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What is the optimal timing for cryopreservation and thawing of cells?
During in vitro culture, cells typically progress through three phases: the lag phase, the exponential growth phase, and the plateau phase. The lag phase generally occurs immediately after passaging, when cells begin to adhere to the substrate and extend their cytoskeleton; metabolic activity is primarily directed toward the expression of adhesion proteins and cytoskeletal proteins, and cell numbers remain virtually unchanged during this period.
Subsequently, the cells enter the exponential growth phase, characterized by vigorous transcription and translation of nucleic acids, frequent DNA unwinding and base pairing, rapid intracellular transport of macromolecules, and a rapid increase in cell number. If cryopreservation is performed during this phase, the cells are effectively frozen at a specific metabolic state, inevitably causing mechanical damage to unwound DNA as well as to RNA and proteins involved in transcription and translation, thereby increasing the risk of errors at individual steps in these processes.
As the cell population expands, cell confluence in the culture vessel reaches over 90%. Most cells, due to contact inhibition, cease rapid metabolism and proliferation, and intracellular activities become more quiescent.
Therefore, we recommend cryopreserving cells as soon as they enter the plateau phase, which allows you to obtain an adequate cell yield while minimizing mechanical damage.
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