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Role of various constituents of media and supplements used in cell culture Physical properties of media e.g. pH, temperature, osmomolarity, viscosity, surface tension, foaming Essential chemical constituent of media: salt, amino acids, vitamins, minerals, organic supplements, hormones, growth factors, serum Role of serum as a media supplement and advantage& disadvantage of using serum as a supplement Importance of using complete, synthetic media without serum supplements Factors influencing the choice of media for different types cell culture Selection of appropriate media for the cultivation of a particular cell type
Media for specialized objectives ( for particular cells) Cell type T-cell Skin explant Liver cells Fibroblast Ganglia Growth factors IL-2 Epidermal gowth factor (EDGF) Gly-His-Lys ( liver growth factor) Fibroblast growth factor( FGF) Nerve growth factor ( NGF-beta)
Development of Media for cell culture Initial attempt to culture cells were performed in natural media based on tissue extracts and body fluids
Chemically defined were introduced in the 1950s - Eagles Basal Medium - Eagles Minimal Essential Medium ( MEM) - Dulbeccos modification of MEM ( DMEM) - Supplemented with serum, protein hydrolysates or embyo extracts - Roswell Park Memorial Institute ( RPMI 1640)
Natural media :
Plasma clots These are of limited use, traditionally this is for avian tissue culture Biological fluids Serum, tissue extracts, embryo extracts, amniotic fluid, aquous humour, insect hemolymph, chick embryo extracts
The choice of culture medium and supplements can have a major impact on growth, function and phenotype of cells in vitro.
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MgSO4,7H2O MgCl2,6H2O NaH2PO4,H2O Na2HPO4,2H2O KH2PO4 Glucose Phenol red NaHCO3 Gas Phase air 1.00 air 1.00 1.00 0.05 1.00 2%CO2 in air 0.05 0.10 0.20 0.21
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Mammalian cell culture media Salts : K, Na, Cl, Mg, Ca -provide osmotic balance, - help regulate membrane potential - as cofactors for cell attachment Carbohydrates - mostly glucose and galactose but also maltose or fructose Trace elements : Se, Zn, Cr - Se is detoxifier and remove oxygen free radicals 9 essential amino acids : His, Ile, Leu, Lys, Meth, Phe, Thr, Try, Val - some cells require also cys and Tyr Vitamins - Niacin, folic acid, riboflavin, inositol, thiamine, essential for replication but the deficiency does not manifest itself until several cell doublings - vit D, C, E, A
Other Important components: Some nonessential molecules ( Pyruvate, Hypoxanthine, thymidine, adenosine) to improve growth Fatty acids Cholesterol ( usually not included in the mixture of fatty acids) Detergent to emulsify lipids, but may be toxic to some cells Hormones and growth factors Specific factors for growth and differentiation Phenol red as pH indicator
pH :
Most cells grow well at pH 7.4 Some fibroblasts grow at pH 7.4-7.7 Transformed cells pH 7.0-7.4 Epidermal cell some times pH 5.5
Phenol red is commonly used as indicator - Red at 7.4 - Orange at 6.5 - Yellow at 6.3 - Purple at 7.8 Most often pH is maintained by CO2 /bicarbonate buffer
- Cells have to grow in the CO2 incubator
Buffering Agents
CO2 and bicarbobnate Most commonly used method to stabilize pH natural buffer, low cost, non-toxic H2O + CO2 H+ + HCO3Cell grown in open vessels such as dishes need to be incubated in CO2 CO2 level must be set to match the medium Bicarbonates are also energy source
Osmolarity
Cells usually need osmotic pressure of between 280-320 mOsm/litre and ideally osmotic pressure should be maintained around 290-300 mOsm/litre .[ Normal saline 9.0g NaCl/litre]
If HEPES, acid, alkali are used to adjust pH of the medium then equivalent amount of NaCl should be omitted to allow for increase in osmotic pressure. The correct amount of NaCl to be omitted can be calculated by using formula based on : (D-O)/32 = X Assuming that 1 mg NaCl/ml = 32 mOsm increase / decrease D = desired ( mOsm) osmotic pressure O = observed ( mOsm) osmotic pressure X= ml of stock NaCl to be added or omitted to obtain optimum osmotic pressure( provided that 1 ml of stock changes the concentartion of NaCl in the medium by 1mg/ml)
Viscosity The viscosity of the medium is mainly influenced by its serum content, and usually has little effect on cell growth Viscosity is important when the cell suspension is agitated, e.g. in reactor Damage to cells in large scale cultures can be reduced by adding carboxymethyl cellulose , polyvinyl pyrollodine to increase viscosity These are specially important when low levels of serum are used for culturing cells in scaled up stirred reactors
Dynamic surface tension : surface tension of the medium at dynamic situation, i.e, during bubbling with some additives at different bubbling rate. Static surface tension is the surface tension of the media at static condition().
Temperature
The optimal temperature is dependent on body temperature of the animal from which the cells were obtained - Mammals 370 C -Birds 38.50 C - Fish 220 C
Temperature also effects the pH of the medium due to increased solubility of CO2 at lower temp. The pKa of the buffer and the degree of ionization of serum components is also affected by the temp. The pH of of a typical medium at room temp. is around 0.2 units lower than at 370 C.
Fetal Bovine serum ( FBS) Most often used type of serum Each serum batch is produced from over 1000 fetuses to account for gestational stage, gender and biochemical composition Potential source of adventitious agents ( mycoplasma and viruses) Very sensitive to degradation, adsorption and accidental contamination Other type of serum - newborn calf serum or horse serum
Serum/Protein free media are defined and contain Transferrin/ ferrous citrate Lipid concentrate Yeast extract Insulin Bovine serum Albumin Pluronic acid
Serum Free Medium
Low serum and serum free media are particularly valuable where it is vital to control the culture conditions precisely. If the up regulation of immological markers on cells is being studied, it is important to eliminate the undefined growth factors present in the serum If we want produce monoclonal antibodies using hybridoma or lymphomas to produce lymphokines and other factors, serum free media is good.
Methods for optimization of nutrient composition Serum free medium Co culture on feeder layer
Feeder cells Peritoneal exudates cells(PEC) Human dermal fibroblast Thymocytes Mouse embryo fibroblast ( 3T3)
Antibiotics: Reduce the frequency of contamination Encourage the development of antibiotic resistant strains Hide the low level cryptic contaminants Have antimetabolic effect Encourage poor aseptic technique
Can adhere to plastic and glass Most cell need to be attached to special surface to express their physiological phenotype - topography of the surface at the cellular dimension ( 1-5m) scale - negative charged group on the surface Plastic Petri dishes covered with - collagen -poly-Lysine
Majority of vertebrate cells in vitro grow as monolayer on an artificial substrate Most cells need to spread out on a substrate in order to proliferate Overcrowding will inhibit proliferation But cells do not like to be lonely Cell yield is proportional to the available surface area
Contaminants in cell culture: Bacteria ( Pseudomonas sp, Staphylococcus, E. coli) Fungi / yeast ( Aspergillus, Candida, Penicillium ) Mycoplasma ( M. orale, M. fementaris, M. argini, Alcholesplasma leidlavii, M,.hyorhinis ) Culture may become infected with mycoplasma from media, sera, trypsin or the opearator. Detection of Mycoplasma Fluorescent dye : Mycolplasma DNA binds to fluorescent dye ( Hoechst 33258). Under the microscope, cells stained with this dye have fluorescent nuclei. Mycotest : In this test cells are incubated with 6-methyl purine deoxyriboside. This is non toxic to mammalian cells, but mycoplasma cells metabolize this deoxyriboside to form either 6-methyl purine or 6-methyl purine riboside. These are toxic to mammalian cells. Therefore, mammalian cell harboring mycoplasma are killed. Gene Probe : This consist of 3H-labeled DNA probe which will hybridize to mycoplasma DNA but not to mammalian DNA. Hybridized and free 3H-labeled DNA probe are easily separated electrophoretically
Short questions :
Which of the following statements are true or false ( T / F )
a) b) c) d) e) f)
It should be anticipated that normal ( non malignant) epithelial cells when cultured in vitro will grow attached to the surface of the cultured vessel . ( T / F ) Non-malignant human cells will grow indefinitely in vitro and are said to be immortal. ( T / F ) Cells derived from embryos are relatively easy to cultivate in vitro whereas cells derived from adults are often difficult or impossible to culture. ( T / F ) Mature cells derived from haematopoietic stem cells show little or no anchorage-dependence. ( T / F ) When cultivating cells derived from tumours, it is essential to use vessels with large surface area to which the cells may adhere. ( T / F ) The ability to grow animal cells in culture shows that the extracellualr matrix found in natural tissues simply provides a cementing material that binds cell together. ( T / F )