The table below lists some potential problems and possible solutions that may help you troubleshoot your cell culture experiments.  Note that the list below includes only the most commonly encountered problems in cell culture, and provides guidelines to solutions only.  To help evaluate your results more successfully, we recommend that you consult the manuals and product information sheets provided with the products you are using as well as the published literature and books on the subject.

Problem: No Viable Cells After Thawing Stock

Reason
Solution
Cells were stored incorrectly
  • Obtain new stock and store in liquid nitrogen.  Keep the cells in liquid nitrogen until thawing
Home made freezer stock is not viable
  • Freeze cells at a density recommended by the supplier
  • Use low-passage cells to make your own freezer stocks
  • Follow procedures for freezing cells exactly as recommended by the supplier.  Note the freezing procedure recommended by this handbook is a general procedure provided as a guideline only.
  • Obtain new stock
Cells were thawed incorrectly
  • Follow procedures for thawing cells exactly as recommended by the supplier.  Note the thawing procedure recommended by this handbook is a general procedure provided as a guideline only.
  • Make sure that you thaw the frozen cells quickly, but dilute them slowly using pre-warmed growth medium before plating.
Thawing medium is not correct
  • Use the medium recommended but the supplier.  Make sure the medium is pre-warmed
Cells are too dilute
  • Plate thawed cells at high density as recommended by the supplier to optimize recovery.
Cells not handled gently
  • Freezing and thawing procedures are stressful to most cells.  Do not vortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge the cells at high speeds.
Glycerol used in the freezing medium was stored in light (if applicable)
  • If stored in light, glycerol is gets converted to acrolein, which toxic to cells.  Obtain new stock.

Problem: Cells Grow Slowly

Reason
Solution
Growth medium is not correct
Serum in the growth medium is of poor quality
  • Use serum from a different lot.
Cells have been passaged too many times
  • Use healthy, low passage-number cells.
Cells were allowed to grow beyond confluency
  • Passage mammalian cells when they are in the log-phase before they reach confluence.
Culture is contaminated with mycoplasma
  • Discard cells, media, and reagents.  Obtain new stock of cells, and use them with fresh media and reagents.

Gibco Cell Culture Basics Videos

Getting started in 3D - Setting up your lab and reviewing common pitfalls of researchers new to 3D
Part 1: Getting started in 3D: Setting up your lab and reviewing common pitfalls of 3D research Part 2: Monitoring your 3D culture (what's good and bad): How to detect problems and what a successful growth looks like
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