During reconstitution, a vial of cells is removed from storage and placed in an incubator or water bath set at 37 degrees F for one to two minutes. Care is taken to not incubate the vial for too long to prevent cell damage. Cells are then centrifuged to remove the storage media, re-suspended in pre-warmed culture media and transferred to a growth vessel, where is it incubated at optimum temperature, usually 37 degrees F.
Sub-culturing is done to increase the amount of cells while keeping them in the log phase of growth. Cells grow exponentially, depending on the amount of space available. Once space is maximized, cells reach a stage referred to as full confluency, where cells no longer proliferate and start dying. Sub-culturing varies slightly according to cell type and growth rate.
Adherent cells are attached to the bottom of the growth vessel, and are removed after decanting spent media, either mechanically by scraping, or with enzymes such as trypsin, which detaches cells from the surface. In contrast, continuous cell lines circulate within the culture media and are separated during centrifugation. Cells are centrifuged to remove trypsin and media. Cells are then collated in a pellet and re-suspended in culture media. A tiny portion of the re-suspension is stained with Trypan Blue dye, counted, and assessed for viability, either manually using a microscope and hemocytometer, or by an automated cell counter. According to growth rate and lab requirements, a specific cell concentration is placed in a growth vessel containing the appropriate volume of fresh culture media.
Tissue culture cells are stored in either liquid nitrogen or in a freezer at below -130 degrees Celsius. Protective equipment is required when placing or removing cells from liquid nitrogen. A known concentration of viable cells is suspended in storage media such as Dimethyl sulphoxide (DMSO) and glycerol, transferred to an appropriate storage flask and placed in liquid nitrogen or a freezer. The cooling rate is controlled either by electronic freezing units or alcohol-filled containers to ensure temperature decline is not too drastic to cause any cell damage.