INTRODUCTION

This protocol describes the procedure for master cell banking of new cell lines for the collection.

1. On arrival of a new cell line, an accession number is given. The New Cell Lines for Master Bank form is filled in and an entry is created on the computer database.
2. If cells arrive frozen, ampoules are stored in liquid nitrogen tanks until culture can be started (AHC/1998/3/2.5/2). Growing cultures are stored at the appropriate temperature for one day before subculture. If cells are non-viable the depositor is contacted.
3. Cells are subcultured according to the instructions of the depositor (see AHC/1998/3/2.1/2 and appendix). A 25cm² flask containing antibiotics (penicillin (100U/ml) and streptomycin (0.1mg/ml) may be kept as backup. If only one ampoule or growing flask has been received from a depositor a token freeze (up to 6 ampoules) is made from a 25cm² flask within the first three passages.
4. After two passages without antibiotics, a 25cm² flask is tested for mycoplasma contamination by Hoechst stain (AHC/1998/3/3.2/2.1) and by PCR technique (AHC/1998/3/3.2/3.1).
5. Cells are further expanded to 3 or 4 x 175cm² flasks and a master cell bank of 23 ampoules is generated.
6. If cell growth decreases or stops and the production of master cell bank is not possible within 8 to 12 weeks, the depositor should be contacted for further advice. If no response is received within 2 to 4 weeks cells are frozen (2 to 4 x 10⁶ cells/ampoule) generating the maximum number of ampoules possible.
7. After at least one week of storage in liquid nitrogen, one ampoule is resuscitated and the cells are prepared for full QC (AHC/1998/3/2.6/2).
8. The following tests have to be performed for a full QC:

• Cell count and viability (AHC/1998/3/3.1/2.1)
• Isoenzyme analysis (AHC/1998/3/3.1/2.2)
• Microbial detection assays for microbial and fungi contamination (AHC/1998/3/3.3/2.1, AHC/1998/3/3.3/2.2,
• Antibody secretion (AHC/1998/3/3.5/2)

• viability and cell count are acceptable
• cultures are mycoplasma, bacteria and fungi negative
• isoenzyme test identifies the species as described by the depositor.

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998