1. Cell density
Mammalian cells:adherent cultured cells into the logarithmic growth phase, did not meet thestate should be submitted when the passage. Normal cells stop growing when theyreach the confluence (contact inhibition) and take longer to recover afterre-inoculation. Transformed cells continue to increase even when confluent butdegrade after about two doublings. Similarly, cells cultured in suspensionenter the logarithmic growth phase and should also be passaged when they havenot reached the state of confluence. When confluence is reached, cells insuspension culture will agglomerate, and the media will become cloudy when theflask is rotated.
Insect cells: insectcells into the logarithmic growth phase, did not meet the confluence should bepassed on. Immobilized insect cells can also be passaged when they reach thejunction. Although the cells are more likely to dissociate from the incubatorat this time, the number of doublings of the cells will decrease if the insectcells are repeatedly passaged at a density exceeding the confluence, Vitalitywill be reduced, the adherent ability will decline. On the other hand, thepassage of the adherent cultured insect cells before confluence requiresgreater mechanical forces to detach the cells from the monolayer. Repeatedlybefore reaching the confluence state of reading will lead to reduced celldoubling frequency, decreased vitality, poor health.
Mammaliancells: Reduced pH in growth media, usually indicative of lactate accumulation,a byproduct of cellular metabolism. Lactic acid is cytotoxic, and decreasing pHis also a negative factor for cell growth. The rate of pH change usuallydepends on the cell concentration in the culture system. The higher the cellconcentration, the faster the medium is depleted. If a rapid decrease in pH isfound (> 0.1-0.2 pH units) and the cell concentration is increased, thecells should be passaged.
Insect cells:Growth media for insect cell culture is usually more acidic than mammalian cellculture medium. For example, the pH of TNM-FA and Grace medium for Sf9 cellculture is 6.2. Unlike mammalian cell culture, the pH of the medium in insectcells increases gradually but does not exceed 6.4. However, like mammaliancells, the pH of growth media begins to decrease as insect cells reach higherdensities.
Strictlyaccording to the scheduled time for cell passage to ensure that the biologicalbehaviour of cells stable, secure to monitor their health status. Graduallyadjust cell seeding density from a given seeding density until a steady growthrate, and yield is achieved for the cell. Cells that deviate from the definedgrowth pattern usually represent poor cell health (e.g., spoilage,contamination) or dysfunction of a component of the culture system (e.g.,failure to reach optimal temperature, media too old). We strongly recommendthat you keep the detailed cell culture records, record feed and passage times,media used, dissociation methods, seed rating, morphological observations, inoculumconcentrations, yields, and antibiotic usage.
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