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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES
REFERENCE NO: AHC/1998/3/2.2/2
TITLE: MASTER CELL BANKING
INTRODUCTION
This protocol describes the procedure for master cell banking of new cell lines for the collection.
PROCEDURE
- 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/1998/3/2.5/1, 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).
9.1 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
- 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.
Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998
© The CABRI Consortium 1999 -
2023
This work cannot be reproduced in whole or in part without the express written permission of the CABRI consortium.
Site maintained by Paolo Romano. Last revised on February 2023.
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