Explain most problems may occurred during the tissue culture processing

1. Professor assistant Dr. Hadeel Alboaklah
Professor assistant Dr. Muntader Mohamed
Professor assistant Shatha Husain Kadhum

2. Causes of cell death in tissue culture Identifying Problems:
Overview
Some general causes of cell death or other problems:
• Contamination: Infections, chemical, or cell line problems.
• Fixable things: Culture conditions/media components
• Less fixable things: Passage number, primary cell line
problems, messy lab mates, outside factors
• Mystery stuff

3. Identifying Problems:
Contamination
What types of contaminants can be present?
 Easily visible:
• Infectious: Yeast / bacteria / fungus
• Other cells being cultured in the lab: fibroblasts, HeLa, etc.
 Less visible:
• Infectious: Mycoplasma / viruses
• Chemical contaminants: Often sterilizing solution (ethanol or bleach),
but could be a bad batch of chemical used in culture or other factor

4. Identifying Contamination
Identifying yeast contamination:
• Rapid growth and consumption of media nutrients
= rapid colour change
 in solution.
• Cloudy media
• “Baking bread” smell
• Dead/poorly spread cells
Yeast morphology:
“string of pearls”
Treatment
1) Discard cells.
2) Keep an eye on other cell line

5. Identifying yeast contamination

6. Identifying yeast contamination

7. Identifying bacterial contamination:
 Rapid growth and consumption of media nutrients = rapid color
change in solution.
• Cloudy solution
• Dead/poorly spread cells
• Morphology: Highly variable, depending on bacteria type.
Treatment:
1) Discard cells.
2) Keep an eye on other cell lines.
• Note: The source of contamination may be pre-existing in primary
cells, due to a patient’s infection prior to cell retrieval

11. Identifying fungal contamination:
 Rapid growth and consumption of media nutrients: rapid colour change
in solution.
• Cloudy/dark solution
• “Garbage” smell
• Dead/poorly spread cells
• Morphology: fuzz or threads
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
3) Sticky mats/dehumidifiers in the labs may prevent the tracking in and
growth of mold spores.
Note: The source of contamination here may be pre-existing in primary
cells (prior exposure to mold spores)

15. Contamination by other cell lines:
Change in growth rate (needs to be faster, in order for this contamination
to overtake the previous cell type)
• Change in morphology
• Mixed morphology
• Change in response to stimuli
Treatment
1) Discard cells.
2) Keep an eye on other cell lines.
Note: The source of contamination here may be pre-existing in cell
lines (ex: HeLa contamination of many CDC cancer cell samples).
Note: Some culture techniques require intentional co-cultures.

17. Less visible contaminants:
 Mycoplasma:
– May be seen by staining with DAPI
– Can be tested for (NBTC has kits)
– Results in changes in cell behavior and/or cell death
• Treatment:
1) Discard cells.
2) Keep an eye on other cell lines

19.  Viruses:
May see some slight granularity (caused by viruses-viruses are not directly
visible by light microscopy).
Usually results in rapid cell death.
 Treatment:
1) Discard cells.
2) Keep an eye on other cell lines.