AssignmentVBT-604 ANIMAL CELL CULTURE: PRINCIPLES &APPLICATIONS

TOPIC; Different type of cell culture media

PRESENTED BYDr.Rajashekar BaldhuM.V.SC 1st year Hyderabad Veterinary College

Cell culture media

Cell culture media generally comprise an appropriate source of energy and compounds which regulate the cell cycle.

A typical culture medium is composed

Amino acids

vitamins

Inorganic salts

Glucose and serum

Media Type Examples

Natural media

Biological Fluidsplasma, serum, lymph, human placental cord

serum, amniotic fluid

Tissue Extracts

Extract of liver, spleen, tumors, leucocytes and

bone marrow, extract of bovine embryo and

chick embryo

Clots plasma clots

Artificial media

Balanced salt solutions PBS, DPBS, HBSS, EBSS

Basal media MEM DMEM

Complex media RPMI-1640, IMDM

Natural Media

Natural media consist solely of naturally occurring biological fluids.

Natural media are very useful and convenient for a wide range of animal cell culture.

The major disadvantage is

poor reproducibility due to lack of knowledge of the exact composition of these natural media.

Embryo Extract Embryo extract is a crude homogenate of 10-day-old chick embryo

that is clarified by centrifugation.

The crude extract was fractionated into

Low-molecular-weight fraction

promoted cell proliferation

may contain peptide growth factors

Embryo Extract

High-molecular-weight fraction

promoted pigment and cartilage cell differentiation.

It should always be frozen and thawed at least twice to ensure thatthere is no carryover of live cells from the embryo.

SERUM

Serum contains

Growth factors

Minerals

lipids

Hormones

many of which may be bound to protein.

The sera used most in tissue culture are bovine calf, fetal bovine, adult horse, and

human serum.

Calf (CS) and fetal bovine (FBS) serum are the most widely used, the latter

particularly for more demanding cell lines and for cloning.

PROTEINS

proteins are required as carriers for minerals, fatty

acids, and hormones.

Albumin as a carrier of lipids, minerals, and globulins.

Fibronectin (cold-insoluble globulin), which promotes cell

attachment

α2-macroglobulin, which inhibits trypsin.

Fetuin in fetal serum enhances cell attachment.

Transferrin binds iron, making it less toxic and

bioavailable.

Protein also increases the viscosity of the medium,

reducing shear stress during pipetting and stirring.

Growth Factors

Natural clot serum stimulates cell proliferation more than

serum from which the cells have been removed physically

This increased stimulation due growth factors.

PDGF - platelet derived growth factor, from the platelets

during clotting.

PDGF is one of a family of polypeptides with mitogenic

activity and is probably the major growth factor in serum.

PDGF stimulates growth in fibroblasts and glia.

Other growth factors

fibroblast growth factors (FGFs)

epidermal growth factor (EGF)

Endothelial cell growth factors such as vascular endothelial growth factor (VEGF) angiogenin

Hormones

Insulin promotes the uptake of glucose and amino acids and may owe its mitogenic effect to this property or to activity via the IGF-1 receptor.

IGF-I and IGF-II bind to the insulin receptor but also have their own specific receptors, to which insulin may bind with lower affinity.

IGF-II also stimulates glucose uptake Growth hormone may be present in serum—particularly fetal serum—and, in conjunction with the somatomedins (IGFs), may have a mitogenic effect.

Advantages of serum in media Disadvantages of serum in media

Serum contains various growth factors and hormones

which stimulates cell growth and functions.Lack of uniformity in the composition of serum

Helps in attachment of the cellsTesting needs to be done to maintain the quality of each

batch before using

Acts as a spreading factor May contain some of the growth inhibiting factors

Acts as a buffering agent which helps in maintaining the

pH of the culture mediaIncrease the risk of contamination

Functions as a binding proteinPresence of serum in media may interfere with the

purification and isolation of cell culture products

Minimizes mechanical damages or damages caused by

viscosity

Artificial Media

Artificial or synthetic media are prepared by adding nutrients (both organic and inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates, cofactors.

Different artificial media have been devised to serve one or more of the following purposes:

1) immediate survival (a balanced salt solution, with specific pH and osmotic pressure);

2) prolonged survival (a balanced salt solution supplemented with various formulation of organic compounds and/or serum);

3) indefinite growth;

4) specialized functions.

Chemically defined media

These media contain contamination-free ultra pure inorganic and

organic ingredients, and may also contain pure protein additives,

like growth factors.

Their constituents are produced in bacteria or yeast by genetic

engineering with the addition of vitamins, cholesterol, specific amino

acids, and fatty acids.

Conditioned media

conditioning medium adds undefined components and should be eliminated if

possible after the active constituents are Determined

Hauschka & Konigsberg showed that the conditioning of culture medium that

was necessary for the growth and differentiation of myoblasts was due to

collagen released by the feeder cells.

Using feeder layers and conditioning the medium with embryonic fibroblasts or

other cell lines remains a valuable method of culturing difficult cells

Protein free media Protein-free media do not contain any protein and only contain non-protein

constituents.

Compared to serum-supplemented media, use of protein-free media promotes

superior cell growth and protein expression and facilitates downstream

purification of any expressed product

Formulations like MEM, RPMI-1640 are protein-free and protein supplement

is provided when required.

Basic Components of Complete Media

Components vary among cell lines, and these differences are partly responsible for the extensive number of medium formulations

Each component performs a specific function:

Buffering systemsRegulating pH is critical for optimum culture conditions and is generally achieved by one of the two buffering systems:

Natural buffering system

In a natural buffering system, gaseous CO2 balances with the CO3/HCO3 content of the culture medium.

Cultures with a natural buffering system need to be maintained in an air atmosphere with 5-10% CO2, usually maintained by an CO2 incubator

Natural buffering system is low cost and non-toxic

HEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Chemical buffering using a zwitterion, HEPES, has a superior buffering capacity in the pH range 7.2-7.4 and does not require a controlled gaseous atmosphere

HEPES is relatively expensive and toxic at a higher concentration for some cell types.

HEPES has also been shown to greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light

The essential amino acids are required by cultured cells,

Plus cysteine and/or cysteine Arginine Glutamine and tyrosine

Other nonessential amino acids are often added as well

The concentration of amino acids usually limits the maximum cell concentrationattainable, and the balance may influence cell survival and growth rate.

Glutamine is required by most cells, although some cell lines will utilizeglutamate;

Glutamine is also used by cultured cells as a source of energy and carbon

Glutamine is unstable in culture medium with a half-life of between 3 and 5days, and it generates ammonia, which can be toxic as a by-product.

Glutamax (Invitrogen) is an alanyl-glutamine dipeptide that is stable anbioavailable due to the action of dipeptidase

Glutamine

Eagle’s MEM contains only the water-soluble vitamins excluding biotin

Biotin is present in most of the more complex media, including the serum free recipes.

p aminobenzoic acid (PABA) is present in M199, CMRL 1066 and RPMI 1640.

All the fat-soluble vitamins (A, D, E, and K)are present only in M199, whereas vitamin A is

present in LHC-9 and vitamin E in MCDB 110

Amino acids, vitamin requirements have been derived empirically and often relate to the

cell line originally used in their development;

The salts are chiefly those of Na+, K+, Mg2+, Ca2+, Cl−,SO42−, PO43−, and HCO3− and are the major components contributing to the osmolality of the medium.

Ca2+, are required by some cell adhesion molecules, such as the cadherins

Ca2+ also acts as an intermediary in signal transduction and the concentration of Ca2+ in the medium can influence whether cells will proliferate or differentiate

Calcium is reduced in suspension cultures in order to minimize cell aggregation and attachment

Na+, K+, and Cl− regulate membrane potential.

SO4 2−, PO4 3−, and HCO3 − have roles as anions required by the matrix and nutritional precursors for macromolecules, as well as regulators of intracellular charge.

The sodium bicarbonate concentration is determined by the concentration of CO2 in the gas phase and has a significant role in buffering capability.

Glucose is included in most media as a source of energy.

It is metabolized principally by glycolysis to form pyruvate, which may be converted to lactate or acetoacetate and may enter the citric acid cycle and be oxidized to form CO2 and water.

The most commonly used proteins are

1)Albumin, Albumin binds to water, salts, free fatty acids, hormones, and

vitamins, and transport them between tissues and cells. Also remover of toxic substance from media.

2)Aprotinin Aprotinin is a protective agent in cell culture systems, stable at

neutral and acidic pH and resistant to high temperatures and degradation by proteolytic enzymes.

Inhibit proteases such as trypsin

3)Fibronectin. Fibronectin -in cell attachment.

4)Transferrin

Fatty Acids and LipidsThey are particularly important in serum-free media

Trace elements are often supplemented to serum-free media to replace those normally found in serum.

Trace elements like copper, zinc, selenium are needed in minute amounts for proper cell growth.

These micronutrients are essential for many biological processes, e.g. the maintenance of the functionality of enzymes.

Hormones and Growth Factors Hormones and growth factors are not specified in the

formulas of most regular media, although they are frequently added to serum-free media

Antibiotics were originally introduced into culture media to reduce the frequency of

contamination.

antibiotics have a number of significant disadvantages:

(1) They encourage the development of antibiotic-resistant

organisms.

(2) They hide the presence of low-level, cryptic contaminants that can become fully

operative if the antibiotics are removed, the culture conditions change.

(3) They may hide mycoplasma infections.

(4) They have anti metabolic effects that can cross-react with mammalian cells.

Most of the commercially available culture media include phenol red as a pH indicator, which allows constant monitoring of pH During the cell growth, the medium changes colour as pH is changed due to the metabolites released by the cells.

At low pH levels, phenol red turns the medium yellow, while at higher pH levels it turns the medium purple.

Medium is bright red for pH 7.4, the optimum pH value for cell culture.

LOW PH

HIGH PH

Disadvantages of using Phenol red

1) Phenol red mimics the action of some steroid hormones, particularly estrogen .Thus

it is advisable to use media without phenol red for studies using estrogen-sensitive cells

like mammary tissue.

2) Presence of phenol red in some serum-free formulations interferes with the sodium-

potassium homeostasis.

3) Phenol red interferes with detection in flow cytometric studies.

Criteria for Selecting Media

Cell Lines

The choice of cell culture media is extremely important, and significantly affects the success of cell culture experiments.

The selection of the media depends on the

Type of cells to be cultured

The purpose of the culture

Resources available in the laboratory .

Different cell types have highly specific growth requirements, therefore, the most suitable media for each cell type must be determined experimentally

EX; MEM for adherent cells and RPMI-1640 for suspension cells.

BALANCED SALT SOLUTIONS

Balanced salt solution (BSS) is composed of inorganic salts and may include sodium bicarbonate and, in some cases glucose.

HEPES buffer (5–20 mM)may be added to these solutions if necessary and the equivalent amount of NaCl omitted to maintain the correct osmolality.

BSS forms the basis of many complete media

commercial suppliers will provide Eagle’s MEM with Hanks’s salts or Eagle’s MEM with Earle’s salts, indicating which BSS formulation was used;

BSS USES

BSS is also used as a diluent for concentrates of amino acids and vitamins to make complete media

As an isotonic wash or dissection medium, and for short incubations up to about 4 h (usually with glucose present).

Used to maintain pH and osmatic pressure of the medium

Presence of serum in the media lead to misinterpretations in immunological studies;

serum-free media have been developed .

These media are specifically formulated to support the culture of a single cell type and incorporate defined quantities of purified components.

Definition of Standard MediumGiven pure constituents are used, a given medium formulation can be standardized regardless

of where it is used and by whom.

Selective Media One of the major advantage-control over growth promoting activity by serum-free media

This ability to make a medium selective for a particular cell type. Fibroblastic overgrowth can be inhibited (in breast and skin cultures) by using MCDB 170 and

153

Regulation of Proliferation and Differentiation

DISADVANTAGES OF SERUM-FREE MEDIA

(1) Multiplicity of media. Each cell type appears to require a different

recipe, and cultures from malignant tumors may vary in requirements from

tumor to tumor, even within one class of tumors.

(2) Reagent purity. The removal of serum also requires that the degree of

purity of reagents and water. as the removal of serum also removes the

protective, detoxifying action that some serum proteins may have.

(3) Cell proliferation. Growth is often slower in serum-free media, and

fewer generations are achieved with finite cell lines.

The essential factors in serum have been described and include

(1) adhesion factors such as fibronectin; (2) peptides, such as insulin, PDGF, and TGF-β, that regulate growth and differentiation; (3) essential nutrients, such as minerals, vitamins, fatty acids, and intermediary metabolites (4) hormones, such as insulin, hydrocortisone, estrogen, and triiodothyronine.

All these constituents regulate membrane transport, differentiation, and the constitution of the cell surface.

some of these constituents are included in the formulation of serum-free media, others are not and may require addition and optimization.

1.MCDB 131 (Sigma) for endothelial cells 2.LHC-9 (Invitrogen) for bronchial epithelium

Serum SubstitutesA number of products have been developed commercially to replace all or part of the serum in conventional media

Iron, copper, and a number of minerals have been includedin serum-free recipes, although evidence that some ofthe rarer minerals are required is still lacking.

Selenium(Na2SeO3), at around 20 nM, is found in most formulas

There appears to be some requirement for lipids or lipidprecursors such as choline, linoleic acid, ethanolamine, orphosphoethanolamine.

Hormones that have been used to replace serum includegrowth hormone (somatotropin) at 50 ng/mL, insulin at 1 to10 U/mL, which enhances plating efficiency in a number of different cell types.

Hydrocortisone, which improves the cloning efficiency of glia and fibroblasts and has been found necessary for the Maintenance of epidermal keratinocytes

Tiiodothyronine (T3) as a necessary supplement for MDCK (dog kidney) cells,

Various combinations of estrogen, androgen, or progesterone with hydrocortisone and prolactin at around 10 nM can be shown to be necessary for the maintenance of mammary Epithelium

The inclusion in medium of proteins such as bovine serum albumin (BSA), 0.5 to 10 mg/mL.

Tissue extracts include bovine pituitary extract used in conjunction with keratinocyte serum-free media, but it may be possible to replace this with defined recombinant growth factors.

BSA, fatty acid free, is used at 1 to 10 mg/mL. Transferrin, at around 5 to 300 ng/mL, is required as a carrier for iron and may

also have a mitogenic role. Recombinant transferrin is now available OR can be substituted with lactoferrin,

also recombinant,

One of the actions of serum is to increase the viscosity of the medium.

This is particularly important in stirred suspension culture as it helps to

minimize shear stress.

Carboxy methyl cellulose (CMC) at 1.2 to 30 mg/mL , poly vinyl

pyrrolidone (PVP) at 1 mg/Ml, and the non ionic surfactant Pluronic F-

68 at 1 mg/mL have all been used to minimize mechanical damage.

If the reason for using a serum-free medium is to promote the selective growth of a particular type of cell, then that reason will determine the choice of medium, such as

MCDB 153 for epidermal keratinocytes,

LHC-9 for bronchial epithelium

HITES for small-cell lung cancer

MCDB 130 for endothelium

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