Quality Control Considerations in Cell Culture

Cook Book Sept 2010 Volume 12, Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

Introduction

Quality is important in all aspects of tissue culture. The quality of materials used (cell lines, media and other reagents) will affect the quality of the cultures and the subsequent scientifi c data 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

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Reagents and Materials

Potential sources of contamination are reagents and materials, in particular bovine serum which has been identifi ed 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 such as Sigma will carry out a range of quality control tests including screening for mycoplasma and BVDV and supply a Certifi cate of Analysis with their products. These state the product and lot numbers and form 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 foetal bovine serum to ensure that they are ‘fi t for purpose’ due to batch-to-batch variation.

Manufacturers of sterile plastic ware (fl asks, centrifuge tubes, pipettes) designed for tissue culture use also supply Certifi cates of Analysis for each batch produced, which should be kept for future reference.

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Provenance and Integrity of Cell Lines

The sourcing of cell lines can have an important effect on quality; freshly imported cell lines are a major source of contamination. The advantages of obtaining cell lines from a recognised source such as a culture collection are that the cultures will be:

  • Contaminant free
  • Fully characterised and authenticated in terms of DNA profi le 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 confi rming the identity of cultures.

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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 suffi cient to guarantee quality of product (cell stock or culture products); it is necessary to demonstrate quality throughout the production process and also in the fi nal product. Routine screening aids the early detection of contamination since all manipulations are a potential source of contamination.

The three 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 colour 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 fi rst signs of contamination become apparent. In addition, specifi c 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 on page 54).

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 0.3μm in diameter and can be observed as fi lamentous 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 in cell cultures. These include:

  • Altered 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 diffi cult to detect requiring the use of specialist techniques (see Protocol 9 on page 56 - Isolation by Culture and Protocol 10 on page 58 – 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. ECACC tests cultures for the presence of mycoplasma on a routine basis and offers a mycoplasma testing service.

Viral Contamination

Some cell lines contain endogenous viruses and secrete virus particles or express viral antigens on their surface (e.g. Epstein-Barr Virus (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.

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Environmental Monitoring

It is good practice to monitor the laboratory environment where cell cultures and their products are prepared. Class 2 microbiology safety cabinets, fi tted with HEPA fi lters, should be tested every 6 months to ensure that they are working effi ciently, i.e. the level of airfl ow across the fi lter should be tested. However, it is also advisable to monitor the level of ingress of contaminants into the cabinet by periodically placing open settle plates (Tryptone Soya Bean 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 32°C and 22°C 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 defi ned in terms of “alert” levels and “action” levels, the actual values being dependent on the containment classifi cation of the work area, the criticality of the work and the levels of cleanliness that can be achieved under normal operating conditions.

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Aseptic Technique and Contamination Control

Personal Hygiene

When entering the laboratory it is important to wash hands since this will remove dry skin and loosely adherent microorganisms which could potentially contaminate cell cultures. Gowns and surgical gloves must be worn. Gloves should be frequently swabbed with 70% (v/v) sterile isopropanol. Other personal protective equipment includes head caps and face masks, but these are not always necessary, particularly when a class 2 microbiological safety cabinet is being used. Long hair should be tied back to remove obstruction and reduce the risk of contamination.

Working within the Microbiological Safety Cabinet

When working within the cabinet the operator should remember that the air-fl ow does not make the environment sterile but keeps it clean. Before any practical procedure is conducted the cabinet should be stocked with all the materials required for the experiment. In doing so the operator restricts the number of times that their hand/arm is removed from the cabinet into a non-clean environment. When stocking up the cabinet it is essential that a clutter free condition is maintained. Each item within the cabinet should be positioned to minimise movement and traffi c over the area where cell culture operations are performed. Both the rear and front of the cabinet should be cleared to achieve maximum airfl ow. Flasks and dishes should be the last items to enter the cabinet. All items that enter the cabinet must be sprayed with 70% (v/v) sterile isopropanol to prevent dust and particulate from entering the cabinet. Twenty minutes should elapse before any tops or containers are opened to allow the airfl ow to purge the work area of particulates that may have been introduced.

Pipetting and Prevention of Aerosols

Disposable plastic pipettes (1ml, 2ml, 5ml, 10ml and 25ml) are the easiest forms to use for cell culture. Microbial and cellular contamination can arise through pipetting errors such as spillage of material. Adherence to the following guidance can minimise contamination and safety risks associated with pipetting:

  • Never mouth pipette
  • Use automatic pipette aids, with one pipette aid designated to each cabinet. Ensure the pipette fi ts into the pipette aid without any force. To avoid contamination disinfect the pipette aid regularly and ensure that fi lters are changed regularly (weekly).
  • Use plugged pipettes when transferring medium
  • Avoid drawing liquid into the pipette plug. Use an individual pipette once only.
  • To avoid generating aerosols do not create bubbles in the medium or pipette. Aerosols can spread contaminating microorganisms and by introducing cells into the air increases the potential risk of crosscontamination.
  • Clean spills that arise immediately with 70% (v/v) sterile isopropanol.

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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 diffi cult 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 it is 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 recognised source.

Viral infections are virtually impossible to remove from cultures since they do not respond to antibiotic treatment. Also, as 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.

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Did You Know?

  • Mycoplasmas are the smallest free-living selfreplicating prokaryotes. They lack a cell wall and do not have the ability to synthesize one.
  • The main source of microbial contamination in the cell culture laboratory is the operator.

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