Cooperstein Marta A, Nguyen Phuong A H, Canavan Heather E
Department of Chemical and Nuclear Engineering, Center for Biomedical Engineering, 1 University of New Mexico, MSC01 1141, Albuquerque, New Mexico 87131-0001.
Biointerphases. 2017 Apr 12;12(2):02C401. doi: 10.1116/1.4979920.
Although there is a great deal of research focused on cell sheet engineering from polymers such as poly(N-isopropyl acrylamide) (pNIPAM), the biocompatibility of pNIPAM surfaces and the nature of cellular detachment from this polymer is still unclear. The most extensive study of the mechanism of detachment proposed a two-step process, with a first (passive) phase involving hydration of pNIPAM chains, and the second (active) phase involving cellular metabolism. However, a number of studies performed successful cell sheet detachment from pNIPAM-grafted surfaces at low temperatures which calls this hypothesis into question. Furthermore, although it has been demonstrated that low-temperature cell sheet detachment using pNIPAM-grafted surfaces is less destructive than other methods of detachment, it has not been investigated if cell sheet detachment removes a portion of pNIPAM from the surfaces as well. It is essential to know if any fragments of the polymer are removed along with the cells, as small polymer fragments could have cytotoxic effects on the cells. This is especially important if these cells are used for the generation of tissues used for transplantation. In this work, the mechanism of cell detachment from pNIPAM coated surfaces is investigated by testing how temperature and presence of an adenosine triphosephase inhibitor affect cellular detachment. Surface initiated atom transfer polymerization (ATRP) was utilized to synthesize thermoresponsive atrpNIPAM surfaces. pNIPAM surfaces were labeled to assess whether cell sheet detachment from pNIPAM is accompanied by the removal of pNIPAM from the substrate itself. Using a semipermeable superstrate, cell sheets were transferred to a secondary culture dish to assess whether cell detachment resulted in any pNIPAM removal. In addition, the function of the transplanted bovine aortic endothelial cells was assessed by determining whether they would proliferate and grow on a new secondary substrate.
尽管有大量研究聚焦于基于聚（N-异丙基丙烯酰胺）（pNIPAM）等聚合物的细胞片工程，但pNIPAM表面的生物相容性以及细胞从该聚合物上脱离的本质仍不清楚。关于脱离机制的最广泛研究提出了一个两步过程，第一步（被动）阶段涉及pNIPAM链的水合作用，第二步（主动）阶段涉及细胞代谢。然而，一些研究在低温下成功地从pNIPAM接枝表面分离出细胞片，这对该假设提出了质疑。此外，尽管已经证明使用pNIPAM接枝表面进行低温细胞片分离比其他分离方法的破坏性更小，但尚未研究细胞片分离是否也会从表面去除一部分pNIPAM。了解聚合物的任何片段是否与细胞一起被去除至关重要，因为小聚合物片段可能对细胞具有细胞毒性作用。如果这些细胞用于生成移植用组织，这一点尤为重要。在这项工作中，通过测试温度和三磷酸腺苷抑制剂的存在如何影响细胞脱离，研究了细胞从pNIPAM涂层表面脱离的机制。利用表面引发原子转移聚合（ATRP）合成了热响应性的atrpNIPAM表面。对pNIPAM表面进行标记，以评估从pNIPAM分离细胞片是否伴随着从底物本身去除pNIPAM。使用半透性覆盖物，将细胞片转移到二次培养皿中，以评估细胞脱离是否导致任何pNIPAM去除。此外，通过确定移植的牛主动脉内皮细胞是否会在新的二次底物上增殖和生长来评估其功能。
