Canavan Heather E, Cheng Xuanhong, Graham Daniel J, Ratner Buddy D, Castner David G
National ESCA and Surface Analysis Center for Biomedical Problems, University of Washington Engineered Biomaterials, and Departments of Bioengineering and Chemical Engineering, Box 351750, University of Washington, Seattle, Washington 98195-1750, USA.
Langmuir. 2005 Mar 1;21(5):1949-55. doi: 10.1021/la048546c.
The temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) directly affects the attachment and detachment of cells cultured on these surfaces. At culture temperatures, cells behave similarly to those on tissue culture polystyrene (TCPS), while at room temperature, cells cultured on pNIPAM spontaneously detach as a confluent sheet. In comparison, cells grown on TCPS remain attached indefinitely after the temperature drop, requiring enzymatic or mechanical removal. In this work, we present an examination of the response of bovine aortic endothelial cells (BAECs) and extracellular matrix (ECM) proteins to plasma polymerized NIPAM (ppNIPAM) surfaces using X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and immunostaining. Immunoassay results reveal that, although fibronectin, laminin, and collagen closely associate with the cell sheet, some collagen may be associated with the surface, as well. Our XPS results indicate that ppNIPAM surfaces after cell liftoff differ from their blank counterparts, the primary distinction being the presence of amide and alcohol species on ppNIPAM surfaces used for cell culture, possibly owing to the presence of a proteinaceous film. Finally, a comparison between ppNIPAM-treated surfaces used for cell culture versus control surfaces by principal component analysis of the ToF-SIMS data confirms that the surfaces differ; the presence of molecular ion fragments from amino acids (e.g., alanine, glycine, and proline) is the chief reason for this difference. Therefore, from our surface characterization of ppNIPAM-coated TCPS after cell liftoff, we conclude that although low-temperature liftoff of the BAEC monolayer is accompanied by the majority of the components of the ECM, some of the ECM proteins still remain at the surface.
聚（N-异丙基丙烯酰胺）（pNIPAM）的温度响应行为直接影响在这些表面上培养的细胞的附着和脱离。在培养温度下，细胞的行为与在组织培养聚苯乙烯（TCPS）上的细胞相似，而在室温下，在pNIPAM上培养的细胞会作为汇合片自发脱离。相比之下，在TCPS上生长的细胞在温度下降后会无限期附着，需要酶解或机械去除。在这项工作中，我们使用X射线光电子能谱（XPS）、飞行时间二次离子质谱（ToF-SIMS）和免疫染色，对牛主动脉内皮细胞（BAECs）和细胞外基质（ECM）蛋白对等离子体聚合NIPAM（ppNIPAM）表面的反应进行了研究。免疫测定结果表明，尽管纤连蛋白、层粘连蛋白和胶原蛋白与细胞片紧密结合，但一些胶原蛋白也可能与表面结合。我们的XPS结果表明，细胞脱离后的ppNIPAM表面与其空白对应物不同，主要区别在于用于细胞培养的ppNIPAM表面存在酰胺和醇类物质，这可能是由于存在蛋白质膜。最后，通过对ToF-SIMS数据的主成分分析，比较用于细胞培养的ppNIPAM处理表面与对照表面，证实了表面存在差异；氨基酸（如丙氨酸、甘氨酸和脯氨酸）分子离子碎片的存在是造成这种差异的主要原因。因此，从我们对细胞脱离后ppNIPAM包被的TCPS的表面表征中，我们得出结论，尽管BAEC单层的低温脱离伴随着ECM的大部分成分，但一些ECM蛋白仍留在表面。
