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.

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). 

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. 

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.

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