Description About the cell culture media and laboratory set up

1. By:
Dr. Fahad Khan
Assistant Professor
Department of Biotechnology
Noida Institute of Engineering & Technology, Greater
Noida
Basic of Animal Cell Culture
PART-II

2. *Media is generally defined as the medium which contains all the
necessary elements requires for the growth of cell or tissue
culture.
*Besides meeting the basic nutritional requirement of the cells, the
culture medium should also have any necessary growth factors,
regulate the pH and osmolality, and provide essential gases (O2
and CO2).
*The ‘food’ portion of the culture medium consists of amino acids,
vitamins, minerals, and carbohydrates. These allow the cells to
build new proteins and other components essential for growth &
function as well as providing energy necessary for metabolism.
*Two types of media used in culture technique:
1. Natural Media 2. Artificial Media

3. Cell
Culture
Media
Natural
Media
Artificial
Media
Balanced Salt
Solutions
Ex-Eagle’s BS,
Hank’s BS,
Tissue Extracts
Ex-Extract of liver,
spleen,
tumors, leucocytes and
bone
Biological Fluids
Ex-plasma, serum,
lymph
Basal media
Ex-MEM, DMEM
Complex media
Ex-RPMI-1640,
IMDM

4. *Culture media (as a powder or as a liquid) contains:
Amino acids
Sugar
Inorganic Salts
Vitamins
Natural buffering system Ex-HEPES
Phenol red
Proteins and Peptides (important in serum-free media).
Trace Elements

5. *The essential amino acids are required by cultured cells.
*Other non-essential 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.
*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.

6. *Amino acids, vitamin requirements have been derived
empirically and often relate to the cell line originally used in
their development.
*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.
*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

7. *The salts are chiefly those of Na+, K+, Mg2+, Ca2+, Cl−, SO4
2-, PO4
3-,
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.

8. *Regulating pH is critical for optimum culture conditions and is generally
achieved by one of the two buffering systems: Natural buffering system and
Chemical 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.
*Chemical buffering using a zwitterion, HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid).
*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.

9. 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.

10. *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.

11. *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.

12. Antibiotics and Antimycotics
• Antibiotics and/or antimycotic agents are added 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.
• Antibiotics can mask contamination by mycoplasma and resistant bacteria.
• Further, they can interfere with the metabolism of sensitive cells.
Serum
• Sera serve as a source for amino acids, proteins, vitamins , carbohydrates, lipids,
hormones, growth factors, minerals, and trace elements.
• Serum buffers the culture medium, inactivates proteolytic enzymes, increases medium
viscosity, and conditions the growth surface of the culture vessel.
• 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 factors and is appropriate for cell cloning and for the
growth of fastidious cells.
• Calf serum, because of its lower growth-promoting properties, is used in
contact-inhibition

13. *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;

14. *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

15. *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:
1. Type of cells to be cultured
2. The purpose of the culture
3. 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.

16. *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.
Advantages:
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

17. *Culture medium is available in three forms from the commercial suppliers:
 Powdered form: It need to prepared and sterilized.
 Concentrated form: Needs to be diluted.
 Working solution: Used directly without further manipulation.
 Powder medium is least expensive but needs to be sterilized.
It is advisable to filter and sterilize it prior to addition of serum as the
foaming that occurs in presence of serum denatures protein.
Serum should be added after filter sterilization.
 Media should be tested for sterility by placing it in a incubator for 72 hours
prior to utilization to ensure that the lot is contamination free.
*Media after preparation should be stored at 40C.

18. *pH:
Most normal mammalian cell lines grow well at pH 7.4, and
there is very little variability among different cell strains.
*Carbon Dioxide:
The growth medium controls the pH of the culture and buffers the cells in culture against changes
in the pH. Usually, this buffering is achieved by including an organic or CO2-bicarbonate based
buffer. Because the pH of the medium is dependent on the delicate balance of dissolved carbon
dioxide (CO2) and bicarbonate changes in the atmospheric CO2 can alter the pH of the medium.
Therefore, it is necessary to use exogenous CO2 when using media buffered with a CO2-
bicarbonate based buffer, especially if the cells are cultured in open dishes or transformed cell
lines are cultured at high concentrations.
*Temperature:
The optimal temperature for cell culture largely depends on the body temperature of the host from
which the cells were isolated, and to a lesser degree on the anatomical variation in temperature
(e.g., temperature of the skin may be lower than the temperature of skeletal muscle). Overheating
is a more serious problem than underheating for cell cultures; therefore, often the temperature in
the incubator is set slightly lower than the optimal temperature, 36 -37’C optimal for growth.

19. *Eagle’s Minimum Essential Medium (EMEM)
*Dulbecco’s Modified Eagle’s Medium (DMEM)
Low glucose
High glucose
*RPMI-1640
*Ham’s Nutrient Mixtures
*DMEM/F12
*Iscove’s Modified Dulbecco’s Medium (IMDM)

20. *Laminar-Flow Hood-
• It is used to provide sterile environment and protect
the laboratory worker from exposure to aerosols from
cell culture.
• Air is filtered through a HEPA (high efficiency
particulate air) filter before exiting the cabinet. They
are classified at levels I, II, and III.
• Class I cabinets are the simplest and easiest to
maintain but offer least sterile protection to the cell
culture.
• Class II cabinets are the most widely used for cell
culture work and offer good protection to both the
operator and cell cultures since air passing over the
working area is HEPA filtered.
• Class III cabinets are completely sealed units and
are used for more hazardous types of work.
• Cabinet’s surface should be wiped with ethanol
both before and after use.
• Cabinets are also equipped with a UV light that can
be used to sterilize the surfaces of the cabinet.

21. *Humid CO2 Incubator
• It Is required to maintain the optimum temperature for cell
growth.
• A controlled atmosphere is achieved by using a
humidifying tray .
• It draws air from the incubator into a sample chamber,
determines the concentration of CO2, and injects pure CO2
into the incubator to make up any deficiency.
• Air is circulated around the incubator by natural
convection or by using a fan to keep both the CO2 level and
the temperature uniform.

22. *Refrigerators and freezer (-20 °C)
*Both items are very important for storage of liquid media at 4 °C and
for enzymes (e.g., trypsin) and some media components (e.g.,
glutamine and serum) at -20 °C.
*A refrigerator or cold room is required to store medium and buffers.
*A freezer will be needed for keeping pre-aliquoted stocks of serum,
nutrients and antibiotics.
*Reagents may be stored at a temperature of -20 °C but if cells are to be
preserved it may be necessary to provide liquid nitrogen or a -80 °C
freezer.
Refrigerator -20 °C Freezer

23. Microscopes:
*A simple inverted microscope is essential so that cultures can be
examined in flasks and dishes.
*It is vital to be able to recognize morphological changes in cultures
since these may be the first indication of deterioration of a culture.
*An inverted microscope will also be needed for examining flasks
and multi-well dishes from underneath.

24. *Tissue culture ware
*A variety of tissue culture plasticware is available, the most
common being specially treated polystyrene.
*Although all tissue culture plasticware should support cell
growth adequately, it is essential when using a new supplier or
type of dish to ensure that cultures grow happily in it.
*Cells can be maintained in Petri dishes or flasks (25 cm2 or 75
cm2) which have the added advantage that the flasks can be
gassed and then sealed so that a CO2 incubator need not be
used.
6-well Plate T-75 Flask

25. Liquid Nitrogen:
*Invariably for continuous and finite cell lines, samples of cultures will need
to be frozen down for storage.
*It is important to maintain continuity in cells to prevent genetic drift and to
guard against loss of the cell line through contamination and other disasters.
*The procedure for freezing cells is general for all cells in culture.
*They should be frozen in exponential phase of growth with a suitable
preservative, usually dimethylsulfoxide (DMSO).
*The cells are frozen slowly at 1 °C/min to -50 °C and then kept either at -196
°C immersed in liquid N2 (in sealed glass ampoules) or above the liquid
surface in the gas phase (screw top ampoules).

26. *Filter sterilization:
*Media that cannot be autoclaved must be sterilized through a 0.22 μm
pore size membrane filter.
*These are obtainable in various designs to allow a wide range of
volumes to be filtered (e.g., Millipore, Gelman).
*Culture media, enzymes, hormones, cofactors and bicarbonate buffers
are examples of non-autoclavable substances.