ECACC Handbook

Fundamental Techniques in Cell Culture . . . a Laboratory Handbook

1.0   The ECACC and its Collections 2.0   Design and Equipment for the Cell Culture Laboratory 3.0   Safety Aspects of Cell Culture 4.0   Sourcing of Cell Lines 5.0   Main Types of Cell Culture 6.0   The Cell Environment 7.0   Cryopreservation and Storage of Cell Lines 8.0   Good Cell Banking Practices 9.0   Quality Control Considerations 10.0   Authentication of Cell Lines 11.0   Alternative Cell Culture Systems 12.0   Cell Culture Protocols Glossary of Terms Who to Contact

8.0   Good Cell Banking Practices

It is bad practice to maintain a cell line in continuous or extended culture for the following reasons:

• Risk of microbial contamination
• Loss of characteristics of interest (i.e. surface antigen or monoclonal antibody expression)
• Genetic drift particularly in cells known to have an unstable karyotype (i.e. CHO Prod. No. 85050302-1v1, BHK 21 Prod. No. 85011433-1v1)
• Loss of cell line due to exceeding finite life-span e.g. human diploid cells such as MRC-5 (Prod. No. 84101801-1v1)
• Risk of cross contamination with other cell lines
• Increased consumables and staff costs

All of these potential risk factors may be minimized by the implementation of cell banking system as described below. This type of system is known as a tiered banking system or Master Cell Banking system. On initial arrival into the laboratory a new cell culture should be regarded as a potential source of contamination from bacteria, fungi and mycoplasma and should be handled under quarantine conditions until proven negative for such microbial contaminants. Following initial expansion 3-5 ampules should be frozen as a token stock before a Master Bank is prepared. One of the token stock ampules should then be thawed and expanded to produce a Master Bank of 10-20 ampules depending upon the anticipated level of use.

Ampules of this bank (2-3) should be allocated for quality control comprising confirmation that the cell count and viability of the bank is acceptable and that the bank is free of bacteria/fungi and mycoplasma. Additional tests (such as viral screening and authenticity testing) may also be required. Once these tests have been completed satisfactorily an ampule from the Master Bank should be thawed and cultured to produce a Working Bank. The size of this bank will again depend on the envisaged level of demand. Quality control tests (cell count and viability and the absence of microbial contaminants) are again required prior to using the cultures for routine experimentation or production. It is also important at this stage to confirm that the Master and Working Banks are genetically identical by DNA profiling techniques.

Implementation of this banking system ensures:

• Material is of a consistent quality
• Experiments are performed using cultures in the same range of passage numbers
• Cells are only in culture when required
• The original cell line characteristics are retained

Click here for Figure 3. Schematic Representation of a Tiered Cell Banking System

Notes

1. The number of ampules prepared for Master and Working Banks depends upon the forecast demand for their use.
2. The number of ampules sampled for quality control is dependent upon the size of bank. Ideally 5-10% of the bank should be tested before use.
3. Ampules from the Working Cell Bank should be used sequentially keeping cells in culture for not more than a predetermined number of cell doublings. This number will be least in the case of cell lines having a finite life-span (e.g. diploid lines).
4. The Working Bank should be replenished from an ampule of the Master Bank. This should be done in sufficient time to allow the quality control to be completed.
5. A new Master Bank should be prepared before the number of original Master stock drops below five ampules.
6. The panel of quality control tests performed depends upon the use intended e.g. regulatory authorities may require additional tests such as viral screening and karyotypic studies.

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