This document provides information on different types of cell culture media. It discusses natural media including biological fluids and tissue extracts. It also discusses artificial/synthetic media including balanced salt solutions, basal media, and complex media. Specific components of media are outlined including amino acids, vitamins, inorganic salts, glucose, serum, proteins, growth factors, hormones, and antibiotics. Different types of specialized media are also mentioned such as serum-free, chemically defined, conditioned, and protein-free media. Criteria for selecting the appropriate media for specific cell lines is discussed.

1. Assignment
VBT-604 ANIMAL CELL CULTURE: PRINCIPLES &APPLICATIONS
TOPIC; Different type of cell culture media
PRESENTED BY
Dr.Rajashekar Baldhu
M.V.SC 1st year
Hyderabad Veterinary College

2. 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

3. Media Type Examples
Natural media
Biological Fluids
plasma, 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

4. 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.

5. 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

6.  Embryo Extract
 High-molecular-weight fraction
 promoted pigment and cartilage cell differentiation.
 It should always be frozen and thawed at least twice to ensure that
there is no carryover of live cells from the embryo.

7. 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.

8. 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

9.  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.

10. 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.

11. Other growth factors
fibroblast growth factors (FGFs)
epidermal growth factor (EGF)
Endothelial cell growth factors such as vascular
endothelial growth factor (VEGF) angiogenin

12. 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.

14. 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 cells
Testing 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 media
Increase the risk of contamination
Functions as a binding protein
Presence of serum in media may interfere with the
purification and isolation of cell culture products
Minimizes mechanical damages or damages caused by
viscosity

15. 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.

16. 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.

17. 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

18. 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.

19. 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 systems
Regulating pH is critical for optimum culture conditions and is generally achieved by one of the
two buffering systems:

20. 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

21. HEPES
4-(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

22. 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 concentration
attainable, and the balance may influence cell survival and growth rate.

23.  Glutamine is required by most cells, although some cell lines will utilize
glutamate;
 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 5
days, and it generates ammonia, which can be toxic as a by-product.
 Glutamax (Invitrogen) is an alanyl-glutamine dipeptide that is stable an
bioavailable due to the action of dipeptidase
Glutamine

24.  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;

25.  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

26.  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.

27. 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.

28. 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

29. 3)Fibronectin.
 Fibronectin -in cell attachment.
4)Transferrin
Fatty Acids and Lipids
They are particularly important in serum-free media

30.  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.

31.  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

32.  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.

34.  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

35. 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.

36. 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.

38. 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;

39. 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

41.  Presence of serum in the media lead to mis
interpretations 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.

42. Definition of Standard Medium
Given 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

43. 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.

44.  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.

45. 1.MCDB 131 (Sigma) for endothelial cells
2.LHC-9 (Invitrogen) for bronchial epithelium
 Serum Substitutes
A number of products have been developed commercially
to replace all or part of the serum in conventional media

47.  Iron, copper, and a number of minerals have been included
in serum-free recipes, although evidence that some of
the 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 lipid
precursors such as choline, linoleic acid, ethanolamine, or
phosphoethanolamine.

48.  Hormones that have been used to replace serum include
growth hormone (somatotropin) at 50 ng/mL, insulin at 1 to
10 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

49.  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,

50.  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.

51.  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

53. THANK YOU