Tissue Culture Uses For Endotoxin-Free Water

Tissue and cell culture are important tools in numerous fields of biomedical research. Most steps of the tissue culture process make use of water, including media and buffer preparation and glassware washing. Hence, water quality may play an important role in experimental outcomes. Cardiomyocyte isolation and culture are presented here as an illustration of the potential impact of water contamination on tissue culture experiments.

What are Endotoxins?

Endotoxins are key components of the outer membrane of most gram-negative bacteria. They are a class of lipopolysaccharides (LPS). The endotoxin molecule includes a long polysaccharide chain that carries the endotoxic feature of the molecule (O-antigen) and a lipid component (Lipid A) as shown in Figure 1.

Figure 1. Structure of lipopolysaccharides

Based on the bacterial strain, LPS molecular weight may range from 5kDa to 20kDa. In aqueous solution LPS forms vesicles or micelles as large as one million as shown in Figure 2. Endotoxins stimulate macrophages and phagocytes to produce pro-inflammatory cytokines. They also impact the growth, function and cloning efficiency of many other cell types, including cells lacking endotoxin receptors.

Figure 2. Illustration of a micelle

How to Remove Endotoxins from Water

Although ultra pure water is considered to be an inhospitable environment, it can nevertheless harbor some endotoxin-producing bacteria. Filtration or autoclaving may be used to remove bacteria by autoclaving or filtration, but few sterilization methods can destroy the rest of the endotoxins.

BioPak™ ultrafiltration cartridges contain thin polysulfone hollow fibers designed to remove endotoxins and other macromolecules from water as shown in Figure 3.

Ultrafiltration is a pressure-driven purification process in which water and low-molecular weight substances permeate a membrane while colloids, particles and macromolecules are retained. Figure 4 shows how endotoxins spiked into water are retained by the ultrafiltration cartridge.

Large endotoxin quantities were added to ultrapure water (up to 1,000EU/ml). Both the "challenge" water and the purified water ("output") were analyzed using the Limulus Amebocyte Lysate assay (QCL-1000^® Chromogenic LAL Endpoint Assay, Cambrex Corporation, Walkersville, MD, U.S.A). The ultrafiltration process was used for removing most of the endotoxin (more than 99.9 %). When the Biopak™ cartridge was used under normal conditions (no endotoxin challenge), no endotoxin activity was detected.

Figure 3. Scanning electron microscopy (SEM) photo showing a cross-section of one of the ultrafiltration hollow fibers contained in a BioPak ultrafiltra

Figure 4. Endotoxin removal from water using the Biopak™ ultrafiltration cartridge

Cardiomyocyte Culture - A Practical Illustration of the Effect of Endotoxins on Tissue Culture

A significant loss of cardiac myocytes, the muscle cells of heart tissue characterizes congestive heart failure. Strategies for heart repair using cardiomyocytes are currently under study and isolated cardiomyocytes are a valuable tool for studying the heart muscle.

Cardiomyocytes were isolated from adult rat cardiac tissue after perfusion with Liberase™, a blend of collagenase and neutral protease (Blendzyme 3, Roche Applied Science, Mannheim, Germany) in Krebs-Henseleit buffer (Sigma- Aldrich, St. Louis, MO, U.S.A.).

The freshly dissociated ventricular cardiomyocytes were washed in a HEPESbased buffer containing 25mM HEPES, 0.13mM sodium chloride, 4.5mM potassium chloride, 5mM glucose, 1.2mM potassium phosphate monobasic, 20 g/l bovine albumin and 10mM 2,3-butanedione monoxime (all from Sigma-Aldrich). Cells were suspended in minimum essential medium and plated on laminin-coated plates.

Ultrapure water, containing very few ions (resistivity 18.2 MΩcm) and an extremely low level of organic contaminants (total organic carbon < 15ppb) was used for preparing buffers for the culture and the extraction procedure.

• Water A: Water purified by ion exchange and activated carbon, followed by ultra-filtration at the point of use [with a Simplicity^® water purification system and a Biopak™ ultra-filtration cartridge (EMD Millipore Corporation, Billerica, MA, U.S.A.)]. Only freshly produced water was used.
• Water B: Water purified by reverse osmosis, ion exchange and activated carbon [with a Milli-U™ water purification system (EMD Millipore Corporation, Billerica, MA, U.S.A.)]. This water was stored in a carboy before use, and was not treated by ultrafiltration.

Cardiomyocytes are sensitive to endotoxins both in vivo (as in the case of systemic infection) and in vitro. Bacteria and endotoxin levels in the water stored in the carboy were measured.

The concentration in bacteria was approximately 4,000 CFU/ml and the endotoxin concentration was 8 EU/ml. The endotoxin level present in freshly produced ultrapure water is below the detection limit of commercially available endotoxin test kits. 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 as shown in Figure 5.

However, when ultrapure water was stored before use and not treated with ultrafiltration (Water B), few viable cells were obtained. Therefore, the levels of endotoxin present in Water B were high enough to have a deleterious effect on cardiomyocyte cells during the isolation process.

Figure 5. Photomicrographs of isolated cardiomyocytes courtesy of Drs. C. Plin and R. Zini

Conclusions

High resistivity purified water (18.2 MΩ.cm) with low organic content (total organic carbon < 15 ppb) and no endotoxins should be used when performing tissue culture experiments. Storage of the water after purification should be avoided as the water quality may degrade and bacterial contamination may occur, which leads to the production of endotoxins.

Endotoxins may cause obvious changes in cell growth and morphology, as was the case for cardiomyocytes. They also may have more subtle effects and only influence cell function. Unknowingly working with endotoxin-contaminated cultures may lead not only to inaccurate or erroneous results, but also result in loss of time, money and effort. Placing an ultrafiltration cartridge at the water purification system's point of use will eliminate this potential risk.

About EMD Millipore - Lab Water Business Unit

Water is the most commonly used solvent in laboratories and constitutes often more than 99% of the mass of solutions used in experimentations. The quality of water used in the lab is therefore critical for the success of the tests performed.

This information has been sourced, reviewed and adapted from materials provided by EMD Millipore - Lab Water Business Unit.

For more information on this source, please visit EMD Millipore - Lab Water Business Unit.