Biophysical Chemistry of Proteins || Cell Culture
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Chapter 17 Cell Culture Cells from plants and animals – including humans – can be grown in culture if their requirements are met with respect to medium composition and temperature. The following paragraphs will focus on the culture of mammalian cells, even though the culture of insect and plant cells is of considerable economic and scientific interest. More detailed information can be found in [131, 260]. The medium needs to be isotonic to the cells and have an ionic composition similar to the interstitial fluid. It also needs to supply nutrients like glucose and amino acids. For buffering the medium contains bicarbonate and the incubators have to maintain a partial pressure of CO 2 similar to that in tissue (5 % CO 2 ). Other media, designed to be used in closed bottles, have a different buffer composi- tion. Phenol red is added to most media so that the pH can be checked by eye (red neutral, yellow acidic, purple basic). The addition of 20 mM Hepes can increase the buffering capacity of the medium, but some cell types are inhibited by it. Some small organic molecules like pyruvate are known to improve cell growth rate. Many hormones, growth factors, and other unknown compounds are required for optimal growth; these are added to the medium in the form of fetal bovine serum (FBS, also known as fetal calf serum FCS). Attempts have been made to identify the required compounds and replace the FBS with a chemically defined cocktail. Such serum-free media are available but are more expensive and support less growth than media containing 5–10 % serum. Apart from such nutrients, antibiotics like penicillin, streptomycin, and anti- fungals like amphotericin B are sometimes routinely added to media to prevent yeasts and bacteria from growing. These additions are contentious, however: Steril- ity should be maintained by aseptic working technique rather than antibiotics. Incubators maintain a body-like temperature of 37 °C and a high humidity. They also form an enclosed environment where sterility is easier to maintain. Only few mammalian cell lines can be grown in suspension, most need to attach themselves to a surface. Cell culture dishes are specially treated to make attachment easier, hence they can be used only once. The number of cells grown in culture is limited by the surface area of the dish. Some products are available to increase this surface area, like polysaccharide beads or polyester matrixes. Even using those however the cell density in culture is much lower than that in a body with its efficient supply of nutrients and removal of waste products. E. Buxbaum, Biophysical Chemistry of Proteins: An Introduction to Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 17, © Springer Science+Business Media, LLC 2011 187
Transcript of Biophysical Chemistry of Proteins || Cell Culture
Chapter 17Cell Culture
Cells from plants and animals – including humans – can be grown in culture if theirrequirements are met with respect to medium composition and temperature. Thefollowing paragraphs will focus on the culture of mammalian cells, even though theculture of insect and plant cells is of considerable economic and scientific interest.More detailed information can be found in [131, 260].
The medium needs to be isotonic to the cells and have an ionic compositionsimilar to the interstitial fluid. It also needs to supply nutrients like glucose andamino acids. For buffering the medium contains bicarbonate and the incubatorshave to maintain a partial pressure of CO2 similar to that in tissue (�5 % CO2).Other media, designed to be used in closed bottles, have a different buffer composi-tion. Phenol red is added to most media so that the pH can be checked by eye (redneutral, yellow acidic, purple basic). The addition of 20 mM Hepes can increase thebuffering capacity of the medium, but some cell types are inhibited by it.
Some small organic molecules like pyruvate are known to improve cell growthrate. Many hormones, growth factors, and other unknown compounds are requiredfor optimal growth; these are added to the medium in the form of fetal bovine serum(FBS, also known as fetal calf serum FCS). Attempts have been made to identify therequired compounds and replace the FBS with a chemically defined cocktail. Suchserum-free media are available but are more expensive and support less growth thanmedia containing 5–10 % serum.
Apart from such nutrients, antibiotics like penicillin, streptomycin, and anti-fungals like amphotericin B are sometimes routinely added to media to preventyeasts and bacteria from growing. These additions are contentious, however: Steril-ity should be maintained by aseptic working technique rather than antibiotics.
Incubators maintain a body-like temperature of 37 °C and a high humidity. Theyalso form an enclosed environment where sterility is easier to maintain.
Only few mammalian cell lines can be grown in suspension, most need to attachthemselves to a surface. Cell culture dishes are specially treated to make attachmenteasier, hence they can be used only once. The number of cells grown in cultureis limited by the surface area of the dish. Some products are available to increasethis surface area, like polysaccharide beads or polyester matrixes. Even using thosehowever the cell density in culture is much lower than that in a body with its efficientsupply of nutrients and removal of waste products.
E. Buxbaum, Biophysical Chemistry of Proteins: An Introductionto Laboratory Methods, DOI 10.1007/978-1-4419-7251-4 17,© Springer Science+Business Media, LLC 2011
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188 17 Cell Culture
Cells are grown until they cover the available surface area (exchanging the mediaevery 2–3 days). Then they are removed by treatment with trypsin (to digest theextracellular matrix) and EDTA (to remove the bivalent cations necessary for cellattachment). The suspended cells are then seeded into 5–10 new dishes and grownagain until confluence. This is called a passage. If cells are harvested for studyingmembrane proteins, the trypsin should be left out (EDTA alone is able to detachcells, it just takes a little bit longer).
Cells which are not needed can be kept frozen in liquid nitrogen. For this pur-pose they are suspended in a special medium with high serum concentration andwith 10–20 % glycerol or dimethyl sulphoxide (DMSO) to prevent the formation ofice crystals. Cells need to be frozen slowly (at about 1 °C per min), while thawingshould be rapid.
17.1 Cell Types
Cells can be grown directly from tissue samples obtained under sterile conditionsfrom animals or humans. This is called primary cell culture. In most cases the cellsof interest can be kept alive only for a few days, either because they die or becausethey are overgrown by fibroblasts.
However, with luck and skill it is sometimes possible to expand an interestingcell type into a stable cell line. These can then be grown for about 20–30 passagesuntil the cells stop multiplying (finite life span of higher organisms, HAYFLICK-limit [148] due to shortening of telomeres during cell division). For that reason itis important to keep frozen stocks at low passage numbers. Stocks of establishedcell lines can be bought, for example, from the American type culture collection(ATCC), its European counterpart (ECACC), or obtained from colleagues. Scientificethics requires that researchers make their cell lines available to colleagues, this isbest done by submitting them to type culture collections.
Some cancer cells, and established cells treated with certain virus, can be grownindefinitely as they do not stop multiplying at a certain passage number. We speakof transformed cell lines. HeLa (from a human cervical cancer) and CHO (from aChinese hamster ovary carcinoma) are probably the most well known transformedcell lines. Transformation frequently leads to the loss of specialised functions in acell, which makes them less useful in research. Note, however, that even such im-mortal cells will change their properties if grown to high passage numbers, resultsobtained with such cells may differ from those obtained with low passage numbercells. In some cases, researchers are interested in immortalising a cell with interest-ing functions. This can be achieved by fusing the cell with a transformed cell, whichneeds to be genetically similar (for example from the same mouse strain). One ex-ample of this hybridoma technique is the production of monoclonal antibodies froma fusion product of a B-cell with a lymphoma cell (see p. 100).
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17.1.1 Contamination of Cell Cultures
Because cell cultures are maintained for a long time with frequent handling, andbecause in many laboratories more than one cell line is grown, cross-contaminationof cell cultures is an all too common problem. In a recent review of 22 studies on2328 cell lines, 366 (15:6 %) were from a different tissue than stated, 86 (3:7 %)were even from a different species [172], including exotic mix-ups as bird withmanta ray. In particular, HeLa cells have a considerable propensity of showing up inplaces where they do not belong. Therefore, identity of cell lines should be verified(e.g., by short tandem repeat DNA profiling). For research and production of mate-rials regulated by the FDA this is mandatory, some journals now also require it forpublication.
In addition, contamination of cell cultures with mycoplasma continues to be aproblem; these can be detected easily by PCR. Simpler, but less reliable, is stainingof cells with DNA intercalating fluorescent dyes like DAPI, which give infected cellsa spotted appearance. Ciprofloxacin or Rifampicin may be used to cure valuable celllines from mycoplasma infection.
