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Cell Culture Guidelines

Several factors should be considered before a cell line is put into culture

  • Are the cells coming from established, well-defined lines?
  • Are the nutrient and CO2 requirements known?
  • Are the growth patterns known?
  • Is the species known?
  • Has mycoplasma testing been done?

After consideration of all the above, thoughtful planning should be given to other factors

  • Will the cells be cultured in plates, flasks, bioreactors, etc.

  • Who will do the tissue culture?
  • Who will prepare, test and maintain stocks of media, regents and supplies?
  • What volume/number of cells will be required?

The following are general guidelines for culturing cells.

If hybridoma cells are received in a tissue culture flask (or tissue culture plate), information regarding the optimal type of transfer should be available. Once cells can be maintained in one type of culturing vessel, it should be easy to adapt them to other cultureware.

If tumor cells are received it is necessary to know if they are of fibrosarcoma or lymphoma origin. Fibrosarcoma cells will generally be adherent to plastic and require the use of trypsin or EDTA to remove them. Tumor cells also usually require less fetal calf serum in their media.

If cells are received frozen, in an ampule or cryovial, they should be washed free of DMSO prior to culturing. If you do not know how to properly thaw cells, you may refer to our protocol by clicking here.

Establish a routine for the passage/transfer of the cells based on the rate at which they form a monolayer. For example, culture the cells in 2 wells of a 24-well plate. On Monday, cell should be resuspended gently with a pasteur pipet or 1mL pipet and enough cells transferred, dropwise, into duplicate wells of fresh, warmed media so that 4 days later, a monolayer is again observed and the transfer is repeated. It may be useful to determine the cell concentration at various days in culture in order to make projections and appropriate expansions.

If cells require the addition of growth factors, dilutions must be made carefully and good tissue culture technique maintained.

Some cells can easily be adapted to various kinds of media. Adaptation should take place overtime, with adequate post-adaptation analysis of proliferation, secretion, function, etc. During the period of adaptation the original culture should be maintained

Successful culturing of cells requires frequent monitoring of growth, media color, contamination, etc. The greater the familiarity with a cell line, the greater the expectation for a problem-free culture.

To view a sample protocol for the culturing of cells, click here