Cell Culture media

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

Broadly cell culture media could be classified into four groups

Media designed to achieve immediate, short term survival of cells [Immediate survival media should provide a source of energy and correct osmolarity and this is achieved by a defined media of balanced salt solution( BSS) containing glucose] Media designed to achieve prolonged survival of cells [For prolonged survival and growth more complex with variety of factors are needed] Media for long term growth of the cell

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)

The Most Common Types of Media

Medium 199 MEM CMRL 1066 DMEM Hams F-12 RPMI 1640 Morgan et.al,1950 Eagle 1959 Parker et.al,1957 Dulbecco 1959 Ham 1965 Moore et. Al, 1967

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

SERUM AVAILABILITY in MARKET

a) Whole serum : b)Serum ultra filtered : Ultra filtration removes large molecules( protein bound cellular growth factors) from the serum and some of these macromolecules are essential for cellular growth. So the ultrafiltered serum does not support growth and main use of this type serum is to provide a supplementary source of nutrients. c) Dialysed serum : Dialysed serum is used in semi synthetic media in which low M.W.components are defined and in this case serum provide essential large molecules.

The role of cell culture media

Provide essential nutrients: amino acids, fatty acids, trace elements, salts, vitamins and cofactors Maintain proper chemical Environment: pH(7.4) and osmolarity(290-300 mOsm/litre) mostly through ions and bicarbonates Provide energy ( carbon) source

The choice of culture medium and supplements can have a major impact on growth, function and phenotype of cells in vitro.

Components of cell culture media

Components Water Balanced Salt Solution Energy Sources Glutamine Amino Acids Vitamins Serum Advanced Additives Issues Source; Purification; Seasonal Variation Osmotic & Ionic Equilibrium; Metal Ions; Buffer Sugars; Biosynthesis; Lactate Buildup ATP Production; NH3 Production; Biosynthesis Biosynthesis; Chelator; Buffer; Energy Source Metabolic Functions; Lipid Synthesis; e- Transport Cell Attachment; Growth; Buffer; Toxin Inactivation Serum Replacement; Lipids; Attachment Proteins

Balance Salt solution ( BSS)

These are simple synthetic media which are designed to maintain pH and osmotic pressure as well as provide adequate concentration of essential inorganic ions. There are different formulations and some are as follows. Substance Ringer Tyrode Simms Earle Hanks Dulbeco
g/l NaCl KCl CaCl2 9.00 0.42 g/l 8.00 0.20 g/l 8.00 0.20 g/l 6.80 0.40 g/l 8.00 0.40 g/l 8.00 0.20

0.25

0.20

0.14

0.20 0.10

0.14 0.10 0.10 0.06 0.06 0.06

0.10

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

0.10

0.20 0.20 0.20

1.00 0.05 2.20 5% CO2 in air

1.00 0.02 0.35 air air

Balanced Salt Solution ( BSS)

Choice of BSS to make a defined media depends on The type of atmosphere in which cells are grown( BSS is used as diluents for Essential Medium which is used for equilibration with CO2) Whether medium is to be used for disaggregating or monolayer dispersal. For this application medium should be free from Ca+2 and Mg+2 ions, Dulbeccos phosphate buffered solution is suitable for this purpose Whether medium is used for the growth of adherent cell or suspension culture. Reducing the Ca+2 ion in the medium helps to reduce cell aggregation and attachment.

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

Role amino acid in the cell culture media

The concentration of amino acids influences cell yield and also affects the survival and growth rate. Differentiation may lead to reduction in cell capacity to synthesize amino acids Failure to synthesize a particular amino acid in vitro may represent the lack of correct precursor or cofactor in artificial environment ( Monkey kidney cells have reduced ability to convert folic acid to folinic acid and the later is required as cofactor for the conversion of Ser to Gly) Monolayer cells divide at a very active pace, thus insufficient amounts can be synthesized Amino acid deficiency inhibit cell division, induces chromosomal damage, increases lysosomal activity and cell death Imbalance of amino acids may also produce karyotype change. The most rapid amino acid use occurs at lag phase of growth cycle and Cys, Glu, Ile, Ser are used very quickly.
Non essential AA: Ala,Asn,Asp.Glu,Gly,Pro,Ser Essential AA: Arg,Lys,His,Iso,Leu,Cys,Meth,Phe,Thr,Trp,Tyr,Val,Gln

Glutamine is also used as source of N as well as C source.

Support cell growth and amino acid uptake Support glucose utilization A source of energy Support protein turn over It is most unstable essential amino acid and decomposes in a time and temperature dependent manner , at 370C half life-8 days; at 40C half life-40 days Gln decomposes to pyrrolidin carboxylic acid ( C4H8NCOOH) and NH3 in solution
N-COOH

* Glutamine must be added added at last and replenished regularly

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

Physicochemical properties of mammalian cell culture media

pH Osmolarity Viscosity Surface tension and foaming Temperature

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

Other buffering systems:

Culture media must be buffered under two conditions - open dishes when pH rises - over production of CO2 and lactic acid at high cell concentration - HEPES in addition to bicarbonate / CO2 buffer Phospahte buffer saline ( Dulbeco medium or PBS) is widely used by virologist Tris-citrate buffer also used for the growth of HeLa, BHK21 cells Other buffers used in cell culture having pKa value around pH 6.8-8.0 MOPS ( pKa 7.2), TES ( pKa 7.6) HEPES ( pKa 7.6), DIPSO 9 pKa 7.6)
HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) DIPSO: 3-[bis(2-hydroxyethyl)amino]-2-hydroxypropane-1-sulfonic acid MOPS: 3-(N-morpholino)propanesulfonic acid

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

Surface tension ( static and dynamic) and foaming

Lowering the surface tension in the medium can be used to promote adherence of cells to the substratum when these are required Foaming occurs especially in medium containing serum and can cause the denaturation of proteins and inactivation of enzymes. If foam reaches the neck of the culture vessel, microbial contamination becomes a problem Silicone antifoaming agents are used in suspension cultures where CO 2/air mixtures are bubbled through the medium

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.

Protective agents used in cell culture

These agents protect cells from damage by shear, osmotic pressure, toxic metals and oxidative injury Methyl cellulose Polyvinyl pyrolidine Dextran Pluronic F-68 Polyethylene glycol Antioxidant

Oxygen level in the medium

Oxygen consumption of the cells varies from 0.05-0.47 m mol/l/hr for 106 cell/ml Cells in culture rely mostly on Glycolysis Rely on dissolved oxygen Too much oxygen leads to the induction of free radicals In lab scale cell culture in general oxygen depletion does not occur, but large scale cultivation oxygen has to be supplied in different ways by which cell will not be damaged but enough oxygen will be supplied.

Some times reducing agent ( -mercaptoethanol) is used

-mercaptoethanol form of glutathione stimulate cysteine uptake forming mixed -S-Shelp to prevent peroxide damage by restoring the reduced

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.

Advantage of serum free or low serum medium

Reproducibility between the cultures is improved The risk of introducing contaminants ( bacteria,viruses, mycoplasma) is reduced Serum cytotoxicity and protein interference is reduced Serum-free medium can be less expensive Culture products are easier to purify Fibroblast overgrowth in primary culture is prevented ( serum promotes fibroblast growth in wound healing)

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)

Used for Hybridoma Epithelial cells Hybridoma Several cell type

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

*In general penicillin + Streptomycin are used 0.5mg/ml

Some cells require special surface :

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

Choice of cell culture container:

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

Alternative or specialized ways to cell culture

Tissue culture on feeder layer

Three dimensional matrices Microcarriers ( for anchorage dependent cells in suspension)

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 )