Development of novel co-culture system of parenchymal cells and nonparenchymal cells on thermoresponsive polymer-grafted dish surfaces

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This research focused on the design and characterization of novel patterned dual thermoresponsive surfaces for possible patterned co-culture and recovery of co-cultured cell monolayers by temperature modulation. Thermoresponsive polymers of poly(N-isopropylacrylamide) and its hydrophobic derivatives containing n-butyl methacrylate (BMA) as hydrophobic units were pattern grafted using electron beam irradiated polymerization. In this fiscal year, several types of the patterned dual thermoresponsive surfaces were prepared by applying limited electron beam irradiated polymerization of BMA onto PIPAAm-grafted surfaces through aluminum masks having different sizes of circular holes. In case of the 100μmφ-patterned masks, irradiated electron beam was diffracted with the mask and meniscus of the monomer solution, resulting in the formation of hydrophobic patterns with >100μmφ in size, and we did not obtain patterned dual thermoresponsive surfaces reproducibly. Using metal masks with larger pat … More tern sizes (0.5mmφ and 1.0mmφ), we successfully prepared patterned, dual thermoresponsive surfaces. Patterned co-culture of hepatocytes (HCs) and endothelial cells (ECs) were then performed by modulation of surface hydrophilic/hydrophobic property alteration with temperature. HCs were seeded and cultured at 27℃ to culture only onto relatively hydrophobic domains, followed by ECs culture at 37℃ to realize patterned co-culture of these two cell types. By lowering culture temperature to 20℃ at which entire surfaces become hydrophilic, co-cultured cells spontaneously detach from the culture surfaces in a single cell sheet maintaining patterned structure. We then investigate the effect of the pattern sizes on hepatic function changes in patterned co-culture systems, in terms of the albumin production and urea syntheses by ammonium metabolism. Both physiological functions increased with decreasing patterned domain sizes at constant HCs/ECs ratio than homotypic culture of HCs. More functional augmentation was observed with smaller patterned domains with longer culture periods. These results are consistent with the previously obtained results, in which HC monolayers were covered with EC monolayers obtained by using PIPAAm-grafted dishes. Thus, the control of the distance between HCs and ECs has preferential influences to co-cultured cell functionalities including cellular viabilities. Less