QUALITY CONTROL, MAINTENANCE, PRESERVATION, STORAGE AND ACCEPTANCE OF CELL LINES

LABORATORY PROCEDURES FOR PLANT CELL LINES

REFERENCE NO.: PC/1998/3/2

TITLE: QUALITY CONTROL, MAINTENANCE, PRESERVATION, STORAGE AND ACCEPTANCE OF CELL LINES - FROZEN CULTURES -

INTRODUCTION

The following procedure describes the handling of plant cell lines provided to the collection centre in the form of frozen ampoules or as living cultures together with a brief description of a cryopreservation method for these cells.

If the living cultures or reactivated samples of frozen cultures passed the preliminary quality checks (PC/1998/2/2 and PC/1998/2/3) they are further propagated to check whether cultivation can be managed at the collection centre and to produce material for final quality check and authentication. In case of cell lines which should be stored in the frozen state it also has to be tested whether the cryopreservation procedure given by the depositor can be successfully carried out and whether the cultures recovered from freezing are identical to the original cell line. When these tests have been carried out and the cultivation and cryopreservation conditions are acceptable for the collection centre the cell line is finally included into the public collection.

PROCEDURE

1. Living cultures or cells recovered from the frozen state are propagated until enough material has been produced for characterisation or at least for three transfer cycles.

2. Growth and morphological characters are collected by the standard procedure (PC/1998/3/1.1) and stored in the database.

3. 2 g of callus or suspension cells (fresh weight) are extracted with methanol according to the standard procedure (PC/1998/3/1.2).

4. The extracts are analysed with HPLC (PC/1998/3/1.3) and the HPLC profiles are stored in a data base.

5. Additionally UV spectra of the unpurified extracts may be measured and stored in a data base (PC/1998/3/1.4)

6. The cell line is further propagated and the cryopreservation procedure as well as the recovery of frozen cells is carried out as described by the depositor.

7. If cells fail to regrow after freezing the cryopreservation procedure is repeated once more. In case the regrowth of cells can again not be achieved the accession procedure is halted and the depositor is informed (PC/1998/3/2 Appendix 1).

8. The quality check and authentication is repeated using cells regrown from samples cryopreserved and recovered at the collection centre as described in points 2 - 5 of this procedure.

9. If the results of the second quality and authentication check are identical to the results of the first one’s the cell line is accepted for inclusion into the public collection.

9.1 All data are entered into the data base

9.2 40 ampoules are frozen. 20 ampoules each are stored in the "Working Cell Bank" and the "Master Cell Bank" (see PC/1998/3/4.1).

10. If the results of the of the second quality and authentication check differs from the results of the first the procedure may be repeated as often as acceptable dependent upon the importance of the cell line.

11. If no better results can be achieved after repeated trials a sample is sent back to the depositor. He is asked to decide if the culture sample is identical to his original culture or whether the differences are acceptable (Standard Letter PC/1998/3/2 Appendix 2).

12. If the depositor decides that the cryopreservation procedure has been successful to an acceptable level the culture is included into the public collection and it proceeds as described under Point 9.

Guidelines prepared for CABRI by DSMZ, 20 Jan. 1998; updated August 1999
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