INTRODUCTION HISTORY PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIA pH CO2, BICARBONATE AND BUFFERING OXYGEN TEMPERATURE OSMOLALITY BALANCED SALT SOLUTIONS CONSTITUENTS OF CULTURE MEDIA AMINO ACIDS VITAMINS SALTS GLUCOSE OTHER ORGANIC SUPPLEMENTS ANTIBIOTICS SERUM PROTEINS NUTRIENTS AND METABOLITES HORMONES AND GROWTH FACTORS LIPIDS MINERALS INHIBITORS APPLICATIONS OF CULTURE MEDIA CONCLUSION REFERENCES

1. Constituent of animal tissue culture media
and their specific application
By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. • INTRODUCTION
• HISTORY
• PHYSICOCHEMICAL PROPERTIES OF CULTURE MEDIA
• pH
• CO2, BICARBONATE AND BUFFERING
• OXYGEN
• TEMPERATURE
• OSMOLALITY
• BALANCED SALT SOLUTIONS
• CONSTITUENTS OF CULTURE MEDIA
• AMINO ACIDS
• VITAMINS
• SALTS
• GLUCOSE
• OTHER ORGANIC SUPPLEMENTS
• ANTIBIOTICS
• SERUM
• PROTEINS
• NUTRIENTS AND METABOLITES
• HORMONES AND GROWTH FACTORS
• LIPIDS
• MINERALS
• INHIBITORS
• APPLICATIONS OF CULTURE MEDIA
• CONCLUSION
• REFERENCES
15-May-20 2

3. • The nutrient media used for culture of animal
cells and tissues must be able to support their
survival as well as growth, i.e., must provide
nutritional, hormonal and stromal factors.
• Most cells require a surface or an artificial
substrate (adherent or monolayer culture)
whereas others can be grown free floating in
culture medium (suspension culture).
15-May-20 3
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

4. Year Scientists Event
1907 Ross
Granville Harrison
Frog embryo nerve fiber outgrowth in vitro.
1911 Lewis and Lewis First liquid media consisted of sea water, serum,
embryo extract, salts and peptones (Natural
media).
1950 Earle and co-
workers
Artificial media have been in use for cell culture to
produce vaccin.
1965 Ham Introduced the first serum-free medium which
was able to support the growth of some cells.
1967 Stanley Cohen’s and
Rita Levi-Montalcini
Epidermal growth factor.
1978 Sato MCDB selective, serum-free media
1980-87 - Development of many specialized cell lines.
15-May-20 4APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

5. CATEGORIES OF ANIMAL - CELL CULTURE MEDIA
CATEGOR
Y
DEFINATION TYPE EXAMPLE
Natural
media
Consisting of
natural
biological
substances, such
as plasma,
serum, and
embryo extract
Coagulant
or clots
Plasma separated from heparinized blood, serum and fibrinogen
Tissue
extracts
Extracts of chicken embryos, liver & slpleen & bone marrow extract
Biological
fluids
Plasma, serum, lymph, amniotic fluid and pleural fluid
Synthetic
media
Composed of a
basal medium
and
supplements,
such as serum,
growth factors,
and hormones
Serum‐cont
aining
media
Human, bovine, equine, or other serum is used as a supplement
Serum‐free
media
Crude protein fractions, such as bovine serum albumin or α‐ or
β‐globulin, are used as supplements
Xeno‐free
media
Human‐source components, such as human serum albumin, are
used as supplements but animal components are not allowed as
supplements

6. 15-May-20 6APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

7. 1. PH
Most animal cells can grow at a pH in the range of 7.0 – 7.4.
The indicator phenol red is most commonly used for
visible detection of pH of the media. The coloration at the
different pH is –
At pH 7.4 – Red
At pH 7.0 – Orange
At pH 6.5 – Yellow
At pH 7.8 – Purple

8. Carbon di-oxide in the medium is in a dissolved state,
the concentration of which depends on the
atmospheric CO2 tension and temperature. CO2 in the
medium exists as carbonic acid (H2CO3), and
bicarbonate (HCO3
-) and H+ ions.
CO2 + H2O ↔ H2CO3 ↔ H⁺ + HCO3
-
Addition of sodium bicarbonate (as a component of
bicarbonate buffer) neutralizes bicarbonate ions.
NaHCO3 ↔ Na+ + HCO3
-
15-May-20 8
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

9. • The 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.
 The optimal temperature for a given cell culture is
dependent on the body temperature of the organism,
serving as the source of the cells.
Humans and warm blooded animals – 37°C
Birds – 38.5°C
15-May-20 9
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA
4. TEMPERATURE

10. • The osmolality for most of the cultured cells (from
different organisms) is in the range of 260 – 320 mosm/kg.
• Whenever there is an addition of acids, bases, drugs etc. to
the medium, the osmolality gets affective.
• The viscosity of a culture medium is influenced mainly by
the serum content and in most cases will have little effect
on cell growth. Viscosity becomes important, however,
whenever a cell suspension is agitated (e.g., when a
suspension culture is stirred) or when cells are
dissociated after trypsinization.
15-May-20 10
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA
6. VISCOSITY

11. The balanced salt solutions (BSS) are primarily composed
of inorganic salts.
Sometimes, sodium bicarbonate, glucose and HEPES
buffer may also be added to BSS.
Phenol red serves as a pH indicator.
The important functions of balanced salt solutions are
listed here under.
• Supply essential inorganic ions.
• Provide the requisite pH.
• Maintain the desired osmolality.
• Supply energy from glucose.
15-May-20 11
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

12. The composition of two most widely used BSS
namely Earl’s BSS and Hank’s BSS is given in table :
15-May-20 12
Composition (g/l) of balanced salt solutions (BSS)
Ingredient Earle’s BSS Hank’s BSS
NaCl 6.68 8.0
KCl 0.4 0.4
CaCl2
(anhydrous)
0.02 0.14
MgSO4·H2O 0.02 0.1
NaHCO3 2.2 0.35
NaH3PO4·H2O 0.14 –
Na2HPO4·7H2O – 0.09
KH2PO4 – 0.06
D – Glucose 1.0 1.0
Phenol red 0.01 0.01
HEPES, Na salt
(buffer)
13.02 2.08
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

13. • The term complete medium implies a medium that has
had all its constituents and supplements added and is
sufficient for the use specified.
EMEM – Eagle’s minimal essential medium.
DMEM – Dulbecco’s modifications of Eagle’s medium.
GMEM – Glasgow’s modification of Eagle’s medium.
RPMI 1630 and RPMI 1640 – Media from Rose well
Park Memorial Institute.
The older important culture media are Ham’s E10,
and F12, TC 199 and CMRL 1060.
15-May-20 13
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

14. 15-May-20 14
Composition of three commonly used culture media(mg/l)
Component Eagle’s MEM RPMI 1640 Ham’s F12
Amino acids
L - Alanine 8.91
L - Arginine HCl 105 200 211
L - Asparagine H2O 50 15.0
L – Aspartic acid 20 13.3
L – Cystine 24 50 24.0
L – Glutamic acid 20 14.7
L – Glutamine 292 300 146.2
Glycine 10 7.51
L - Histidine HCl H2O 31 15 21.0
L – Isoleucine 52 50 3.94
L – Leucine 52 50 13.12
L – Lysine 58 40 36.54
L – Methionone 15 15 4.48
L – Phenylalanine 32 15 4.96
L – Proline 20 34.5
L – Serine 30 10.51
L – Threonine 48 20 11.91
L – Tryptophan 10 5 2.042
L – Tyrosine 36 20 5.43
L – Valine 46 20 11.7
Glutathione 1
L - Hydroxyproline 20

15. 15-May-20 15
Vitamins
D – Biotin 0.2 0.007
Ca D – pantothenate 1 0.25 0.26
Choline chloride 1 3.0 13.96
Folic acid 1 1.0 1.32
i - Inositol 2 18.02
Nicotinamide 1 35 0.037
P – Aminobenzoic acid 1.0
Pyridoxine HCl 1 0.062
Pyridoxine HCl 1
Riboflavin 0.1 0.2 0.038
Thiamine HCl 1 1.0 0.34
Vitamin B12 0.005 1.36

16. 15-May-20 16
Inorganic salts
CaCl2·2H2O 200 44.1
CaNO3·4H2O 100
CuSO4·5H2O 0.0025
FeSO4·&H2O 0.83
KCl 400 400 223
MgSO4·7H2O 220 100 133
NaCl 6800 6000 7599
NaHCO3 2000 2000 1176
Na2HPO4·7H2O 1512 268
NaH2PO4·2H2O 150
Other components
D – Glucose 1000 2000 1801
Phenol red 5.0 1.2
Sodium pyruvate 110
Lipoic acid 0.21
Linoleic acid 0.084
Hypoxanthine 4.08
Putrescine 0.16

17. Depending on the medium, the quality and quantity of the ingredients vary.
AMINO ACIDS
• All the essential amino acids (which cannot be synthesized by the cells) have
to be added to the medium. In addition, even the non – essential amino acids
(that can be synthesized by the cells) are also usually added to avoid any
limitation of their cellular synthesis. Among the non – essential amino acids,
glutamine and/or glutamate are frequently added in good quantities to the
media since these amino acids serve as good sources of energy and carbon.
VITAMINS
• The quality and quantity of vitamins depends on the medium. For instance,
Eagle’s MEM contains only water soluble vitamins (e.g. B – complex, choline,
inositol). The other vitamins are obtained from the serum added. The
medium M 199 contains all the fat soluble vitamins (A, D, E and K) also.
15-May-20 17

18. The salts present in the various media are basically those found
in balanced salt solutions (Eagle’s BSS and Hank’s BSS). The salts
contribute to cations (Na+, K+, Mg2+, Ca2+ etc.) and anions (Cl-,
HCO3-, SO4
2-, PO4
3-), and are mainly responsible for the
maintenance of osmolality.
There are some other important functions of certain ions
contributed by the salts.
Ca²+ ions are required for cell adhesion, in signal transduction,
besides their involvement in cell proliferation and
differentiation.
Na+, K+, and Cl- ions regulate membrane potential.
PO4
3-, SO4
2- and HCO3- ions are involved in the maintenance of
intracellular charge, besides serving as precursors for the
production of certain important compounds e.g. PO4
3- is
required for ATP synthesis.
15-May-20 18

19. 15-May-20 19
GLUCOSE
• Majority of culture media contain glucose which serves as an
important source of energy. Glucose is degraded in glycolysis
to form pyruvate/lactate. These compounds on their further
metabolism enter citric acid cycle and get oxidized to CO2.
Other organic supplements
• Several additional organic compounds are usually added to
the media to support cultures. These include certain
proteins, peptides, lipids, nucleosides and citric acid cycle
intermediates.
Antibiotics
• In the early years, culture media invariably contained
antibiotics. The most commonly used antibiotics were
ampicillin, penicillin, gentamycin, erythromycin, kanamycin,
neomycin and tetracycline. Antibiotics were added to reduce
contamination.

20. 15-May-20 20
SERUM
• Serum is a natural biological fluid, and is rich in various components to
support cell proliferation. The most commonly used sera are calf serum
(CS), fetal bovine serum (FBS), horse serum and human serum.
Approximately 5 – 20% (v/v) of serum, is mostly used for supplementing
several media.
PROTEINS
• The in vitro functions of serum protein are not very clear. Some of them
are involved in promoting cell attachment and growth e.g. fetuin,
fibronectin. Proteins increases the viscosity of the culture medium,
besides contributing to buffering action.
NUTRIENTS AND METABOLITES
• Serum contains several amino acids, glucose, phospholipids, fatty acids,
nucleosides and metabolic intermediates (pyruvic acid, lactic acid
etc.).These constituents do contribute to some extent for the nutritional
requirements of cells. This may however, be insignificant in complex
media with well supplemented nutrients.

21. 15-May-20 21
GROWTH FACTORS
•There are certain growth factors in the serum that
stimulate the proliferation of cells in the culture.
•Platelet – derived growth factor (PDGF).
•Fibroblast growth factor (FGF).
•Epidermal growth factor (EGF).
•Vascular endothelial growth factor (VEGF).
•Insulin – like growth factors (IGE – 1, IGF – 2).
HORMONES
•Hydrocortisone promotes cell attachment, while insulin
facilitates glucose uptake by cells. Growth hormone, in
association with somatomedins (IGFs), promoters cell
proliferation.
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

22. 15-May-20 22
LIPIDS
Limoleic acid, oleic acid, ethanolamine, and
phosphorethanolamine are present in serum in small amounts,
usually bound to proteins such as albumin.
MINERALS
Serum replacement experiments have also suggested that trace
elements and iron, copper, and zinc may be bound to serum
protein, probably albumin.
INHIBITORS
Serum may also contain cellular growth inhibitors. Majority of
them are artefacts e.g. bacterial toxins, antibodies. The natural
serum also contains a physiological growth inhibitor namely
transforming growth factor β (TGF – β).
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

23. 15-May-20 23
 The nutrient media used for culture of animal
cells and tissues must be able to support their
survival as well as growth, i.e., must provide
nutritional, hormonal and stromal factors.
 Scientists or researchers can control the growth
of cultured cells as required by changing the
composition of the media.
 Media can be designed using specific factors,
which will help in differentiation of cultured cells
with specific desired functions.
APPLICATIONS AND CONSTITUENTS OF CULTURE MEDIA

24. 15-May-20 24
Culture of animal cells and tissue is rather more difficult
than that of microorganisms and plants because the later
synthesize certain chemical constituents from inorganic
substances.
However, the culture media provide the optimum growth
factors (e.g. pH, osmotic pressure, etc) and chemical
constituents (unlike microbes).
The selection of the appropriate medium for a given type of
cell , depends on their types and source.

25. 15-May-20 25
 Culture of animal cells – R. Ian Freshney (sixth edition)
http://www.biotechnologynotes.com/animals/animal-cell-
culture-history-types-and-applications/671
https://biocyclopedia.com/index/biotechnology/animal_biotec
hnology/animal_cell_tissue_and_organ_culture/biotech_cultur
e_media.php