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Growing cells in vitro requires that optimal conditions are maintained to ensure peak cell proliferation. Though they vary for each cell type, basic culture conditions include controlled temperature, an appropriate growth medium, growth factors, gases such as carbon dioxide and oxygen, and a suitable vessel. Cells used in culture systems can be broadly classified as adherent cells and nonadherent cells. Since adherent cells are anchorage dependent, they require attachment substrates such as tissue culture surfaces or microcarriers. However, nonadherent cells grow free-floating in suitable media for suspension cell culture.

Characteristics of adherent and suspension cell cultures

Most cells from vertebrate sources require a suitable substrate for cell adhesion and spreading. However, it’s sometimes possible to adapt adherent cells to grow in suspension. Many cell lines like insect cell lines support suspension culture.

Dissociation reagents Suspension cell culture
Appropriate for most cell types and primary cultures Used for nonadherent cell lines and cells adapted to suspension culture
Easily viewed under an inverted microscope Easy to passage
Cells require dissociation mechanically or through a dissociation reagent such as porcine trypsin or ADCF recombinant trypsin Doesn’t require cell dissociation
Surface area limits growth and product yields Cells are surrounded by media, which supports higher cell density and product yields
Common applications include cytology, research, and biologic production when nonadherent cells are not available Grown in bioreactors for biologic production and in spinner flasks for research

Cell dissociation

To grow adherent cells, such as epithelial cells, outside their natural environment, they’re seeded onto tissue culture-treated surfaces or microcarriers. These cells then attach to the surface and each other as they grow and proliferate. Before adherent cells are ready for subculturing or most downstream analyses, they’re released from the growing surface through cell dissociation buffers and reagents or other means. Using cell dissociation reagents, you can generate single-cell suspensions for cell counting, reseeding subcultures, and cellular assays.

Types of dissociation

Here are ways to release adherent cells using dissociation reagents and other means:

  • Mechanical dissociation: This process uses physical methods to dislodge cells and is the least gentle option. A cell scraper is often used in research.
  • Enzymatic dissociation: Enzymatic dissociation uses specific protein and buffered dissociation reagents – proteases or other enzymes such as collagenase – to disrupt attachments between the attachment surface and cells.
  • Chemical dissociation: This nonenzymatic method involves the use of chemical compounds like ethylene glycol tetraacetic acid (EGTA) that bind calcium ions needed for cell attachment.

How long do you trypsinize cells?

Always check the manufacturer’s instructions for the dissociation reagent you’re using and note that different cell lines may need different conditions. In general, to remove cells from tissue culture surfaces aseptically remove the medium, then add prewarmed porcine or recombinant trypsin. Incubate the culture vessel for 2 to 3 minutes at 37°C, then check it under a microscope. If less than 90% of cells detach, incubate for 2 minutes while observing under a microscope at 30-second intervals. Avoid prolonged exposure to trypsin – greater than 15 minutes. Neutralize the trypsin with serum or an ADCF alternative.