cell culture medium

common nutrient requirements

macroelements (macronutrients)

  • C, O, H, N, S, P, K, Ca, Mg, and Fe

  • required in relatively large quantity

  • builds cell structures and help with metabolism

micronutrients (trace elements)

  • Mn, Zn, Co, Mo, Ni, and Cu

  • required in trace amounts to help enzymes function

culture media

  • contain all the nutrients required by the organism for growth

  • classification

    • physical nature: liquid, semisolid, solid

    • chemical composition: defined, complex

    • functional type: supportive(general purpose), enriched, selective, differential

media components

peptones - water soluble proteins prepared by partial digestion of various protein sources (e.g. milk) using pepsin

extracts - aqueous extracts, usually of beef or yeast to supply nucleic acids, fats, polysaccharides, as well as vitamins and trace minerals

what is in culture medium

water

  • keeps cells hydrated

  • water is the solvent for the nutrients, salts and oxygen that the living cells need

  • the medium is isotonic such that there is no salt imbalance with the intracellular content

amino acids

  • protein synthesis

  • essential amino acids must be added as cells are not able to synthesize themselves

  • glutamine - acts as a precursor in a series of biochemical reactions in living cells that generates energy

carbohydrates

glucose and fructose as energy sources

Serum

promotes cell growth

contains

  • lipids e.g. cholesterol

  • trace elements/nutrients e.g. insulin

  • adherence factors that help cell adhesion

  • protease inhibitors

plasma vs serum

plasma - clear non cellular portion of blood (contains proteins for blood clotting)

serum - plasma without proteins that are involved in blood clotting such as fibrin

types of serum

  • FBS (Fetal Bovine Serum) - fetal calf serum preferred because they will not contain antibodies other than those obtained from its mother

  • bovine calf

  • older calf

  • horse

  • human - for human cell lines but need to be screened for viruses such as HIV and hepatitis B

Antibiotics

to reduce frequency of contamination (penicillin and streptomycin commonly used)

addition of antibiotics may

  • encourage development of antibiotic resistant microorganisms

  • hide presence of low level contaminants that grow when antibiotics are removed

  • encourage poor aseptic techniques

sodium bicarbonate

most cells require pH ranging 7.2 - 7.4

used as buffering system

despite limited buffering capacity, NaHCO3 used due to low cost nontoxic and providing additional nutritional benefits to the cells

dissociates to form

how sodium bicarbonate maintains pH

  • during cell culture incubator is set to 5%-10% CO2 level

  • Sodium bicarbonate dissociates, releasing CO2 into atmosphere and hydroxide ions into medium

  • in freshly prepped medium, enriched CO2 atmosphere in incubator helps to equilibrate association of ions to maintain pH 6.9-7.0

  • when cells metabolize, wastes such as CO2 is released, turning medium acidic (e.g. carbonic acid)

  • hydroxide ions neutralise acidity in culture medium and maintain a near neutral pH

pH indicator

Phenol red is added so pH status is constantly indicated by colour

culture medium should be changed/replenished if colour turns yellow (acid) or pinkish purple (alkali) - culture medium should be red (neutral)

nutrients

carbohydrates - glucose and fructose as energy sources

lipids - fatty acids and cholesterol

salts (ions) - involved in cell function and metabolism

nucleic acids - thymidine, uracil, adenine

vitamins, hormones, growth and adherent factors

preparing cell culture medium

contains various ingredients to mimic physiological fluids (blood, lymph) - provide conditions for cell growth

all ingredients have to be added in the right quantity