Selecting the medium and serum in
animal Biotechnology
Most of the media described in our
previous post- Serum – as a growth medium in animal tissue culture were
developed to support particular cell lines or conditions. Many were developed
with L929 mouse fibroblasts or HeLa cervical carcinoma cells, and Ham’s F12 was
designed for Chinese hamster ovary (CHO) cells; all now have more general applications
and have become classic formulations. Among them, data from suppliers would
indicate that RPMI 1640, DMEM, and MEM are the most popular, making up about 75%
of sales. Other formulations seldom account for more than 5% of the total; most
constitute 2–3%, although blended DMEM/F12 comes closer, with over 4%. Eagle’s
Minimal Essential Medium (MEM) was developed from Eagle’s Basal Medium (BME) by
increasing the range and concentration of the constituents. For many years,
Eagle’s MEM had the most general use of all media. Dulbecco’s modification of
BME (DMEM) was developed for mouse fibroblasts for transformation and virus
propagation studies. It has twice the amino acid concentrations of MEM, has four
times the vitamin concentrations, and uses twice the HCO3 −and CO2
concentrations to achieve better buffering. α-MEM has additional amino acids
and vitamins, as well as nucleosides and lipoic acid; it has been used for a
wide range of cell types, including hematopoietic cells. Ham’s F12 was
developed to clone CHO cells in low-serum medium; it is also used widely,
particularly for clonogenic assays and primary culture. CMRL 1066, M199, and
Waymouth’s media were all developed to grow L929 cells serum-free but have been
used alone or in combination with other media, such as DMEM or F12, for a
variety of more demanding conditions. RPMI1640 and Fischer’s media were
developed for lymphoid cells—Fischer’s specifically for L5178Y lymphoma, which has
a high folate requirement. RPMI 1640 in particular has quite widespread use,
often for attached cells, despite being designed for suspension culture and
lacking calcium. L15 medium was developed specifically to provide buffering in
the absence of HCO3 − and CO2. It is often used as a transport
and primary culture medium for this reason, but its value was diminished by the
introduction of HEPES and the demonstration that HCO3 − and CO2
are often essential for optimal cell growth, regardless of the requirement for
buffering.
Information regarding the
selection of the appropriate medium for a given type of cell is usually
available in the literature in articles on the origin of the cell line or the
culture of similar cells. Information may also be obtained from the source of
the cells. Cell banks, such as ATCC and ECACC, provide information on media
used for currently available cell lines, and data sheets can be accessed from
their websites. Failing this, the choice is made either empirically or by
comparative testing of several media, as for selection of serum.
Many continuous cell lines (e.g.,
HeLa, L929, BHK21), primary cultures of human, rodent, and avian fibroblasts,
and cell lines derived from them can be maintained on a relatively simple
medium such as Eagle’s MEM, supplemented with calf serum. More complex media
may be required when a specialized function is being expressed or when cells
are sub-cultured at low seeding density (<1×103 /mL), as in
cloning. Frequently, the more demanding culture conditions that require complex
media also require foetal bovine serum rather than calf or horse serum, unless
the formulation specifically allows for the omission of serum.
If information is not available,
a simple cell growth experiment with commercially available media and multiwell
plates can be carried out in about two weeks. Assaying for clonal growth and
measuring the expression of specialized functions may narrow the choice further
[you may soon find the protocol for this cell growth experiment in our upcoming posts, or just mail us at BiotechExplorer@gmail.com].
Autoclavable media are available
from commercial suppliers (you may search it on web). They are simple to
prepare from powder and are suitable for many continuous cell strains. They may
need to be supplemented with glutamine for most cells and usually require
serum.
Note a
simple trick: The cost of serum should be calculated on the
basis of the volume of the medium when cell yield is not important, but if the
objective is to produce large quantities of cells, one should calculate serum
costs on a per-cell basis. Thus, if a culture grows to 1×106 /mL in
serum A and 2×106 mL in serum B, serum B becomes the less expensive
by a factor of two, given that product formation or some other specialized function
is the same.
If foetal bovine serum seems essential,
try mixing it with calf serum. This may allow you to reduce the concentration
of the more expensive foetal serum. If you can, leave out serum altogether, or
reduce the concentration, and use a serum-free formulation.
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Did you read these few relative topics ?
Balanced Salt Solution in Animal Tissue Culture
Maintenance of Sterility in Animal Tissue Culture Labs
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