LOB, Ecole Polytechnique, CNRS, INSERM, Institut Polytechnique de Paris, Palaiseau, France.
Laboratoire Softmat, Université de Toulouse, CNRS UMR 5623, Université Toulouse III - Paul Sabatier, Toulouse, France.
Biophys J. 2024 Aug 20;123(16):2476-2483. doi: 10.1016/j.bpj.2024.06.011. Epub 2024 Jun 13.
Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time.
胰蛋白酶是细胞培养中常用的一种酶，通过切割负责细胞黏附的蛋白质来收获细胞。然而，胰蛋白酶也会对细胞产生不良影响，例如改变膜蛋白和细胞骨架，改变细胞质和细胞体积的组成，甚至在使用不当时导致细胞死亡。我们使用太赫兹域中的衰减全反射、共聚焦显微镜和碘化丙啶测试，实时定量分析了胰蛋白酶蛋白水解对 Madin-Darby 犬肾上皮细胞细胞质内容物的诱导变化。我们观察到从胰蛋白酶作用的最初几秒钟开始，细胞质就发生了变化，这是由于细胞膜的力学重新平衡导致细胞体积的变化。我们发现细胞质的改变与小分子溶质（电解质和代谢物）的转移有关。我们还发现，太赫兹传感监测到的副作用与细胞高度之间存在非常好的非线性相关性，而与细胞高度对胰蛋白酶浓度和暴露时间的依赖性无关。
