Three-dimensional organoid culture using the CellRaft microwell technology

Project SummaryOrganoids are minitissue structures that are revolutionizing in vitro studies due to the fact that they arederived from normal or diseased tissues from human donorsinduced pluripotent stem cellsiPSCsand nearlyeverymodelorganismTheuseoforganoidsindiseasemodelinghasbecomeapowerfulmethodtoreplicatepathophysiologyusing straightforwardcellcultureconditionsVirtuallyeverytissuetypenowhasaninvitroorganoidcorrelateAsthe nameimpliesorganoidsarerepresentationsoftissuelayerstypicallyepitheliumthat have a specific function in an organismThey are typically sphericaland are stem cell drivengiven thempotential to produce all the differentiated cell types found in any given tissueThis has led to inaccurate resultsfailureinpredictingdrugefficacyandlossofmillionsofdollarsandmanhoursfollowinglinesofresearchbased on artifactChallenges associated with rapidly establishing organoid cultures are largely associated withinitiatingaclonalcolonyfroma singlecelltrackingcolony growthandinducingdifferentiationprocessesInadditionsystemssupportingorganoidculturemustallowtimecoursebasedanalysisofphysiologicalphenotypesCellMicrosystemshasdevelopedtheCellRaftTechnologyamicrowellarraybasedplatformwheresinglecellscanbeseededinsmallculturechambersgrownintoclonalcoloniesandtrackedovertimeusing virtually any imaging modalityThese capabilities have shown initial promise in addressing many of thechallenges associated with organoid cultureScott MagnessPhD of the University of North Carolina at ChapelHillhaspublishedmethodsusingCellRaftArraysforestablishinggrowingandanalyzingorganoidsderivedfromvariousentericstemcellsTheresearchproposedherebuildsonbothinnovativearraydesignsandmaterials by Cell Microsystemsas well as the workflows established by DrMagnessUnder this collaborativeprojectwe will develop a novel CellRaft ArraytheDCytoSort Arraywith larger microwells than are currentlymanufacturedmicrons andmillimeter squarewith greater depthmicrons instead of the standardmicronsto facilitate the growthdifferentiation and analysis of various types of organoidThese arrays will bevalidatedforusewithCellMicrosystemsAIRSystemanautomatedplatformfortheimagingsortingandisolationofcellsorcoloniesfromtheCellRaftArrayThisinstrumentwillallownotonlytheimagingoforganoidsfortemporalanalysisbutalsoisolationoforganoidsfordownstreammolecularanalysisvianextgenerationsequencingorothermolecularanalysismodalityDrMagnessteamwilltranslatetheircurrentorganoidculturemethodstothenewlargerDCellRaftArraySuccessfulrecapitulationoftheirpreviouslypublisheddatausingthenewarrayandtheAIRSystemwillserveasaninitialvalidationoftheproduct sperformanceasaplatformforbroadorganoidculturemethodsPendingcompletionoftheSpecificAimsproposed herea Phase II project will be proposed that will expand the range of organoid types with the goal ofexpanding the utility of the AIR System software for organoidspecific analysisProject NarrativeThreedimensionalcellcultureallowsinvestigatorstousestandardinvitromethodstomorecloselyreplicateinvivophysiologyGiventhebroadrangeoforganoidculturemethodsthathaveemergedthereexistsanunmetneedforaflexibleplatformforautomatingthegrowthimagingandisolationofindividualorganoidstopairdetailed phenotypicobservationswithdownstreammolecular methodsHereweproposeanovel microwell array based on the Cell Microsystemsproprietary CellRaft TechnologytheDCytoSort Arrayto accelerate these powerful studies