Tissue Culture Tips

Tissue culture is the propagation of cells in a liquid growth medium in the lab for various scientific purposes. For example, tissue cultures are used in diagnosis of genetic disease, as a medium for growing intracellular pathogens such as bacteria or viruses or in investigations into the cells themselves.

Tissue Culture Cells

• Some cells adhere to the bottoms of petri dishes or remain suspended in the liquid. Cells can be of plant or animal origin. Cells used in tissue cultures can be primary cells, meaning they were harvested directly from an organism, or of a cell line that has been maintained in culture. Cell lines are often cancer cells, and this must be considered when planning or interpreting experiments.

Treat Cells Gently

• Adherent cells are removed from their petri dish for passage by scraping or with a protease, generally trypsin and EDTA. Scraping kills many cells and should be used only when preparing cell lysates (liquid preparations of cell contents for analysis) or when trypsin/EDTA absolutely must be avoided.

  Trypsinizing removes not only the proteins that adhere cells to the dish but most other proteins on the membrane of the cell, too. Cells ball up and take some time, usually overnight, to reestablish a bond with the dish. Adding a minimal amount of trypsin--around 1mL for a 25cm2 flask--rocking the flask for five seconds or less to coat the monolayer and then pouring off or aspirating the excess trypsin will reduce the amount of trypsin to which the monolayer is exposed. Let the cells rest in the incubator during trypsinization.

  After the cells slide off the bottom of the dish, add media with serum and aspirate the lysate up and down the pipet gently. Allow a little media to remain in the dish as you pipet up and down several times to break apart clumps and get an even suspension.

  Do not suck bubbles into the pipet. Also, do not vigorously mix or vortex the cell suspension; the cells are very fragile in this balled-up state.

To Heat Inactivate or Not To Heat Inactivate

• When cell culture techniques were in their infancy, fetal bovine serum (FBS) was known to contain complement proteins, which are immune system proteins that lyse cells. Complement proteins are not desirable in cell culture. Heat-inactivating FBS at 56 degrees Celsius for 30 minutes will render complement proteins inactive. Prewarming FBS to 37 degrees C also is enough to inactivate complement proteins.

  In the early days, heat inactivating FBS also killed mycoplasma and other contaminants. At the time, filter sterilization was performed with 0.45um filters. Today, we filter sterilize FBS and media through 0.1um or even 0.04um filters. No mycoplasma will slip through that.

  This leaves us the question whether or not to heat inactivate.

  Heat inactivation degrades all components of the FBS--such as vitamins, amino acids and growth factors--possibly below the threshold for some finicky cell lines.

  If you are working with a slow-growing or finicky cell line, consider merely filter sterilizing your FBS. You might find this increases the robustness of your cells.