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The Thawing and Recovery of Cells

Cells can be stored in liquid N2, or in the gas phase over the liquid N2 for long periods of time. However, valuable cells should be thawed and brought back into culture once a year to ascertain viability and function. Thawing is as important as the freezing process to maintain cell viability, and thawing should be carried out according to a rigorous protocol.

Frozen vials with cells are placed on ice directly from the liquid N2 storage tank. The vials should be immersed in a water bath at 37 °C and thawed leaving a small lump of ice in the vial before transfer of the vial to melting ice again. When fully thawed, transfer the contents of the vial into a tube and add slowly cold medium to dilute the DMSO in the freezing medium. It is very important that the cell suspension is kept cold during this procedure.

Spin the cells carefully down in a table top centrifuge, remove the DMSO containing medium and add a small volume of complete medium to resuspend the cells. Take a small sample to monitor cell viability by trypan blue exclusion and light microscopy, or similar viability test (see Sect. 9.5). If cell viability is below 70 % it may turn out difficult to obtain proper cell growth. Reduced cell viability is most likely due to improper handling of cells either before freezing, during freezing or thawing of the cells.

Dilute with complete medium to the desired cell concentration, and add the cell suspension to a suitable culture vessel. Place in incubator. Monitor cell morphology and cell growth during the following days.

Cell Viability Testing

Assessment of cell viability is fundamental in all cell culture work. Cell viability will provide information about culture conditions, in general, and may also represent the final read-out system for a cytotoxicity experiment. A large number of viability assays are reported in the literature, ranging from the simple trypan blue exclusion test to complex analysis of individual cells with Raman spectroscopy.

Cell viability assays will often overlap with cell proliferation assays. They are divided into assays that monitors viability at the single cell level and those that analyses viability at the population level. The simplest and cheapest viability test exploits the vital stain trypan blue. Trypan blue is excluded from cells and tissues with intact membranes, and the staining of dead cells is easy to detect in a standard light microscope.

Prepare a 0.8 mM trypan blue solution in phosphate-buffered saline (PBS). Mix cells and trypan blue solution in a 1:1 ratio. Inspect and count dead and alive cells under a light microscope in a haemocytometer. Dead cells will stain blue due to trypan blue uptake, while live cells appear transparent. Cells should not be exposed to trypan blue solution for more than 20 min. Prolonged incubation will increase cell death and reduce viability.

A variety of fluorescent dyes can also be used to monitor cell viability, typical in the combination with flow cytometry. Propidium iodide and 7-actinomycin D are commonly used for this purpose.

Assessing cell viability at the population level has become very popular. These assays are typically carried out by using different tetrazolium compounds. Among the most popular are MTT ((3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), MTS, XTT and WST-1. Among these MTT is positively charged and readily penetrates viable cells, while the others are negatively charged and are excluded from viable cells. The MTT assay is perhaps the most popular to assess cell viability and proliferation in a population of cells. It was the first assay to be developed for high trough put screening in a 96-well format. Viable cells convert MTT into a purple colored formazan product. When cells die, they lose ability to convert MTT into formazan. The mechanism behind this process is not well understood, although many publications suggest that the MTT assay reflects changes in mitochondrial activity. Commercial kits containing MTT and a solubilization reagent can be obtained from several vendors like Sigma Aldrich, Promega and Millipore, among others.

 
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