Description of cell culture basics and cell contamination

1. Mustapha Umar Imam, PhD
Basics of Cell Culture

2. Introduction
• Cell culture is the process by which cells
(prokaryotic, eukaryotic or plant) are grown
under controlled conditions.
• pH: 7.5
• 37°C,
• relative humidity: 95%
• CO2: 5%

3. What is cell culture used for?
• Model systems for studying:
• basic cell biology
• physiological processes
• pathological bases
• mechanistics bases of interventions
• Toxicity testing for drugs/extracts
• Virology
• Genetic Engineering
• production of commercial proteins

4. Equipment and reagents required
• Biosafety cabinet (laminar flow)
• horizontal
• vertical

5. Equipment and reagents required
• CO2 incubator (temperature, humidity, CO2)
• Sterility (cleaning, cupper sulfate)
• Flask: 25cm3, 75cm3
• 6, 12, 24, 48 and 96-well plate
• Cell culture media
• Enzymes: trypsin, dipase, collagenase
• Cell scapper

6. Equipment and reagents required
• Serological pipette (1, 2, 5, 10, 25mL)
• Pipette boy (girl)
• Suction machine
• Antibiotics
• Antifungals
• Fetal bovine serum
• PBS
• Special nutrients: eg. glutamine
• DMSO

7. Equipment and reagents required
• Fridge
• Freezer (-20, -80 degrees)
• Autoclave
• Liquid nitrogen tank (dewar)
• microscope

8. Relevant terms
• Passage/sub-culture
• Confluent
• cryopresevation

9. Primary culture
• Culture of cells that are surgically or enzymatically removed
from a live organism
• Limited life span (become senescent after several passages)
• heterogeneous population of cells

10. Continuous (secondary) cell lines
• Cells that have been induced to grow constinuously
• Grow very fast

11. Cell morphology
• Epithelial like: attached and appear flattened and
polygonal in shape
• Lymphoblast like: remain suspended with a
spherical shape
• Fibroblast like: attached and appear elongated and
bipolar

12. General Requirements for cell culture
• Growth requirements
• nutrient medium
• surface on which to grow
• growth factors
• Temperature
• pH (buffered)
• Prevent contamination
• (aseptic technique, antibiotic, fungicide)

13. Culture media
• depends on cell type
• Commonly ones are :
• RPMI-1640 (Roswell Park Memorial Institute)
• MEM: minimum essential medium
• DMEM: Dulbecco's Modified Eagle Medium, etc.
• Media is supplemented with antibiotics/fungicides/FBS
• Prepared media is filtered and incubated at 4 C

14. Examples of cell lines
• Human cell lines
• MCF-7 breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney
• HeLa Henrietta lacks
• Primate cell lines
• Vero African green monkey kidney epithelial cells
• Cos-7 African green monkey kidney cells

15. Contaminantion
• Adulteration of cultures with an external substance
• Chemical:
• may not stop growth
• difficult to detect
• may change behaviour of cells
• Biological:
• mycoplasma, yeast, bacteria or fungus
• cross-contamination from other cell lines
• inhibit growth (nutrient consumption and waste secretion)

16. Biological contamination
www.eppendorf.com

17. Biological contamination
• Adulteration of cell cultures with living
organisms:
• Bacteria
• Yeast
• Viruses
• Mycoplasmas
• Protozoa
• Cross-contamination from other cell lines

18. Biological contamination
Çelik-Uzuner & Uzuner (2017)

19. Bacterial contamination
• Rapid colour change
• Cloudy media
• Morphology depends on
bacteria
www.eppendorf.com

20. Yeast contamination
• Rapid colour change
• Cloudy media
• Characteristic “baking bread”
smell
• “Strings of pearl” morphology

21. Fungal contamination
• Rapid colour change
• Cloudy media
• Smells like “garbage”
• Morphology looks like thread
www.eppendorf.com

22. Contamination by other cells
• Mixed morphology
• Abrnormal response
to stimuli
• Continuous
authentication of cell
lines
Routray et al. (2016)

23. Mycoplasma contamination
• Less visible
• Cell death or change in
response to stimuli
• DAPI (4′,6-diamidino-2-
phenylindole) staining
• ELISA/PCR testing

24. Viral contamination
• Less visible
• Cytopathic changes:
• morphology
• detachment
• reduced growth etc)
• Rapid cell death

25. Other things that may look like
contamination
• Incubator (CO2 or temprature) problems
• Over-passage
• Lack of nutrients
• Over-confluence

26. Basic aseptic conditions
• Always use labcoat and glove
• Sterilise consumables and reagents
• UV light
• Autoclave
• Filteration
• Swab all surfaces, bottle tops & necks with 70% ethanol
• Avoiding placing caps & pipettes down on the bench
• Work either left to right or vice versa
• Clean up spills immediately & always leave the work place
neat & tidy
www.sigmaaldrich.com

27. Basic aseptic conditions
• Do not pour liquids, instead dispense by pipette,
autodispenser
• Never pass hands over open vessels
• Do not block the vents (laminar flow)
• Tie back long hair, or wear a cap.
• Avoid excessive talking.
• Obtain supplies from reputable vendors
www.sigmaaldrich.com

28. Basic aseptic conditions
• Regularly inspect/service/sterile instruments (laminar
flow, incubator, cell culture room, microscope,
autoclave, centrifuge)
• Cover caps with aluminium/parafilm during storage
• Avoid using water bath.
• Wipe items with 70% alcohol before entry into culture
room.
www.sigmaaldrich.com

29. Routine testing to prevent
biologcal contamination
• Aerobic nutrient broth (Tryptone Soy Broth)
• Anaerobic nutrient broth (Thioglycollate Medium)
• ELISA/PCR/microscopy for mycoplasma
• Short tandem repeat (STR) profiling for
authentication

30. Take home message
• Cell culture is growing of cells artificially in a laboratory
• Can be used to study biological and pathological or doscover
alternative treatments
• Biological conditions are recreated to optimize growth
• nutrients
• temperature
• pH
• O2, CO2

31. Take home message
• Cell culture contamination (especially biological) can
significantly affect in vitro experiments
• Cell death (bacteria, fungi, yeast, virus)
• Morphology/metabolism (virus, mycoplasma, cells)
• Routine aseptic techniques should be adopted in any culture
lab to prevent contamination
• Routine testing is recommended for early detection of
contamination
• microbiological culture
• ELISA/PCR/microscopy
• STR analysis

32. References
• Çelik-Uzuner, S., & Uzuner, U. (2017). An Extensive Method for Maintenance of Sterility in
Mammalian Cell Culture Laboratory Routine. Challenges, 8(2), 26.
• Routray, I., Mahmood, A., Ngwa, N. E., Tasleem, M., Sahin, K., Kucuk, O., & Ali, S. (2016). Cell
line cross-contamination and accidental co-culture. J Stem Cell Res Ther, 1(5), 00031.
• https://handling-solutions.eppendorf.com/cell-handling/contamination/scientific-
background/bacterial-contamination/
• https://www.sigmaaldrich.com/NG/en/technical-documents/technical-article/cell-culture-
and-cell-culture-analysis/mammalian-cell-culture/cell-culture-troubleshooting-
contamination

33. THANK YOU