MEDIA

Basic constituents of media

Inorganic salts

Carbohydrates
Amino acids

Vitamins
Fatty acids and lipids Proteins and peptides

Mammalian cell culture media

Salts: K+, Na+, Cl-, Mg2+, Ca2+

Provide osmotic balance

Help regulate membrane potential of the cells Some are required as cofactors for cell attachment factors Carbohydrates Mostly glucose and galactose but also maltose or fructose

Trace elements: Se2+, Zn2+, Cr2+

Selenium is a detoxifier and removes oxygen free radicals

Mammalian cell culture media

Nine essential Amino Acids
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine Most cell require also cysteine and tyrosine

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

Antioxidants and differentiation agents

Other constituents
Some nonessential molecules (Pyruvate, Hypoxanthine thymidine, adenosine) to improve growth Fatty acids Cholesterol (usually not included in the mixture of Detergent to emulsify lipids Toxic to some cells Hormones and growth factors fatty acids)

Specific factors for growth and differentiation

Phenol red as pH indicator

Development of media
Initial attempt to culture cells were performed in natural media based on tissue extracts and body fluids Chemically defined media were introduced in the 1950s

Eagles Basal Medium

Eagles Minimal Essential Medium (MEM) Dulbeccos modification of MEM (DMEM) Supplemented with serum, protein hydrolysates or embryo extracts

Optimization of media
Practically all cells can grow in Minimum Essential Media Optimization goes in the direction of replacing serum or more selective media appropriate for a particular cell type

RPMI 1640 for lymphoblastoid cell lines

Hams F-12 with higher content of vitamin and amino acids

Leibovitz L-15 for cells grown without CO2

Physicochemical properties of mammalian cell culture media

pH Osmolarity Surface tension and foaming

pH
pH 7.4 to pH 7.7 Phenol red is commonly used as an indicator Red at 7.4

Orange at 7.0
Yellow at 6.5 Purple at 7.8 Most often maintained by CO2/bicarbonate buffer

Osmolality
The total osmolality of aqueous solutions is determined by comparative measurements of the freezing points of pure water and of solutions. While water has a freezing point of 0 C, a solution with saline concentration of 1 Osmol/l has a freezing point of -1.858 C.

Osmol is a standard unit of osmotic pressure based on a one molal concentration of an ion in a solution

Osmotic pressure: The pressure that is produced by the

CO2 and bicarbonate

Most commonly used method to stabilize pH

natural buffer, low cost, non-toxic

H2O + CO2H+ +HCO3 Cells 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 buffers
Culture media must be buffered under two conditions

Open dishes when pH rises

Overproduction of CO2 and lactic acid at high cell concentration

HEPES in addition to bicarbonate/CO2 buffer

To stabilize pH when cells are out of the incubator Serum also has considerable buffering capacity

ENDOTOXIN
A complex lipopolysaccharide (LPS) which is a major component of the outer membrane of most gram-negative bacteria. A single Escherichia coli contains about 2 million LPS molecules per cell. Bacteria shed endotoxin into their environment in small amounts when they are actively growing, and in large amounts when they die.

Potential Sources of Endotoxin in Cell Culture

Water used for glassware washing or making media and solutions; Commercially obtained media and sera;

Media components and additives;

Laboratory glassware and plasticware

Depending on cell type and culture conditions, endotoxin can have a variety of effects on cell growth and function

What is serum?
Fluid that is left when blood clots The cells are enmeshed in fibrin and the clot retracts because of the contraction of platelets. It differs from plasma in having lost various proteins involved in clot formation (fibrinogen, prothrombin, various blood-clotting factors such as Hagemann factor, Factor VIII etc.) In addition it contains various platelet-released factors, notably platelet-derived growth factor.

Serum
Most widely used additive to cell culture medium
Source of identified and unidentified growth factors, metabolites, hormones etc. promotes cell proliferation promotes cell attachment source of minerals, lipids and hormones eg Calf serum and Fetal Bovine serum Least defined and most variable component of tissue culture Variable from lot to lot Contaminants or degradative enzymes

Infectious agents (viruses, prions)

Expensive Increases buffering capacity

Able to bind and neutralize toxinase

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, breed 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

Characteristics of bovine serum

pH 6.85 to 7.05

Osmolarity (mOsm/l)
Haemoglobin content (ug / ml) Protein content (mg / ml) Albumin content (mg / ml) Endotoxin content (ng / ml)

250- 295
< 120 60 80 30 50 < 15

Special treatment to serum

Gamma irradiation source Exposure to radiation from a 60 0 C

Heat inactivation
Gamma globulin removal Dialysis

Incubation at 560C for 30 min

Involves ethanol fractionation usually against 0.15 M NaCl until the level of glucose < 5 mg%. This reduces the low molecular weight components of the serum

Defined Fetal Bovine Serum

The highest quality FBS available Widely used by cell culturists who have a concern for viral contaminants and require an extensive biochemical profile

Filtered through serial 40 nm (0.04 m) pore-size rated filters, the most retentive filters used in commercial FBS production
Over 50 components are analyzed and the results are included on the biochemical assay

Characterized Fetal Bovine Serum

Filtered through three sequential 100 nm (0.1m) pore-size rated filters

Standard Fetal Bovine Serum

Filtered through three sequential 100 nm (0.1m) pore-size rated filters Endotoxin and hemoglobin are tested and reported

Antibiotics
Amphotericin B Ampicillin Gentamycin sulphate Kanamycin sulphate Nystatin 2.5 mg /l Fungi and yeasts 100 mg / l 50 mg / l G+ and G- and mycoplasma 100 mg/l G+ and G- and mycoplasma 50 mg / l Fungi and yeasts G+ and G- bacteria

Penicillin G
Polymyxin B sulphate Streptomycin sulphate Tylosin

100 U / ml
100 U / ml 100 mg / l 100 mg / l

G+ bacteria

G- bacteria G+ and G- bacteria G+ and mycoplasma

Serum/protein free media

are more defined and contain Transferrin/ferrous citrate Lipid concentrate Yeast extract Insulin Bovine Serum Albumin

Completely mammalian origin free (MOF)