Cell Culture Techniques - The “Do’s and Don't” of Cell Culture

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The best practices of cell culture techniques
Modified from the text in
Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition, Cook Book Volume 12
Table of Contents

Given below are a few of the essential "do's and don'ts" of cell culture. Some of these are mandatory, e.g., use of personal protective equipment (PPE). Many of them are common sense and apply to all laboratory areas. Some of them are specific to cell culture.

Cell Culture Best Practices

Cell Lines, Cell Culture Media and Cell Culture Reagents

  • Only handle one cell line at a time. This common-sense approach will reduce the possibility of cross contamination. It will also reduce the spread of bacteria and mycoplasma due to the generation of aerosols across opened media bottles and flasks in the cabinet.
  • Test cells for the presence of mycoplasma on a regular basis.
  • Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes media that has been purchased commercially.
  • Maintain separate bottles of media for each cell line in culture.
  • Quality control all media and reagents prior to use.

Laboratory Practices

  • Ensure that incubators, cabinets, centrifuges and microscopes are cleaned and serviced at regular intervals.
  • Ensure that equipment requiring calibration is calibrated on a regular schedule, or if its performance is suspect.
  • Correctly label reagents including flasks, media and ampoules with contents and date of preparation.
  • Use personal protective equipment (PPE), (laboratory coat/gown, gloves and eye protection) at all times. In addition, thermally insulated gloves, a full-face visor and a splash-proof apron should be worn when handling liquid nitrogen.
  • Wear dedicated PPE for the tissue culture facility and keep separate from PPE worn in the general laboratory environment. The use of different colored gowns or laboratory coats makes this easier to enforce.
  • Use disposable head caps to cover hair.
  • Clean the work surfaces with a suitable disinfectant, e.g. 70% isopropanol, between operations and allow a minimum of 15 minutes between handling different cell lines.
  • Keep all work surfaces free of clutter.
  • Keep cardboard packaging to a minimum in all cell culture areas.

Common Mistakes to Avoid in Cell Culture

Cell Lines, Cell Culture Media and Cell Culture Reagents

  • Do not handle cells from unauthenticated sources in the main cell culture suite. They should be handled in quarantine until quality control checks are complete.
  • Do not use antibiotics continuously in culture medium as this can lead to the appearance of antibiotic resistant strains and may mask underlying contamination.
  • Avoid keeping cell lines continually in culture without returning to frozen stock.
  • Avoid cell cultures from becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on the ECACC cell culture data sheet.
  • Do not allow media to go out of date. The shelf life is only 4 - 6 weeks at +4°C once glutamine and serum are added.

Laboratory Practices

  • Do not allow essential equipment to be out of calibration. Especially ensure that microbiological safety cabinets are tested regularly.
  • Do not allow water baths to become contaminated; clean on a regular basis
  • Do not allow waste to accumulate, particularly within the microbiological safety cabinet or in the incubators.
  • Do not have too many people in the lab at any one time.