Department of Molecular and Cellular Bioanalytics, Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany.
Institute of Neuroanatomy & Developmental Biology INDB, Eberhard Karls University Tuebingen, Tuebingen, Germany.
Biotechnol Bioeng. 2022 Jul;119(7):1728-1739. doi: 10.1002/bit.28095. Epub 2022 Apr 11.
Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.
前沿的生物医学应用需要越来越复杂和精细的细胞系统，例如各种类型的原代或干细胞。然而，它们的培养方法与 30 年前相比几乎没有什么变化。这尤其适用于使用不分青红皂白的蛋白酶进行非特异性细胞分离。一种更为温和的替代方法是通过基于温敏聚合物的涂层来改变细胞培养基质的粘附特性。这种聚合物在 37°C 时介导细胞黏附，但在降至 32°C 等细胞相容的温度时会变得排斥。虽然这种方法的高功能已经得到了很好的证明，但它也必须易于应用且具有可重复性。在这里，我们强调了通过喷涂涂覆有温敏微凝胶的标准细胞培养材料在常规培养和其他方面的潜力。在这些表面上，我们成功地培养和分离了各种细胞类型，包括诱导多能干细胞和无血清培养的细胞。此外，我们评估了与粘附促进蛋白的兼容性，这些蛋白对于例如干细胞或神经元细胞是必不可少的。最后，我们证明了微凝胶表面不会损害增殖并显示出长期稳定性。我们得出结论，对于细胞分离，温敏细胞培养基质可以通过使用与许多工业处理线兼容的相对简单的方案，完全替代蛋白酶（如胰蛋白酶）。
