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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/2.2/2
TITLE: MASTER CELL BANKING
This protocol describes the procedure for master cell banking of new cell lines for the collection.
AHC/1998/3/2.5/1, 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).
DNA fingerprinting is performed: after cells have been expanded into two 75cm2 flasks, one flask is submitted for DNA fingerprint (AHC/1998/3/3.1/2.3). For hybridoma cell lines a DNA fingerprint is not carried out, however, supernatant is tested for correct antibody class and subclass (AHC/1998/3/3.5/2).
Cells are further expanded to 3 or 4 x 175cm2 flasks and a master cell bank of 23 ampoules is generated.
One ampoule (except for hybridoma cell lines) is submitted for DNA fingerprint (AHC/1998/3/3.1/2.3), one ampoule and 1ml of supernatant is submitted for BVDV testing (AHC/1998/3/3.4/2.2 and AHC/1998/3/3.4/2.3). If cells are of human lymphoid origin, additional testing (such as HIV and HTLV testing) as recommended by the Curator has to be performed.
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/1998/3/3).
- 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 (
9.1 The following tests have to be performed for a full QC:
Isoenzyme analysis (AHC/1998/3/3.1/2.2)
DNA fingerprint (AHC/1998/3/3.1/2.3)
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)
- Cell count and viability (
If the quality control procedures are completed satisfactorily, i.e.:
- viability and cell count are acceptable
- cultures are mycoplasma, bacteria and fungi negative
- DNA fingerprint of master stock profile is identical to profile from the initial growing culture
- 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/1998/3/2.2/3.
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