Culture media

preparation
 Introduction
• Cell culture is the general term used for the removal of cells, tissues or organs from an
animal or plant and their subsequent placement into an artificial environment conducive
to their survival and/or proliferation.
• Basic environmental requirements for cells to grow optimally are:
• controlled temperature, a substrate for cell attachment, and appropriate growth medium
and incubator that maintains correct pH and osmolality.
• The most important and crucial step in cell culture is selecting appropriate growth
medium for the in vitro cultivation.
 Introduction
• A growth medium or culture medium is a liquid or gel designed to support the growth
of microorganisms, cells, or small plants.
• Cell culture media generally comprise an appropriate source of energy and compounds
which regulate the cell cycle.
• A typical culture medium is composed of a complement of amino acids, vitamins,
inorganic salts, glucose, and serum as a source of growth factors, hormones, and
attachment factors. In addition to nutrients, the medium also helps maintain pH and
osmolality.
 Types of Cell Culture Media

1. Natural media
• Natural media consist of naturally occurring biological fluids such as plasma,
serum, lymph, Extract of liver, spleen, tumors, coagulants or plasma clots.
• Natural media are very useful and convenient for a wide range of animal cell
culture.
• The major disadvantage of natural media is its poor reproducibility due to lack of
knowledge of the exact composition of these natural media.
 Types of Cell Culture Media

2. 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.
• Basal media: MEM DMEM, Complex media: RPMI-1640, IMDM
Artificial media are grouped into four categories:

1. Serum containing media

• Fetal bovine serum is the most common supplement in animal cell culture media.
• It is used as a low-cost supplement to provide an optimal culture medium.
• Serum provides carriers or chelators for labile or water-insoluble nutrients, hormones and
growth factors, protease inhibitors, and binds and neutralizes toxic moieties.
Artificial media are grouped into four categories:

2. Serum-free media
• such as DMEM and mTESR medium incorporate defined quantities of purified growth
factors, lipoproteins, and other proteins, which are otherwise usually provided by the
serum.
• These media are also referred to as ‘defined culture media’ since the components in these
media are known.
Artificial media are grouped into four categories:
3. Chemically defined media
• These media contain contamination-free ultra pure inorganic and organic ingredients, and
may also contain pure protein additives, like growth factors.
4. 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.
 3. Basic Components of Culture Media
• Culture media contain a mixture of amino acids, glucose, salts, vitamins, and other nutrients,
and available either as a powder or as a liquid form from commercial suppliers 
3.1 Buffering systems
Regulating pH is critical for optimum culture conditions and is generally achieved by one of
the two buffering systems:
3.1.1 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 a CO2 incubator. A natural buffering
system is low-cost and non-toxic.
 3. Basic Components of Culture Media

3.1.2 HEPES
• 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.
 3. Basic Components of Culture Media

3.1.3 Phenol red

• phenol red as a pH indicator, which allows constant monitoring of pH.
• 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.
 3. Basic Components of Culture Media
3.2 Inorganic salt
• Inorganic salt in the media helps to retain the osmotic balance and help in regulating
membrane potential by providing sodium, potassium, and calcium ions.
3.3 Amino acids
• Amino acids are the building blocks of proteins, and thus are obligatory ingredients of all known
cell culture media.
• Essential amino acids must be included in the culture media as cells can not synthesize these by
themselves.
• They are required for the proliferation of cells and their concentration determines the maximum
achievable cell density. L-glutamine, an essential amino acid, is particularly important.
• Nonessential amino acids may also be added to the medium to replace those that have been
depleted during growth.
3.4 Carbohydrates
• Carbohydrates in the form of sugars are the major source of energy. Most of the media
contain glucose and galactose, however, some contain maltose and fructose.
3.5 Fatty acids and lipids
• They are particularly important in serum-free media as they are generally present in
serum.
3.6 Proteins and peptides
• The most commonly used proteins and peptides are albumin, transferrin, and fibronectin.
• They are particularly important in serum-free media.
3.7 Vitamins
• Many vitamins are essential for growth and proliferation of cells.
• Vitamins cannot be synthesized in sufficient quantities by cells and are therefore important
supplements required in tissue culture.
• The B group vitamins are most commonly added for growth stimulation.
3.8 Trace elements
• Trace elements are often supplemented to serum-free media to replace those normally
found in serum.
• Trace elements like copper, zinc, selenium and tricarboxylic acid intermediates are
chemical elements that 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.
3.9 Media supplements
• which are not present in the basal media and serum such as hormones, growth factors and
signaling substances.
• For most of the cell lines, optimal osmolality should be between 260 mOSM/kg and 320
mOSM/kg.
3.10 Antibiotics
antibiotics are often used to control the growth of bacterial and fungal contaminants.
Eg: Penicillin-streptomycin, amphotericin B (fungizone) and/or fluconazole for fungi;
and pentamidine and/or atovaquone for protozoa.
3.11 Serum in media
• Serum is a complex mix of albumins, growth factors and growth inhibitors.
• Serum is one of the most important components of cell culture media and serves as a
source for amino acids, proteins, vitamins (particularly fat-soluble vitamins such as A,
D, E, and K), carbohydrates, lipids, hormones, growth factors, minerals, and trace
elements.
• Serum 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 is used in contact-inhibition studies because of its lower growth-
promoting properties.
 4. Criteria for Selecting Media

• 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 and also the purpose
of the culture and resources available in the laboratory.
• In general, it’s always good to start with MEM for adherent cells and RPMI-1640 for
suspension cells. 
• 5. Common cell culture media
• Most commonly used culture media include the following.
• Dulbecco’s Modified Eagle’s Medium (DMEM) (3.7 g/L + 5-10% Fetal Bovine Serum
(FBS)
• RPMI-1640 (3.7 g/L).
• Eagle’s Minimum Essential Medium (EMEM) (1500 mg/l)
• Hams’s F-12 medium (3.7 g/L).
• Iscove’s Modified Dulbecco’s Medium (IMDM) (1500 mg/l)
• IMDM is a highly enriched synthetic media well suited for rapidly proliferating, high-
density cell cultures
Cell harvesting methods
 cell harvesting

• The process of isolating target cells from a growth medium is called cell

harvesting.
1. Immunomagnetic Cell Separation

• Immunomagnetic cell separation is a technique whereby magnetic particles are used to

isolate target cells from heterogeneous mixtures.
• To accomplish this, the magnetic particles are bound to specific cell surface proteins on
the target cells via antibodies, enzymes, lectins, or streptavidin.
• The sample is then placed in an electromagnetic field that pulls on the magnetic particles,
bringing the labeled cells with them.
• The unlabeled cells remain in the supernatant, thus creating a physical separation
between target and non-target cells within the sample.
Due to its speed and simplicity, immunomagnetic cell separation is one of the most
commonly used methods and has several advantages, including:
•High purity
•Fast protocols
•Ease of use
•Low equipment cost
•Many cells can be isolated at once
•Potential for automation
•High cell viability
2. Fluorescence-activated Cell Sorting
• Fluorescence-activated cell sorting (FACS) is a method that uses flow cytometry and fluorescent
probes to sort heterogeneous mixtures of cells.
• Fluorophore-tagged antibodies bind to epitopes on specific antigens on the target cells within a
single-cell suspension.
• After tagging, the flow cytometer focuses the cell suspension into a uniform stream of single cells.
• This stream is then passed through a set of lasers that excites the cell-bound fluorophores, causing
light scattering and fluorescent emissions.
• Based on the wavelengths produced by the laser excitation, the resulting photon signals are
converted into a proportional number of electronic pulses that assign a charge to the droplet that is
formed around the cell.
• As each droplet falls between the deflection plates, its charge causes the droplet to either be
deflected into collection tubes or fall into the waste chamber.
2. Fluorescence-activated Cell Sorting
• Immunomagnetic cell sorting is a much faster and simpler procedure than FACS, and
is often the preferred cell isolation method for common cell types. FACS has several
advantages over immunomagnetic cell sorting including the ability to:
• Sort single cells
• Isolate cells based on intracellular markers (e.g. GFP)
• Isolate cells based on surface marker expression levels
• Sort complex cell types with multiple markers at higher purity
3. Cell Harvesting Centrifugation
• One of the most common forms of cell harvesting is centrifugation.
• Centrifugation is the process of spinning a solution continuously until substances separate
themselves.
• By using the density difference between solids and fluids, the process of sedimentation is
accelerated, meaning more dense particles move to the outside while less dense particles
float toward the center of the sample.
Advantages
• This method works quickly and is very easy to perform.
• cell harvest centrifugation can help to maximize quantity in shorter periods of time. 
Disadvantages
• The high levels of acceleration can shear the membranes of cells. This cell rupture,
or lysis, causes organelles and intracellular proteins to leak into the broth.
• It only provides around 90% purification.
4. Filtration
• Filtration is a cell isolation method based on cell size. Using a filtration device specific
to the targeted cell group’s size, smaller cells pass through the device while larger cells
are trapped.
• Membrane filtration is a robust, easy-to-implement, time-tested solution for processing
cell culture media that significantly reduces the risk of bioreactor contamination.
• Filter capacity depends on the filtration membrane structure and the composition of
the cell culture media; the latter vary widely in their constituents and formulations
Advantages
• This process is much gentler than centrifugation.
• There is no extreme speed or harsh pressures on any of the fragile cells.
• Disadvantages
• Depth filtration has limits on the size of sample it can properly sort.
5. Buoyancy-Activated Cell Sorting (BACS™)

• Buoyancy Activated Cell Sorting, also known as BACS, is the process of sorting cells by
using buoyant microbubbles.
• By coating the microbubbles with antibodies, the target cells attach to the surface of the
microbubbles, effectively separating them from the non-target cells.
• Then the microbubbles—composed of a gaseous core that is often shelled by polymers,
lipids, or proteins—float the target cells to the top of the sample.
• the microbubbles are first mixed with the cell sample, where they identify and bind to
the target cells and separate them from the non-target cells.
• After binding with the target cells, the microbubbles float to the top. The target cells
float with them, leaving behind the non-target cells.
• In the final step, the microbubbles and target cells are removed from the top of the
sample.
• Isolating immune cells from peripheral blood
• Recovering cell populations from dissociated tissue
• Capturing circulating tumor cells in blood
• Conducting molecular analysis of specific cell populations
By using BACS, the cells are easily, quickly and gently separated for research purposes.