The impact of ultrapure water quality on biotech research

1st April 2013

The demand for pure water in media preparation and washing materials for mammalian cell culture is well established. J F Pilette reports.

The presence of metal ions and organics can be very harmful to cell culture and can prevent healthy cell growth. There have been various reports regarding the influence of fever-generating compounds (pyrogens) in water.

The importance of water quality on nerve cell survival and growth in serum-free media or media containing a low concentration of blood serum has been well documented. However, a large number of procedures for apure water' preparation remain unclear, and many researchers continue to use deionised water that has been distilled two or three times, guided only by their previous experience.

Water purification via double or triple distillation is time-consuming and is subject to contamination by organics, pyrogens, and other materials from the environment and from storage containers.

A recent study was performed on cell culture growth using different types of water quality. When using purified water from a deionisation system, very poor cell growth quality and quantity were obtained (Fig. 1). When using purified water from the distillation of deionised water, although better, results were still not satisfactory. Poor growth resulted in an incomplete network structure (Fig. 2).

When using ultrapure water from a Milli-Qi Synthesis system combining UV photooxidation and an ultrafiltration cartridge, good cell growth and a uniform network structure were obtained. This is due to low TOC, endotoxin and metal contents (Fig. 3).

This study illustrates the fact that the latest advances in biotechnological research have led to new requirements in water purity. Researchers not only require ultrapure water with constant and low ionic and organic contamination; they also need to target specific parameters such as bacteriological contamination, pyrogens, RNases and DNases.

In order to reach the expected water quality, a correct combination of water purification techniques has to be used.

A complete water purification system needs to typically produce pure (Type 2) water from tap water. The pure water produced should be stored in a reservoir specifically designed to prevent its recontamination. Type 2 water can be used directly for non critical applications or further processed to produce ultrapure (Type 1) water for critical applications.

Millipore's Elix systems process potable tap water in three consecutive steps: pretreatment, reverse osmosis and electrodeionisation.

The reverse osmosis step allows removal of over 95 per cent of the ionic contaminants dissolved in tap water and more than 99 per cent of the organic substances with a molecular weight above 100 dalton. The RO membrane also rejects more than 99 per cent of the colloids, particulates, viruses and bacteria present in tap water.

Electrodeionisation (EDI) will remove the few ions that went through the RO membrane. The resistivity of the pure water produced by such a system is typically above 5 at 25°C, the TOC level is below 30 ppb and there are very low levels of particulates and colloidal contaminants. This type of water can be used directly for non critical applications such as glassware washing and rinsing or preparation of non critical buffers, solutions and culture media. The pure water produced is stored in a reservoir that should answer specific criteria:

€ Made from materials such as polyethylene, to avoid water recontamination.

€ Opaque walls to prevent the development of algae.

€ Smooth inner surfaces to prevent the development of a biofilm.

€ Conical bottom to allow full drainability in order to avoid particulate deposits at the bottom of the reservoir.

€ An overflow system that operates when there is an excess of pure water production and does not allow air from the drain to enter the reservoir.

€ Air vent able to remove ionic (CO2), organic (solvents such as acetone, methanol, toluene), particulate and bacteriological contaminants from the ambient air entering the reservoir when water is drawn.

€ A UV light (254 nm) irradiation module allowing the intermittent automatic irradiation of the water stored inside the reservoir to prevent bacteriological development.

Pure water can be directly sourced from the reservoir through a tap or a delivery pump, or processed further by another system to produce ultrapure water.

The Milli-Q Synthesis is a water polisher that combines a range of advanced purification techniques designed to remove trace contaminants that may affect molecular biology, biochemical and cell biology research.

Ions and heavy metals are removed by high quality mixed bed ion-exchange resins selected for high capacity and fast binding kinetics.

Organic substances are eliminated by a combination of two methods: adsorption on synthetic activated carbon and destruction by photo-oxidation.

A qualified Pyrogard 5000 ultrafiltration cartridge ensures the removal of RNases, DNases, and Pyrogens that might interfere with delicate experimentation. Finally, a 0.22 µm sterilising membrane filter ensures that the delivered water is free of particulates and micro-organisms.

The quality of the water produced by the system is continuously monitored by an in-line resistivity meter for detection of ionic contaminants, and a TOC monitor for detection of organic substances. Both meters are sensitive down to ppb levels and pass the USP24 suitability tests.


J F Pilette is worldwide product manager for the Lab Water Division of Millipore, MA, USA.





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