Canavan Heather E, Graham Daniel J, Cheng Xuanhong, 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, University of Washington, Seattle, Washington 98195, USA.
Langmuir. 2007 Jan 2;23(1):50-6. doi: 10.1021/la062330o.
In the past decade, the temperature-responsive behavior of poly(N-isopropyl acrylamide) (pNIPAM) has come to be recognized as a convenient method for the nondestructive harvest of confluent cell layers. Recently, we have utilized this nondestructive cell harvest method as a means to ascertain the nature of the extracellular matrix (ECM) secreted from cells. In this work, we compare the ECM obtained after cell liftoff to individual ECM proteins adsorbed directly onto RF-plasma-deposited pNIPAM (ppNIPAM). Using X-ray photoelectron spectroscopy, we find that the composition of ppNIPAM post-cell liftoff surfaces is consistent with those of the ppNIPAM post-protein adsorption surface, both of which differ from control surfaces. Using principal component analysis of positive-ion time-of-flight secondary ion mass spectrometry (ToF-SIMS) data, we show that the major ECM proteins examined can effectively be identified from their amino acid compositions. By comparing the positive-ion ToF-SIMS data from each of the ppNIPAM post-protein adsorption surfaces to that of ppNIPAM post-cell liftoff, we find that ppNIPAM post-cell liftoff surfaces are distinctly separate from fibronectin (FN). This result is consistent with our previous observation using immunoassay that FN is clearly associated with the cell sheet after low-temperature liftoff from ppNIPAM.
在过去十年中，聚（N-异丙基丙烯酰胺）（pNIPAM）的温度响应行为已被公认为是一种无损收获汇合细胞层的便捷方法。最近，我们利用这种无损细胞收获方法来确定细胞分泌的细胞外基质（ECM）的性质。在这项工作中，我们将细胞脱离后获得的ECM与直接吸附在射频等离子体沉积的pNIPAM（ppNIPAM）上的单个ECM蛋白进行了比较。使用X射线光电子能谱，我们发现细胞脱离后ppNIPAM表面的组成与蛋白吸附后ppNIPAM表面的组成一致，这两者均与对照表面不同。通过对正离子飞行时间二次离子质谱（ToF-SIMS）数据进行主成分分析，我们表明所检测的主要ECM蛋白可以根据其氨基酸组成有效地识别出来。通过将每个蛋白吸附后ppNIPAM表面的正离子ToF-SIMS数据与细胞脱离后ppNIPAM的正离子ToF-SIMS数据进行比较，我们发现细胞脱离后ppNIPAM表面与纤连蛋白（FN）明显不同。这一结果与我们之前使用免疫测定法观察到的结果一致，即从ppNIPAM上低温脱离后，FN与细胞片明显相关。
