Cell culture media

Introduction

• Animal cell culture

– in vitro maintenance and propagation of animal cells in a suitable

nutrient media.

• Selection of an appropriate growth medium for the in vitro cultivation of

cells is the important and essential step.

• Choice of the medium mostly depends on the type of the cells to be

cultured, and the purpose of the culture (growth, differentiation, production

of desired products).

• Animal cells need either a completely natural medium, or an artificial medium supplemented with some natural products.

Natural media Artificial media

Culture Media

Natural media

• In the early years, natural media obtained from various biological sources were

used.

Body fluid: plasma, serum, lymph, amniotic fluid, etc

These fluids are tested for sterility and toxicity before their utility.

Tissue extract:

• Tissue extract chick embryo extract was most commonly employed.

• The extract of liver, spleen, bone marrow and leucocytes also used as culture

media.

• Plasma clots have been in use for a long time and are now available commercially.

• Bovine embryo extract prepared from bovine embryos of different age ( up to 10

days).

Artificial media• The artificial media containing partly defined components.

The minimal criteria needed for choosing a media for animal cell culture

are listed below:

• The medium should provide all the nutrients to the cell.

• Maintain the physiological pH around 7 with adequate buffering.

• The medium must be sterile, and isotonic to the cells.

The basis for the cell culture media was the balanced salt solution - used

to create a physiological pH and osmolarity required to maintain the cell

in vitro.

• For promoting growth and proliferation of cells, various constituents were added and several media developed. Eg: serum containing media and serum-free media.

Artifical medium

Basic components

• First of all, media have to maintain osmotic pressure (isotonic salt solution)

and pH.

• The latter is checked via an indicator like phenol red.

– Acidic medium is yellow – either too long on the cells or microbial contamination.

– Alkaline medium is purple – too long outside the incubator.

– Medium at appropriate pH of ~ 7.4 is red

• The balanced salt solution contains Na+ + Cl-, but also Mg++, Ca++, K+

and trace metals like Se, Zn, Cr.

• The standard buffer is comprised of the system NaHCO3/ H2 CO3 like in

the body, sometimes assisted by other buffers like HEPES or phosphate.

• Basal medium is completed with vitamins (B-vitamins, cofactors) and

actual nutrition substances, i.e. glucose (low concentration = 1 g/l) as

carbon source and essential amino acids. These components form a

Minimal Essential Medium (MEM) like Eagles´.

• ‘Rich’ media contain extra goodies like non-essential amino acids and

sometimes high glucose (4,5 g/l), examples are e.g. Dulbecco’s

Modification of Eagle’s Medium (DMEM); RPMI, Ham’s F12, or 199.

Basic components

HEPES buffer

• HEPES and other organic buffers can be used with many cell lines to

effectively buffer the pH of the medium.

• Indeed, some standard medium formulations include HEPES.

• However, this compound can be toxic, especially for some

differentiated cell types, so evaluate its effects before use.

• HEPES has been shown to greatly increase the sensitivity of media to

the photo -toxic effects induced by exposure to fluorescent light.

L-Glutamine

• Essential amino acid required by virtually all mammalian and insect cells grown in

culture. It is used for protein production, as an energy source, and in nucleic acid

metabolism.

• It is also more labile in liquid cell culture media than other amino acids.

• The rate and extent of L-glutamine degradation are related to storage temperatures, age

of the product, and pH.

• Because L-glutamine is so labile, it is often omitted from commercial liquid medium

preparations to lengthen the product shelf life.

• In these cases, it must be aseptically added prior to use. L-Glutamine is not as labile in

dry form and most powdered medium formulations do include it

Nonessential amino acids

• All medium formulations contain the ten essential amino acids as well as

cysteine, glutamine, and tyrosine.

• The inclusion of the other non-essential amino acids (alanine, asparagine,

aspartic acid, glycine, glutamic acid, proline, and serine) in some media

formulations reduces the metabolic burden on the cells allowing for an

increase in cellular proliferation.

Sodium pyruvate• Pyruvate is an intermediary organic acid metabolite in glycolysis and the first

component of the Embden-Meyerhof pathway.

• It can pass readily into or out of the cell. Its addition to tissue culture medium provides

both an energy source and a carbon skeleton for anabolic processes.

• Pyruvate may help in maintaining certain specialized cells, in clonal selection, in

reducing the serum concentration of the medium, and in reducing fluorescent light-

induced phototoxicity.

• Cellular metabolism of pyruvate produces carbon dioxide which is given off into the

atmosphere and becomes bicarbonate in the medium.

• Sodium pyruvate is added to give a final concentration of 1 mM in most media, but is

increased to 5 mM in Leibovitz’s L-15 medium primarily to facilitate use in CO2-free

environments.

Media Supplements

• The complete growth media recommended for some cell lines

requires the addition of components not already available in

the base media and serum.

• These components include hormones, growth factors and

signaling substances that sustain proliferation and maintain

normal cell metabolism.

Supplements

• serum: mainly fetal calf serum ; contains among other proteins albumin

(70 g/l), globulins, fibronectin and vitronectin, protease inhibitors,

transferrin (as iron source), growth factors and hormones (insulin), lipids

(LDL, cholesterol), also trace elements

• Additional cell specific growth factors like VEGF (vascular endothelial growth

factor), FGF (fibroblast growth factor), IGF (insulin-like growth factor), ILs

(interleukins - lymphocyte growth factor), CSF, CSFs (colony stimulating

factors), EGF (epidermal growth factor); hormones (glucocorticoid, estrogen,

erythropoietin …); etc.

Supplements

• Glutamine (replace or add fresh; in solution only stable for 2

weeks)

• Penicillin / streptomycin (professionals sometimes work

without; antibiotics inhibit bacterial growth; they are not anti-

fungal, the most frequent infection; against fungi one can use

amphotericin b, which is rather cytotoxic)

• Special serum free media contain at least adhesion factors,

transferrin, insulin, growth factors, nutrients, and lipids

Antibiotics and Antimycotics• Antibiotics and/or antimycotic agents are added to cell culture media as a

prophylactic to prevent contamination, as a cure once contamination is

found, to induce the expression of recombinant proteins, or to maintain

selective pressure on transfected cells.

• Routine use of antibiotics or antimycotics for cell culture is not

recommended unless they are specifically required, such as G418 for

maintaining selective pressure on transfected cells.

• Antibiotics can mask contamination by mycoplasma and

resistant bacteria.

• Further, they can interfere with the metabolism of sensitive

cells. Avoid antimycotics as they can be toxic to many cell

lines.

Antibiotics and Antimycotics

SERUM MEDIA

• Although natural media are very useful and convenient for a

wide range of cases, they suffer from the disadvantages of poor

reproductability due to lack of knowledge of exact composition.

• Reasons 4 using synthetic media:

*Media for Immediate survival.

*Media for Prolonged survival.

*Media for Indefinite growth.

*Media for Specialized functions.

• For the immediate survival, a “balanced diet” solution with defined osmotic

pressure and pH is added. {Combination of inorganic salts and glucose}

• For long survival, serum may be used or the balanced salt solution may be

supplemented with amino acids, O2, vitamins, and serum proteins

• MEM-Minimum Essential Medium [Eagles 1955] -used for mammalian

cell culture

• Role of serum in culture is extremely complex and contains mixture of

many large and small biomolecules {growth promoting and growth

inhibitory factors}

serum

• serve as a source for amino acids, proteins, vitamins(particularly fat-soluble

vitamins suc h as A, D, E, and K),carbohydrates, lipids, hormones, growth

factors, minerals, and trace elements.

• Additionally, serum buffer s the culture medium,inactivates proteolytic

enzymes, increases medium viscosity (which reduces shear stress during

pipetting or stir ring), and conditions the growth surface of the culture vessel.

• The exactcomposition is unknown and varies from lot to lot, although lot-to-

lot consistency has improved in recent year s

Serum• Sera from fetal and calf bovine sources are commonly used to support the

growth of cells in culture. • Fetal serum is a rich source of growth factor s and is appropriate for cell

cloning and for the growth of fastidious cells. • Calf serum, because of its lower growth-promoting proper ties, is used in

contact-inhibition studies with NIH/3T3 cells (ATCC®CRL-1658™). • Horse serum is collected from a closed herd of adult animals ensuring lot-

to-lot consistency. • Horse serum is less likely to car r y the contaminants found in bovine sera

such as viruses and less likely to metabolise polyamines which maybe mitogenic for some cells.

• Horse and bovine calf sera are less expensive and more readily available than fetal bovine ser um.

• The pricing and availability of fetal ser um fluctuates considerably.

Disadvantage:

• Serum may contain inadequate levels of cell-specific GF

which have been supplemented and an over abundance of

others which may be cytotoxic.

• Risk of contamination with virus fungi and mycoplasma

SERUM FREE MEDIA

• Due to some disadvantages associated with the use of serum in media ,Serum Free media have been developed

Disadvantages of Serum in MediaVariable Composition – there is no uniformity in the composition

of serum (highly variable)

Quality Control – to maintain uniform quality of the serum special testes need to be performed with each batch of serum before its use

Contamination – very difficult to get serum from virus contamination

Presence of Growth Inhibitors - in some cases growth inhibitors like TGF- b may dominate and inhibits cell proliferation

Availability and cost - availability is restricted due to its dependence on cattle

Down stream processing - presence of serum in the culture medium interferes with the isolation and purification of cell culture products. For this reason additional steps are needed for the isolation

Serum Free Media :

Advantage and Disadvantage of Serum Free Media

• ADVANTAGE :

*Selection of media with defined composition- Main advantage of serum free media is to control growth of

the cells as desired with a well defined medium.

*Regulation of differentiation – It is possible to use a set of factors to achieve differentiation

of cells with the desired and specialized functions

• DISADVANTAGE:

*Slow cell proliferation

*Need for Multiple media

*Purity of reagents

*Availability and cost:

[Costly than serum media as they use pure chemicals which are expensive ]

GROWTH FACTORS

A large number of Growth Factors that promote in vitro proliferation and differentiation have been developed.

They act synergistically or additively with each other or with other factors

HORMONES : Growth hormones –insulin and hydrocortisone are the most commonly added in to the serum free medium

A combination of steroid hormones hydrocortisone, estrogen and androgen are used for maintaining mammary epithelium

NUTRIENTS : cholin, ethanol amine , linolic acid, iron copper, selenium etc., are added

• PROTEINS : Bovine serum albumin [BSA] is the most commonly added protein. It promote cell survival and growth

• POLYAMINES : promote cellular growth and differentiation

• PROTEASE INHIBITORS : for trypsin mediated subculture

Growth Factors With Serum containing Media :

• Platelet-derived GF [PDGF]

• Fibroblast GF [FGF]

• Epidermal GF [EGF]

• Vascular Endothelial GF [VEGF]

• Insulin GF [IGF-1, IGF-2]

Physio-Chemical Properties of Culture Media:

The culture media is expected to possess certain physiochemical properties ( pH, Oxygen, carbon dioxide, buffering, osmolality, viscosity, temp., etc…) to support good growth and proliferation of cultured cells.

pH: Normally most cells can grow at pH in the range 7-7.4,

although there are slight variations depending on the type of cells.

The indicator phenol red is commonly used for visible detection of pH in the media.

Carbon dioxide, Bicarbonate and Buffering:

Carbon dioxide in the medium is in a dissolved state and the concentration depends on the atmospheric carbon dioxide tension and temperature

Carbon dioxide in the medium exists as carbonic acid and HCO3 and H+ ions.

CO2 + H2O ———› H2CO3 ‹—————›H+ + HCO3ˉ

Concentration of carbon dioxide, bicarbonate and pH are interrelated.

By increasing the atmospheric carbon dioxide, the pH will be reduced making the medium acidic.

• Addition of sodium bicarbonate ( as the component of bicarbonate buffer) neutralize bicarbonate ions.

• The presence of pyruvate in the medium results in the increased endogenous production of carbon dioxide by the cells.

• This is advantageous since the dependence of oxogenous supply of carbon dioxide and bicarbonate will be less. In such case, the buffering can achieved by high concentration.

Oxygen: The scale up of animal cell culture is very dependent up on the

ability to supply sufficient oxygen without causing cell damage.

Oxygen is only sparingly soluble in culture media.

Cultured cells mostly rely on the dissolved O2 in the medium which may be toxic at high concentration due to the generation of free radicals.

adequate quantities of O2 must be supplied so that the cellular requirements are met avoiding toxic affects.

• Free radicals scavengers {glutathione} are sometimes added to nullify the toxicity.

• Addition of selenium to the medium is also advocated to reduce O2 toxicity. [selenium - cofactor for synthesis of glutathione]

Temperature: Animal cells are sensitive to temperature higher than 37c [Culture from birds- 38.5c, Cool blooded animals - 15-25c}

Low gradient heating systems mostly based on the water jacked principle and with a high surface area are favorable.

In a compact loop bioreactor this can be achieved by using a thermo stated draught tube.

Besides directly influencing growth of cells, temperature also affects the solubility of O2 [Higher temperature enhances solubility ]

Osmolality: Osmolality for most of the cultured cells is in the range of

260-320mosm/kg,comparable to the osmolality of human plasma (290mosm/kg)

Once an osmolality is selected for a medium it must be maintained at that level [+/-10mosm/kg]

When ever there is an addition of acids, bases ,drugs etc .to the medium osmolality alters and is measured using Osmometer

Point Osmometer: