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

本研究的重点是设计和表征的新型图案化的双温敏表面可能的图案化的共培养和共培养细胞单层的温度调制的恢复。采用电子束辐照聚合方法对聚N-异丙基丙烯酰胺及其疏水衍生物进行了图案化接枝。在本财政年度，通过具有不同尺寸圆孔的铝掩模，将BMA的有限电子束辐照聚合应用于PIPAAm接枝表面，制备了几种类型的图案化双重温敏表面。在100μmφ图案化掩模的情况下，照射的电子束被掩模和单体溶液的弯月面衍射，导致形成尺寸>100μmφ的疏水图案，并且我们没有可再现地获得图案化的双温敏表面。使用金属面罩， ...更多信息 在0.5mmφ和1.0mmφ两种尺寸下，我们成功地制备了图案化的双温敏表面。然后通过调节表面亲水/疏水性质随温度的变化进行肝细胞（HC）和内皮细胞（EC）的图案化共培养。在27℃下接种并培养毛细胞，使其仅在相对疏水的结构域上培养，然后在37℃下培养内皮细胞，以实现这两种细胞类型的模式化共培养。通过将培养温度降低至20℃，使整个表面变得亲水，共培养的细胞自发地从培养表面分离，形成单个细胞片，保持图案化结构。然后，我们调查的图案尺寸对肝功能的影响，在图案化的共培养系统，在白蛋白的生产和尿素合成的铵代谢。在恒定的HC/EC比下，这两种生理功能都随着图案化结构域尺寸的减小而增加。观察到更多的功能增强与较小的图案化域与较长的文化时期。这些结果与先前获得的结果一致，其中HC单层被通过使用PIPAAm接枝的皿获得的EC单层覆盖。因此，HC和EC之间的距离的控制对共培养的细胞功能包括细胞活力具有优先影响。少

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