DevelopmentofCNSmodelfor drugdiscoveryusing3Dprinted
MatthieuTrigano,Anna-LenaZepernick,EveCorrie,EmmaV.Jones MedicinesDiscoveryCatapult,Block35,AlderleyPark,Cheshire,SK104ZF,UK
Introduction
IntheinvitroneuroscienceteamatMDC,ourgoalistoproduce physiologicallyrelevantcentralnervoussystem(CNS)cellmodels thatwillimprovepredictivityofassaysandtranslationtotheclinic. Onepromisingdirectioninthefieldistheuseofadvanced3D modelswhichbettermimictheinteractionsbetweencellsand extracellularmatrixinvivo.Increasingevidencesuggeststhat3D modelsmaymoreeffectivelyreproducethepathologicaldisease processes.Inthisstudy,wepresenta3DbioprintedCNSmodel generatedfromhumaninducedpluripotentstemcells(iPSCs), embeddedinahydrogelscaffoldusingextrusion-basedbioprinting technology.Thissystemallowsformono-,co-,andtri-cultureof differentneuralcelltypesusingdefineddensityandmatrix conditions.
Methods
OptimizingandCharacterizing3DCo-andTri-Cultures
Calceinlabellingofneuron-astrocytecoculturesat multipletimepointsshowedrobustcellviabilityand increasedculturecomplexityovertime.
2weeks
7weeks
Todistinguishneuronsfromastrocytes,co-culturesweretransducedwith NeuroLightlentivirus,encodinganmRubyfluorescentproteindrivenbythe synapsinpromoter.Theseresultsdemonstratethatthehydrogelmatrixis amenabletoefficienttransductionbylentiviruses.
•The3Dneuron-astrocytemodelwasamenableto immunocytochemistrylabellingandhigh-content confocalimaging(OperaPhenix).
•βIII-tubulin(neurons)andGFAP(astrocytes)labelling revealedanintricateinteractionbetweenthetwocell types.
•Keypre-andpost-synapticproteinsweresuccessfully imaged,enablingtheevaluationofneuronalmaturity.
Conclusion
•Microgliaactivelymigratedintothehydrogelmatrix.
•Cellsremainedviableandembeddedthroughoutculture.
•WeobservedanincreaselevelofIL6andTNFαin3Dtriculture
Acknowledgments
•Establisheda3Dco-andtri-cultureCNSmodel withneurons,astrocytes,andmicrogliaamenable fordrugdiscoverystudies.
•Detectedmaturesynapticproteinexpressionin 3Dcultures,indicatingnetworkformation.
•Observedstrongneuron–astrocyteinteractions.
•Plantocompare2Dvs.3Dculturestoassess cellularandfunctionaldifferencesinmodelofCNS diseases
