1. Cell culture environment
LECTURE OF SUBJECT :
Dr. sharafaldin Al-musawi
College of Biotecholgy
LECTURE: 4
SUBJECT: Animal Tissue culture
LEVEL: 4
Cell culture environment
(in vitro) 1

2. Requirements-
Substrate or liquid (cell culture flask or scaffold material).

3. Nutrients (culture media)

4. Conditions (CO2, temperature 37oC, humidity)

5. Sterility (aseptic technique, antibiotics and antimycotics)

6. Basal Media
Maintain pH and osmolarity
Provide nutrients and energy source.

7. Components of Basal Media
Regulate membrane potential (Na+, K+, Ca2+)
Maintain osmolarity
Ions for cell attachment and enzyme cofactors

8. pH Indicator – Phenol Red Optimum cell growth approx. pH 7.4
Chemical formula
C19H14O5S

9. Buffers (Bicarbonate and HEPES)
Bicarbonate: buffered media requires CO2 atmosphere
Chemical formula:
HEPES: Strong chemical buffer range pH 7.2 – 7.6 (does not require CO2)
Chemical formula
C8H18N2O4S

10. Keto acids (oxaloacetate and pyruvate)
oxaloacetate pyruvate
Intermediate in Glycolysis/Krebs cycle
Keto acids added to the media as additional energy source
Maintain maximum cell metabolism

11. Carbohydrates Energy source Glucose and galactose
Low (1 g/L) and high (4.5 g/L) concentrations of sugars in basal media

12. Vitamins Precursors for numerous co-factors
B group vitamins necessary for cell growth and proliferation
Common vitamins found in basal media is riboflavin, thiamine and biotin
Vitamin B1 (Thiamine)
Vitamin B2 (Riboflavin)
Vitamin B3 or Vitamin P or Vitamin PP (Niacin)
Vitamin B5 (Pantothenic acid)
Vitamin B6 (Pyridoxine and Pyridoxamine)
Vitamin B7 or Vitamin H (Biotin)
Vitamin B9 or Vitamin M and Vitamin B-c (Folic acid)
Vitamin B12 (Cyanocobalamin)

13. Trace Elements
Zinc, copper, selenium and etc…

14. Supplements L-glutamine
Essential amino acid (not synthesised by the cell)
Energy source (citric acid cycle), used in protein synthesis
Unstable in liquid media - added as a supplement

15. Supplements Non-essential amino acids (NEAA)
Usually added to basic media compositions
Energy source, used in protein synthesis
May reduce metabolic burden on cells

16. Supplements Growth Factors and Hormones (e.g.: insulin)
Stimulate glucose transport and utilisation.
Uptake of amino acids.

17. Supplements Antibiotics and Antimycotics
Penicillin, streptomycin, gentamicin, amphotericin B
Reduce the risk of bacterial and fungal contamination.

18. Foetal Calf/Bovine Serum (FCS & FBS)
Growth factors and hormones
Aids cell attachment
Binds and neutralise toxins
Long history of use
Disadvantages
Infectious agents (prions)
Variable composition
Expensive
Regulatory issues (to minimise risk)
Heat Inactivation (56oC for 30 mins) – why?
Destruction of complement and immunoglobulins
Destruction of some viruses (also gamma irradiated serum).