Common Troubleshooting Methods in Cell Culture Techniques

Mittal, Khushi R.; Mani, Shalini

doi:10.1007/978-3-031-19485-6_21

Cited by 1 publication

(2 citation statements)

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“…Non-viable cells appeared in culture due to improper storage, uncorrected thawing or thawing media resulting in damage of cells so frozen cells should be appropriately thawing and diluted slightly before plating in pre warmed growth medium. Also, excessive dilution, improper handling of cells and storage of DEMSO or glycerol in light of liquid freezing resulting in conversion of glycerol to acrolein which is toxic to cells causing deaths of cells [29]. After cell removal from cryopreservation, DMSO should be decanted from media after gentle centrifugation and thawing.…”

Section: Discussionmentioning

confidence: 99%

“…After cell removal from cryopreservation, DMSO should be decanted from media after gentle centrifugation and thawing. Also, cell subculture in specific media is required to adapt the environment as they may lose ability to attach [29]. Numerous factors attributed to slow cell growing such as presence of unsuitable growth medium, multiple passages and confluency past growing and avoiding such matters the recommended pre-warmed growing medium should be used, fewer passages of healthy cells are suggested and passaging of mammalian cells should be applied before confluence during the logphase.…”

Section: Discussionmentioning

confidence: 99%

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Efficiency of MTT and Trypan Blue Assays for Detection of Viability and Recovery of Different Frozen Cell Lines

azzoz,

zaghloul,

sayed

et al. 2024

Egyptian Journal of Veterinary Sciences

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HE quantity of healthy cells in a sample is known as cell viability. For all types of cell culture, measuring cell viability is crucial to ensuring a supply of high-quality cell lines through setup, preparation and storage. Although the cryopreservation technique is used in both scientific and clinical research, there are still certain restrictions. At low temperatures, such as −196 °C (i.e., in liquid nitrogen) cells metabolize is almost nothing, which has unavoidable side effects, such as a genetic drift toward biological variations of cell-associated changes in lipids and proteins that could impair cellular activity and structure. Also, cells can be harmed by cryoprotective agents (CPAs), especially when they were administered in high concentrations in conditions where they would typically be used. So, it is important to use a reliable technique to follow up changes affecting cell viability after cryopreservation which were compared with control freshly subcultured cells. In the present study, the MTT assay was used to correlate cell behavior according to the number of healthy and viable cells. Different kinds of cell cultures were cryopreserved with dimethyl sulfoxide (DMSO) at 10% for variable years in a liquid nitrogen tank (-196 °C). Then, different samples were tested for viability using the MTT assay and counted by the trypan blue assay for comparing both results accuracy and confirmed by visual examination in sub-cultured media twenty four hours post-thawing. The MTT assay was easy, fast and showed more accurate results than the trypan blue assay. The visual examination twenty four hours post-subculture indicated that MTT assay can differentiate between healthy and apparently viable cells which have lost their function whereas the trypan blue assay failed to detect healthy cells in some samples.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%