The document discusses various components of typical cell culture media, including carbohydrates, amino acids, salts, buffers, vitamins and hormones, antibiotics, and serum. It describes the purposes and considerations for each component in maintaining optimal cell growth conditions and metabolism. Key factors include maintaining isotonicity, buffering pH, providing nutrients and growth factors, and preventing bacterial/fungal contamination.

1. Scaling up the production process Oxygen requirements Supply of oxygen to satisfy cell metabolism is one of the major problems associated with culture scale up. O2 consumption rate: 0.06-0.6 mmole/hour for 10^6 cells/ml For small volumes (< 1 litre) O2 diffusion form the headspace through the culture surface

2. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Culture Medium animal cells require chemically complex liquid media suitable for growth of several generations commercially made, supplied as 1x liquid ready for use, 10x concentrated solution, or powdered form dilute concentrate with presterilized distilled water dissolve powdered medium in water and filter sterilize

3. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P56.

4. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 22.

6. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Components of a typical culture medium: Carbohydrates Amino Acids Salts Buffering system Vitamins and hormones Antibiotics Phenol red

7. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 1. Carbohydrates glucose (25 mM) used most often as energy source may use alternative carbohydrate sources such as fructose, galactose, or maltose (reduced lactic acid production, more stable culture pH) precursor for biosynthesis ribose through the pentose phosphate pathway (for nucleic acid synthesis) can also use glutamine (4 mM) for energy

8. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 56.

9. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 2. Amino acids 0.1-0.2 mM added as a source of precursors for protein synthesis glutamine (2-4 mM) used precursor for TCA intermediates, purines, pyrimidines, amino sugars, and asparagine ammonia is released as a by-product of glutamine breakdown, can be inhibitory to growth (half-life of glutamine at 4 o C is 3 weeks) occurs via cellular metabolism or thermal breakdown

10. Specific amino acid consumption in 2 cultures

11. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P57.

12. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media to reduce accumulation of ammonia: -> use continuous feed of low [gln] -> use gln-containing dipeptides that hydrolyze slowly in culture alanine accumulates as a result of ammonia uptake/removal glutamate may be used as a substitute, results in lower ammonia produced

13. Energy metabolism glucose-6P Glucose P-gluconate ribulose-5P Ribose-5P fructose-diP P-enolpyruvate pyruvate Lactate acetyl CoA citrate 2-oxoglutarate succinate oxaloacetate TCA cycle Glutamine glutamate NH 3 NH 3 CO 2 CO 2 CO 2 CO 2 Alanine transamination

14. Radioactive glucose for metabolic flux analysis CH 2 OH C C C O C HC.OH OH OH OH H H H H 1- 14 C 6- 14 C 3- 3 H

15. Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media 3. Salts included to make solution isotonic and to maintain balance with the intracellular content Osmolarity – measure of the total number of particles dissolved in solution a control parameter, can affect property of a culture

16. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 1 Osmolar ( 1 Osm/l) has 6.023x10 23 particles in 1 liter of water ionization increases the number of osmotically active particles based on total number of particles dissolved in solution nature of particles is irrelevant Ex. 1 mM glucose -> 1 mOsm/l 1 mM NaCl -> 2 mOsm/l

17. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media osmolality (mOsm/kg of water) = osmolarity (in dilute solution) osmolarity of standard culture medium (isotonic) ~ 250-350 mOsm/l (300 mOsm -> optimal) -> hypertonic – osmolarity is too high (cells shrink) -> hypotonic – osmolarity is too low (cells burst) osmolarity of medium should be within 10% of optimal value

18. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media higher osmolarity, the lower the freezing point water has a freezing point of 0 o C, a saline solution with an osmolality of 1 Osm/kg has a freezing point of -1.858 o C can use osmometer to determing freezing point of a solution relative to water

19. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 4. Buffering system Bicarbonate is typically included to act as a buffer system in conjuction with a CO 2 atmosphere (5-10%)(enriched air), maintains pH range of 6.9-7.4 Advantage: cheap Disadvantage: cultures may become alkaline very quickly upon removal from incubator alternate buffers may be used -> HEPES (pKa = 7.3 at 37  C) -> MES (pKa = 6.5 at 37  C) -> CHES (pKa = 9.5 at 37  C) 10-20 mM HEPES is optimal, cultures can be grown without an enriched CO 2 atmosphere, expensive

21. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 5. Vitamins and hormones present in micromolar amounts, used as metabolic cofactors varies from one medium formulation to another different cell lines have different requirements

22. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 6. Antibiotics often included in media for short-term cultures in order to reduce the risk of bacterial or fungal contamination often used in combinations as an antibiotic cocktail -> Penicillin G (100 u/mL) inhibits Gram-positive bacteria by inhibiting cell wall synthesis -> Streptomycin (50 mg/L) inhibits Gram-positive and Gram-negative bacteria by inhibiting protein synthesis -> Amphotericin B (25 mg/l) as an antifungal agent by inhibiting ergosterol synthesis

23. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media not used for routine subculture or stock culture low levels of bacteria or fungus may be masked may allow for selective retention of antibiotic resistant contaminants

24. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media 7. Phenol red pH indicator of the medium sensitive to slight pH changes around the growth optimum for cells at lower pH, the phenol red turns orange (pH 7.0) or yellow (pH 6.5) overnight change from red to yellow indicates bacterial contamination

25. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum Serum added as a supplement ( 10% v/v ) supernatant of clotted blood , contains undefined materials essential for cell growth, esp. for anchorage dependent cells -> pH 6.85 – 7.05 -> osmolarity – 250-295 mOsmol/L -> protein content – 60-80 mg/mL -> albumin content - 30-50 mg/mL

26. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum Serum provides: Growth factors Lipids Attachment factors Metal transporting proteins (i.e. transferrin) Protease inhibitors (neutralizes trypsin) High protein content protects against toxins, shear damage by stirring in fermenters

27. Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum cow or horse serum most commonly used fetal calf serum (FCS) most effective due to high content of embryonic growth factors -> newborn calf serum (under 10 days) -> donor calf serum (under 8 months) serum is tested for performance and cytotoxicity effects on plating efficiency and cell growth filtered and tested for bacterial, fungal, and viral contamination

28. Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum Disadvantages of serum: Expensive Chemically undefined High protein content Source of contamination Ethical considerations?

29. Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum want to use serum-free media supplement media with insulin, transferrin, ethanolamine, and selenite specific for different cell types

30. Component characteristics of media formulations Serum based media Serum-free media Protein free media Animal-component free media Chemically-defined media Media formulated goal: Chemically defined Protein-free Animal component-free