Water for Mammalian Cell Culture
Cell culture types, applications and media
Cell culture is a commonly used laboratory technique whereby prokaryotic or eukaryotic cells are grown under controlled conditions. Cells are typically isolated from tissue (primary cells) or from an established immortalized cell line (secondary cells). Primary cells only have a limited lifespan, except if they are derived from tumor tissue, while immortalized cell lines can proliferate indefinitely. It is also possible to fuse primary cells with an immortalized cell line to produce hybridomas, a technique used to produce monoclonal antibodies. Simulating the complex environments for primary and secondary cells and tissues requires specialized culture medias.
Cell culture in research and biotechnology
Large scale mammalian cell culture is widely used in biotechnology to produce vaccines, proteins, enzymes, antibodies and hormones. Cell culture is also used in many research fields including pharmaceutical development, where cells are used as models to study diseases such as cancer or heart disease. Cultured cells are used in genomic analysis, target identification and validation, cell-based assays for high-throughput screening (HTS), permeability assays, and to produce therapeutic proteins. Modern cell culture systems include live cell analysis, 3D cell culture and complex tissue cultures to model organ functions. These cell culture systems could use microfluidics to culture single cells1, or large volume bioreactors to grow millions of cells for diverse applications like the production of recombinant proteins, stem cell therapeutics and even lab-grown 'cultured' meats2.
Cell culture media components
Cultured cells are grown in specific growth media, either as a suspension or as adherent cultures (monolayer). Cell culture media typically contains amino acids, glucose, vitamins, inorganic salts, lipids and may contain animal serum, growth factors, hormones, antibiotics, antioxidants and other components. Types of media include serum based, serum free, animal free, protein free and chemically defined. To keep cells healthy and growing, they need to have sufficient nutrients available, therefore the media needs to be replenished on a regular basis. In addition, when adherent cell lines reach confluency (cover all the surface area available) they need to be sub-cultured to continue to grow. Any preparation or exchange of culture media presents a contamination risk that can affect experimental outcomes or destroy cell stocks.
Impact of water quality on cell culture
Great care must be taken to prevent any risk of cell contamination. Contamination may alter cell growth and characteristics, and unknowingly working with contaminated cells may lead to inaccurate or erroneous results.
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. In addition, water is used in many instruments related to cell culture work, such as autoclaves and incubators. Thus, water quality may play an important role in cell culture experimental outcomes.
Contamination may be biological (bacteria, mold, yeast, mycoplasma, viruses, protozoa, etc.) or chemical (due to contamination of the nutrients, the water, or the additives used to prepare the media, or the plasticware used).
Contamination of cell culture by bacteria, yeasts or molds is always a concern and scientists go to great lengths to avoid them. These contaminations are usually visible by eye or optical microscopy. 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 mammalian cell culture include:
Bacteria
Bacterial contamination can cause sudden changes in media pH and contaminate pure cultures. Bacteria may produce toxins that cause changes in cell growth and function, cloning efficiency, and production of recombinant proteins.
Endotoxins
Endotoxins are lipopolysaccharides (LPS) from Gram-negative bacteria walls. They are the most common type of pyrogens. Macrophages and mononuclear phagocytes release various pro-inflammatory cytokines in response to endotoxin stimulation. LPS are known to generate early toxic effects, like highly damaging free-radicals, and slow toxic actions, like cell dysfunction, apoptosis and necrosis. Endotoxin-free water is required for mammalian cell cultures.
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.
Organic compounds
Small organic molecules are commonly present in natural water (humic acids, tannins, pesticides, endocrine disrupters, etc.) and may remain in tap water. These substances are known to affect cell development and should therefore be removed by suitable processes during the production of laboratory grade water.
Study: Impact of water quality on tissue culture
Cardiomyocytes were isolated from adult rat cardiac tissue. They were washed in a HEPES-based buffer, then suspended in Minimum Essential Medium (MEM) 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 ultrapure water (Water A), and ultrapure water stored before use and not treated with ultrafiltration (Water B). The effect of water quality on cardiomyocyte viability was studied.
When buffers were prepared with freshly purified and ultrafiltered ultrapure water (Water A), many viable cardiomyocytes were obtained, as shown by their elongated and striated shape (Figure 1A). However, when ultrapure water was stored before use and not treated with ultrafiltration (Water B), few viable cells were obtained (Figure 1B).
Figure 1Photomicrographs 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.
During storage of ultrapure water in a plastic carboy container, bacteria are likely to develop, releasing endotoxins. In this study, the bacteria concentration in Water B 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, ultrapure water is recommended for cell and tissue culture. Using freshly purified ultrapure water and placing an ultrafiltration cartridge (Biopak® polisher) 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.
Preparing endotoxin-free water using ultrafiltration
Endotoxins (or LPS) are toxic molecules shed by bacteria. They can affect the growth, function and cloning efficiency of many types of cells, and are therefore a concern for researchers working with cells.
Since LPS consist of a very hydrophobic lipid group (lipid A) covalently bound to a long complex polysaccharide tail, they tend to form large aggregates in aqueous solution. Ultrafiltration is a membrane-based technology that removes endotoxins by a filtration process.
Data shown in Figure 2 demonstrate the efficiency of endotoxin removal by an ultrafilter. The Biopak® polisher is an ultrafiltration device that reliably removes even large endotoxin loads. The ultrafilter is positioned at the outlet of the water purification system. The water delivered is endotoxin-free and bacteria-free and does not require autoclaving.
Figure 2Demonstration of endotoxin removal by an ultrafilter (Biopak® polisher).
Endotoxin-free ultrapure water for cell culture
In conclusion, ultrapure (Type 1) water (with a resistivity of 18.2 MΩ•cm and a low TOC level), purified with an ultrafiltration cartridge, such as the Biopak® polisher, is suitable for preparing cell culture media. It is free from bacteria and endotoxins, as well as chemical contamination. Ultrafiltration is an efficient and simple way to obtain bacteria- and endotoxin-free water on-demand. No autoclaving of water is needed. Ultrapure water purified using ultrafiltration can be safely used for cell culture, including stem cell culture.
A range of water purification solutions adapted to the needs of scientists working with all types of cell culture is available.
Select and configure your optimal water purification system for your cell culture applications or request support from a lab water expert.
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References
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