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ABSTRACT
Tumor spheroids arewidelyused tomodel the tumormicroenvironment andevaluate immune-mediatedcytotoxicity, but
conventionalmethodssuchasultralow-attachmentandhanging-dropplatestypicallyrequire48–72htogeneratestablespheroids,
limitingscalabilityandrapidtesting.Here,wereportasimplemethodforgeneratinguniformtumorspheroidswithin7–8h
usingcontinuousrockinginstandardmultiwellplates.Computationalfluiddynamicssimulationsshowedthatrockingproduced
mildoscillatoryhydrodynamicconditions,withaverageshearstressrangingfrom0.4to0.8dyn/cm2andX-directionshearstress
remainingnear±0.2dyn/cm2,promotingcontrolledcellaggregationwhilemaintainingspheroidintegrity.Usingthisapproach,
Hep3Bcells reproducibly formedspheroids80–150µmindiameteracrossmultipleseedingdensities, yielding thousandsof
spheroidsperwell.Thespheroidswereembeddedinextracellularmatrixandtransferredtoa36PillarPlateforscalable3Dtumor
immunecocultureassays.Todemonstrateutility,Hep3BspheroidswerecoculturedwithNK-92cellsinthepresenceorabsenceof
theimmunomodulatorberberine.BerberinetreatmentenhancedNK-92localizationandspheroiddisruption,resultinginreduced
tumorviability.Thisapproachreducesspheroidpreparationtimefromdaystohoursandenablessame-dayintegrationinto3D
assays,providingapracticalplatformforstudyingtumor-immuneinteractionsandscreeningimmunomodulatorytherapeutics.