The General Guidelines for Cell Culture

Before a cell line is put into culture, several factors should be considered

  • Are the cells coming from established, well-defined lines? Is the species known?
  • Are the nutrient and CO2 requirements known?
  • Are the growth patterns known?
  • Has mycoplasma testing been done?
  • Will the cells be cultured in plates, flasks, bioreactors, etc?
  • What volume/number of cells will be required

Passage cells (10 cm-dishes)

  1. Pour out media from cell culture dish.
  2. Wash it with Hanks 5 ml per dish.
  3. Tilt around then pour out in order to removal the old media, especially blood serum which may prevent the effect of trypsin.
  4. Add 3 ml of Trypsin / EDTA to each dish. Then incubate it in the incubator for about 3-5 minutes.
  5. Add 10% or 20%-complete media 4 ml per dish and tilt.
  6. Scrape (most of the small cells are already released; proliferative cells lift up easy, differentiative cells are more adherent).
  7. Place contents into a15 ml Falcon tube.
  8. Spin it at 1000 RPM for 5 min.
  9. Dump off supernatant.
  10. Add 2 ml enough media per dish.
  11. Each dish should have 8(or 10) ml of completed media, add cells (They are spread by 1 : 3)

Other protocols:

Conventions for Work in the Cell Culture Laboratory (Gumbleton Lab, Cardiff University)
General guidelines and good practice for work in cell culture facility. Highly recommended.

Cell Culture Guidelines (Immunology Applications Core Facility, University of Chicago)
General guidelines for culturing cells.

Guide For Identifying And Correcting Common Cell Growth Problems (Corning)
This 14-page Corning Technical Guide reviews some of the common and not so common cell growth and attachment problems that are often very difficult to identify and eliminate.

Trypsinization of Adherent Cells (Hannun/Obeid Lab, Duke Univ.)
Enzymatic method of passing cells.