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Water for Cell Culture

Water is used in many steps of the tissue or cell culture process. It is the main component of buffers and media, it is used for the dissolution of additives and drugs, and for rinsing bioreactors, plasticware and glassware. Thus, water quality may play an important role in cell culture experimental outcomes.

Contamination of cell culture by bacteria, yeasts or molds is always a concern and scientists go to great lengths to avoid them. These contamination are usually visible to the eye or by optical microscopy. However, contamination from chemicals may also affect the growth, morphology or behavior of cultured cells, yet be undetectable to the eye.

Some compounds naturally present in water that can affect cell culture:

  • Bacteria and endotoxins
    Bacteria contamination can cause sudden changes in media pH and contaminate pure cultures. In addition, most Gram-negative bacteria release endotoxin, a complex lipopolysaccharide (LPS). Macrophages and mononuclear phagocytes release a variety of pro-inflammatory cytokines in response to endotoxin stimulation. Endotoxins affect other types of cells as well, even cells lacking CD14 endotoxin receptors. Effects include changes in cell growth and function, cloning efficiency, and production of recombinant proteins.

  • Inorganic ions
    Heavy metals such as mercury, lead, zinc, nickel, chromium and cadmium have been proven toxic to various cells, including glial and neural cells. Magnesium inhibits glucose-6-phosphate dehydrogenase and DNA nuclease II. Manganese causes DNA polymerase to incorporate ribose instead of deoxyribose into nascent DNA chains.

  • Organic compounds
    Small organic molecules are commonly present in raw water supplies (humic acids, tannins, pesticides, endocrine disrupters, etc.) and may remain in tap water. These substances are known to affect the development of cells and should therefore be removed by suitable processes during the production of laboratory grade water.

    In addition, water is used in many instruments related to cell culture work, such as autoclaves and incubators.

Examples illustrating the impact of water quality on cell culture

(1) Effect of water quality on growth of various cell lines

The ability of three cell lines to grow in medium made using tap water, distilled water and Milli-Q water was studied. Three cell lines were used: mouse Leydig cells (TM3), rat endothelial-like cells (TR-1), mouse melanoma cells (M2R)

Comparison of cell growth in medium prepared with water from different sources. Cells were plated in serum-free medium prepared with tap water (T), distilled (D) or Milli-Q purified water (MQ) supplemented with hormones appropriate for the cell line. Cells were counted on day 4. Results are mean +/- SD (n=3).

In conclusion, quality water is an important factor for serum-free culture media preparation. Different cell lines show different sensitivities to the various water types used. However, regardless of cell type, ultrapure water always resulted in the best cell growth.

(2) Cardiomyocytes
The effect of water quality on cardiomyocyte viability was studied. Cardiomyocytes were isolated from adult rat cardiac tissue. They were washed in a HEPES-based buffer, then suspended in Minimum Essential Medium and plated on laminin-coated plates. Ultrapure water was used to prepare the buffers for the extraction procedure and the culture.

Two types of water were used: freshly purified and ultrafiltered water (Water A), and ultrapure water stored before use and not treated with ultrafiltration (Water B).

Photomicrographs of isolated cardiomyocytes prepared with water A (A) or water B (B).
Images courtesy of Drs. C. Plin and R. Zini, INSERM U660, Créteil School of Medicine, France.

When buffers were prepared with freshly purified and ultrafiltered water (Water A), many viable cardiomyocytes were obtained, as shown by their elongated and striated shape. However, when ultrapure water was stored before use and not treated with ultrafiltration (Water B), few viable cells were obtained.

During storage of ultrapure water in a plastic carboy container, bacteria are likely to develop, releasing endotoxins. In this example, the concentration in bacteria was approximately 4,000 CFU/mL and the endotoxin concentration was 8 EU/mL. The levels of endotoxin present in Water B were high enough to have a deleterious effect on the cardiomyocyte cells during the isolation process.

In conclusion, using ultrapure water is recommended for cell and tissue culture. Using freshly purified water and placing an ultrafiltration cartridge (BioPak) at the point of use of the water purification system eliminates the risk of endotoxin contamination, which may have an important effect on cell and tissue culture outcomes.


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