This document discusses the requirements and components of cell culture media used for growing animal cells in vitro. The key requirements include a substrate or scaffold, nutrients provided in culture media, and sterile conditions with controlled temperature, CO2, and humidity. The basal media contains components like phenol red pH indicator, buffers, keto acids, carbohydrates, vitamins, trace elements, L-glutamine, and non-essential amino acids to maintain pH, osmolarity, provide energy sources, and support cell growth and proliferation. Supplements like growth factors, antibiotics, and fetal bovine serum are also added to further support cell attachment, growth, and prevent bacterial/fungal contamination.

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

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

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. 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
Supplements

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

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

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

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).