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

热响应聚合物接枝培养皿表面实质细胞和非实质细胞新型共培养系统的开发

• 批准号: 13308055
• 负责人: OKANO Teruo
• 金额: $ 33.95万
• 依托单位: Tokyo Women's Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13308055/
• 关键词: poly(N-isopropylacrylamide); patterned thermoresponsive surfaces; pattern co-culture; parenchymal cells; endothelial cells; albumin production; urea synthesis; patterned domain sizes; 温度応答性表面; 細胞シートのマニピュレーション; 相転移挙動

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

本研究的重点是设计和表征新的图案双热响应表面，用于可能的图案共培养和通过温度调节恢复共培养的细胞单层。以甲基丙烯酸正丁酯（BMA）为疏水单元，采用电子束辐照聚合的方法接枝了聚n-异丙基丙烯酰胺及其疏水衍生物的热响应聚合物。在本财政年度，通过具有不同尺寸圆孔的铝掩模，将有限的电子束照射BMA聚合到pipaam接枝表面上，制备了几种类型的图像化双热响应表面。对于100μmφ的掩模，辐照电子束与单体溶液的掩模和半月板衍射，形成了尺寸为100μmφ的疏水图案，我们没有得到可重复性的双热响应图案表面。我们使用更大尺寸（0.5mmφ和1.0mmφ）的金属掩模，成功制备了图像化的双热响应表面。然后通过调节肝细胞（hc）和内皮细胞（ECs）表面亲疏水特性随温度的变化进行模式共培养。将hc在27℃下播种培养，使其仅在相对疏水区域上培养，再将ECs在37℃下培养，实现两种细胞的模式共培养。通过将培养温度降低到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出来，形成单细胞片，保持图案结构。然后，我们研究了模式大小对模式共培养系统中肝功能变化的影响，在白蛋白生产和氨代谢尿素合成方面。与同型培养相比，在恒定的hc /ECs比下，随着模式结构域大小的减小，这两种生理功能都有所增加。随着培养时间的延长，更小的模式域被观察到更多的功能增强。这些结果与先前获得的结果一致，其中HC单层被用pipaam接枝培养皿获得的EC单层覆盖。因此，控制hcc和ec之间的距离对共培养细胞功能（包括细胞活力）具有优先影响。少

项目成果

M Ebara, M Yamato, M Hirose, T Aoyagi, A Kikuchi, K Sakai, T Okano: "Copolymerization of 2-carbozyisoppopylacrylamide with N-isopropylacrylamide accerelates cell detachment from grafted surfaces by reducing temperature"Biomacromolecules. 4. 334-349 (2003)

M Ebara、M Yamato、M Hirose、T Aoyagi、A Kikuchi、K Sakai、T Okano：“2-羧基异丙基丙烯酰胺与 N-异丙基丙烯酰胺的共聚通过降低温度加速细胞从移植表面脱离”生物大分子。

Y Tsuda, A Kikuchi, M Yamato, Y Sakurai, M Umezu, T Okano: "Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces"Journal of Biomedical Materials Research. 69A. 70-78 (2004)

Y Tsuda、A Kikuchi、M Yamato、Y Sakurai、M Umezu、T Okano：“使用温度和热响应性共聚物接枝培养表面控制细胞粘附和脱离”生物医学材料研究杂志。

OH Kwon, A Kikuchi, M Yamato, T Okano: "Accelerated cell sheet recovery by co-grafting of PEG with PIPAAm onto porous cell culture membranes"Biomaterials. 24(7). 1223-1232 (2003)

OH Kwon、A Kikuchi、M Yamato、T Okano：“通过将 PEG 与 PIPAAm 共接枝到多孔细胞培养膜上加速细胞片恢复”生物材料。

M Harimoto, M Yamato, A Kikuchi, T Okano: "Cell sheet engineering : Intelligent polymer patterned surfaces for tissue engineered liver"Macromolecular Symposia. 195. 231-235 (2003)

M Harimoto、M Yamato、A Kikuchi、T Okano：“细胞片工程：用于组织工程肝脏的智能聚合物图案表面”高分子研讨会。

Y.Tsuda, A.Kikuchi, M.Yamato, Y.Sakurai, M.Umezu, T.Okano: "Control of cell adhesion and detachment using temperature and thermoresponsive copolymer grafted culture surfaces"Journal of Biomedical Materials Research. 69A. 70-78 (2004)

Y.Tsuda、A.Kikuchi、M.Yamato、Y.Sakurai、M.Umezu、T.Okano：“使用温度和热响应性共聚物接枝培养表面控制细胞粘附和脱离”生物医学材料研究杂志。