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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/2.2/3
TITLE: MASTER CELL BANKING AT INRC
This protocol describes the procedure for master cell banking of new cell lines for the collection.
- 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.
- 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.
- 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.
- 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).
- Cells are further expanded to 3 or 4 x 175cm2 flasks and a master cell bank of 23 ampoules is generated.
- 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.
- 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).
- The following tests have to be performed for a full QC:
If the quality control procedures are completed satisfactorily, i.e.:
- viability and cell count are acceptable
- cultures are mycoplasma, bacteria and fungi negative
- isoenzyme test identifies the species as described by the depositor.
the cell line is released into the collection and a distribution bank is prepared according to protocol AHC/1998/3/2.3.
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