Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing.

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.

Terahertz Spectroscopy Sheds Light on Real-Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment.

Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+ , ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization.