Quality Control Considerations

Introduction

Quality is important in all aspects of tissue culture since the quality of materials used i.e. media and other reagents) will affect the quality of the cultures and products derived from them. The main areas of quality control that are of concern for tissue culture are:

*The quality of the reagents and materials

*The provenance and integrity of the cell lines

*The avoidance of microbial contamination

1.Reagents and Materials

A potential source of contamination is reagents and materials, in particular bovine serum which has been identified as a source of bovine viral diarrhoea virus (BVDV). Porcine trypsin is also a potential source of Mycoplasma hyorhinis. Good quality reagents and materials are available from numerous manufacturers of tissue culture media and supplements. In addition manufacturers including Sigma will carry out a range of quality control tests including screening for mycoplasma and BVDV and supply a Certificate of Analysis with their products. These state the product and lot numbers and forms a vital part of record keeping and tracking of reagents used in the production of cell stocks. It is advisable to further test key reagents such as FBS to ensure that they are ‘fit for purpose’ due to batch-to-batch variation.

Manufacturers of sterile plastic ware (flasks, centrifuge tubes, pipettes) designed for tissue culture use are also supplied with Certificates of Analysis for each batch produced, which should be kept for future reference.

2.Provenance and Integrity of Cell Lines

The sourcing of cell lines can have an important effect on quality since freshly imported cell lines are a major source of contamination. The advantages of obtaining cell lines from a recognized source such as a culture collection are:

Contaminant free

Fully characterized and authenticated in terms of DNA profile and species of origin

Supplied with a detailed data sheet

Once cell lines have been obtained from a reputable source it is important to implement master and working cell banking procedures and the associated quality control steps such as routine testing for microbial contaminants and confirming the identity of cultures.

3.Avoidance of Microbial Contamination

Potential sources of contamination include other cell lines, laboratory conditions and staff poorly trained in core areas such as aseptic techniques and good laboratory practice. Thus the use of cells and reagents of known origin and quality alone is not sufficient to guarantee quality of product (cell stock or culture products); it is necessary to demonstrate quality throughout the production process and also in the final product. Routine screening aids the early detection of contamination since all manipulations are a potential source of contamination.

The 3 main types of microbial contaminants in tissue culture are:

* Bacteria and Fungi

*Mycoplasma

*Viruses

Bacterial and Fungal Contamination

Bacterial contamination is generally visible to the naked eye and detected by a sudden increase in turbidity and color change of the culture medium as the result of a change in pH. The cell culture may survive for a short time but the cells will eventually die. Daily microscopic observation of cultures will ensure early detection of contamination and enable appropriate action to be taken as soon as the first signs of contamination become apparent (see below). In addition specific tests for the detection of bacteria and fungi should be used as part of a routine and regular quality control screening procedure (see Protocol 8).

Mycoplasma Contamination

Mycoplasmas are the smallest free-living self-replicating prokaryotes. They lack a cell wall and lack the ability to synthesize one. They are 0.35m in diameter and can be observed as filamentous or coccal forms. There are 5 major species that are tissue culture contaminants, namely M. hyorhinis, M. arginini, M. orale, M. fermentans and Acholeplasma laidlawii.

The effects of mycoplasma infection are more insidious than those of bacteria and fungi inducing several long term effects. These include:

Reduced growth rate

Morphological changes

Chromosome aberrations

Alterations in amino acid and nucleic acid metabolism

However, despite these well-documented effects the presence of mycoplasma is often not tested for with the consequence that in such laboratories the majority of cell lines are positive for mycoplasma. Mycoplasma contamination is difficult to detect requiring the use of specialist techniques (see Protocol 9 - Isolation by culture and Protocol 10 – Detection by DNA staining). In the past only specialist laboratories, such as culture collections, have performed these tests. However a variety of commercial kits are now available although the performance characteristics of these kits can be extremely variable. A combination of these should be used as part of a routine and regular quality control screening procedure. Culture collections such as ECACC are able to test cultures if required. Mycoplasma testing products are available from Sigma, refer to pages 489-490 of Life Sciences Catalogue.

Viral Contamination

Some cell lines contain endogenous viruses and secrete virus particles or express viral antigens on their surface (e.g. EBV transformed lines). These cell lines are not considered contaminated. However, bovine serum is a potential source of bovine viral diarrhoea virus (BVDV) contamination. Use of infected serum will lead to contamination of cell lines with the virus. Contamination of cell lines with BVDV may cause slight changes in growth rate but since this virus is non-cytopathic macroscopic and microscopic changes in the culture will not be detected. Suppliers of bovine serum are aware of this and screen sera accordingly and generally serum is sold as BVDV tested.

4.Environmental Monitoring

It is good practice to monitor the laboratory environment where cell cultures and their products are prepared. Class II microbiology safety cabinets should be checked every 6 months to ensure that they are working efficiently. However it is also advisable to monitor the number of contaminants within the cabinet by periodically placing open settle plates (blood agar bacteriological culture plates) on the cabinet work surfaces. In addition settle plates should be used to assess airborne microbial burden at selected points around the laboratory. Plates should be left open for a period of 4 hours. After this time they should be covered, placed in sealed boxes and incubated at 32oC and 22oC for up to 7 days. At the end of this period the plates should be examined for the presence of microbial growth. The position of each plate in the cabinet should be recorded and results stored for trend analysis.

Acceptable limits should be defined in terms of “alert” levels and “action” levels, the actual values being dependent on the criticality of the work and the levels of cleanness that can be achieved under normal operating conditions.

5.What to do in the event of contamination

One hugely under-estimated problem in tissue culture is the routine use of antibiotics. Continuous use of antibiotics is unnecessary and can lead to the development of resistant strains that are difficult to eradicate and may require the use of more exotic antibiotics that may be toxic to the cell cultures. In addition the use of antibiotics may mask a low level of contamination.

Once a contamination has been detected, whether is it due to bacteria, fungi or mycoplasma, the recommended course of action is to discard the culture and continue the work with earlier stocks that are known to be free of contaminants or obtain fresh stocks from a recognized source. However if this is not possible eradication of the contaminant may be attempted with the use of antibiotics. In addition culture collections such as ECACC will attempt to eradicate any contaminants if required. Please contact ECACC for further details.

Viral infections are virtually impossible to remove from cultures since they do not respond to antibiotic treatment. Also, since they are intra cellular parasites it is not possible to remove them by centrifugation or other separation techniques. If virus free stocks or a virus free alternative is not available then a thorough risk assessment should be undertaken prior to continuing work with the infected cell line.