatsi
RecercadelDepartamentd’EconomiaiConeixementdela
Generalitatde Catalunya).C.Twas supportedby
StG-2014-640525_REGMAMKID. P.PwaspartiallysupportedbyMINECO
(SAF2014-59778). F.F.A.wassupportedbyInstitutode Salud
CarlosIII-ISCII(MINECO:PI10-00141andPI10-02038),Redde
Investigacio´n Cardiovacular(RIC)andRedTerCelfrom ISCII
(MinistryofEconomyandCompetitiveness,Spain),andCAM:
S2010/BMD-2420andhasbeenfinanciallysupportedby the
CommissionforUniversitiesandResearchoftheDepartmentof
Innovation,Universities, andEnterpriseoftheGeneralitatde
Catalunya(2014 SGR1442)anddevelopedinthecontextof
ADVANCE(CAT)withthesupportofACCIO´ (CataloniaTrade&
Investment;Generalitatde Catalunya)andtheEuropean
Community undertheCatalonianERDF operationalprogram
(EuropeanRegionalDevelopmentFund)2014-2020.This
workalsowaspartially supportedbytheprojectMINDS
(TEC2015-70104-P),awarded bytheSpanish Ministry
ofEconomyandCompetitiveness,andbyCERCA
Programme/GeneralitatdeCatalunya.N.M wassupportedby
StG-2014-640525_REGMAMKID,MINECO, (SAF2014-59778,
RYC-2014-16242),andbyCERCAProgramme/Generalitatde
Catalunya,andCardioCel(TerCel,InstitutodeSaludCarlosIII)
and2014SGR1442.
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Challengesandfutureperspectives
Researcharea Specificchallenges Futureoutcomes Decellularization Standardizationofdecellularizationprotocolsforeach
specificorgan
Systematicevaluationofimmunologicresponseandthe possibilityofusingdECMscaffoldsfromdifferentspecies
Robustproductionoforgan-deriveddECMscaffolds Determinationoftheneedofimmunosuppressivedrugs aftertransplantation.Increasethetissuesource,overcoming donorshortage
Recellularization Improvedseedingmethodologiesforcomplete re-endothelizationoftheorganvascularbedandparenchyma recellularization
Novelbioreactorsystemswithoptimalcontroloverthe cultureconditions(oxygendelivery,biophysicalstimulation, continuousmonitoringoffunctionalparameters)
Optimalvascularization
Determinationoforganbiochemical/biophysical parametersduringrecellularization,andend-pointsforeach organ
Definitionofcommonguidelinesfortheassessmentof organmechanicsPreservationofbioengineeredorgans beforetransplantation
Functionalevaluationinlargeanimalmodels Humanpluripotent
stemcells
Standardizedprotocolsforexpansionanddifferentiationto largecellnumbers
PurificationofhPSC-differentiatedcellsandidentification ofoptimalcellcultureconditions(growthfactors,cytokines) forcellphenotypemaintenance
IdentificationofhPSC-derivedprogenitorcelltypessuitable forengraftmentandmaturationintodECMscaffolds
Repopulationoforgan-deriveddECMscaffoldswith multiplehPSC-derivedprogenitorcelltypesspecificforeach organcompartment,andinsufficientcellnumbertoachieve comparablenativeorgancelldensities
Generationofpersonalizedhumanbioengineeredorgan equivalentswithoptimalfunctionalactivity
3Dbioprinting Developmentofnovelcell-compatiblehydrogelswith tailoredviscosityandbiochemicalcomposition
Monitoringofbioinkflowratewithautomaticadjustment ofprintingconditions
Presentalignmentcapabilityonpreviouslyprinted constructs
Improvebioprintingfullautomation
Developmentofbioreactorsfortissue/organgrowthand maturationpost-processing
Bioprintingofcell-ladenstructureswithincreased mechanicalstrength,bioactivityandcellviability Increasedprintingfidelity
Step-wisefabricationofcomplexmulticellularstructures Rapidproductionof3Dprintedconstructs,thereby improvingcellviability
Generationoffunctionaltissue/organs
12
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