The use of patterned dual thermoresponsive surfaces for the collective recovery as co-cultured cell sheets

Heterotypic cell interactions are critical to achieve and maintain specific functions in many tissues and organs. We have focused on patterned structure surfaces to enable co-culture of heterotypic cells and recovery of patterned co-cultured cell sheets for applications in tissue engineering. Thermoresponsive polymers exhibiting different transition temperatures in water comprise both poly(N-isopropylacrylamide) (PIPAAm) and n-butyl methacrylate (BMA) co-grafted as side chains to PIPAAm main chains. These copolymers were surface-grafted in patterns to obtain patterned dual thermoresponsive cell culture surfaces using electron beam polymerisation method and porous metal masks. On patterned surfaces, site-selective adhesion on and growth of rat primary hepatocytes (HCs) and bovine carotid endothelial cells (ECs) allowed patterned co-culture, exploiting hydrophobic/hydrophilic surface chemistry regulated by culture temperature as the sole variable. At 27 degrees C, seeded HCs adhered exclusively onto hydrophobic, dehydrated P(IPAAm-BMA) co-grafted domains (1-mm laser dot), but not onto neighbouring hydrated PIPAAm domains. Sequentially seeded ECs then adhered exclusively to hydrophobised PIPAAm domains upon increasing culture temperature to 37 degrees C, achieving patterned co-cultures. Reducing culture temperature to 20 degrees C promoted hydration of both polymer-grafted domains, permitting release of the co-cultured, patterned cell monolayers as continuous cell sheets with heterotypic cell interactions. Recovered co-cultured cell sheets can be manipulated, moved and sandwiched with other structures, providing new useful constructs both for basic cell biology research and preparation of tissue-mimicking multi-layer materials through overlaying co-cultured cell sheets.