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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/2.6/2

TITLE: PREPARATION OF CELL LINES FOR QUALITY CONTROL

INTRODUCTION

This protocol describes the procedure for preparation of cell lines for Quality Control procedures such as microbial detection, isoenzyme analysis, assessment of antibody production by hybridomas etc.

PROCEDURE

  1. Resuscitate one ampoule (AHC/1998/3/2.5/2) in to 4ml of media (culture details on cell line safety datasheet or on catalogue database) for quality control testing. In addition store allocate one ampoule for DNA testing (AHC/1998/3/3.1/2.3).
  2. Perform cell count and viability check (AHC/1998/3/3.1/2.1) on 0.2ml of cell suspension and record the results in the cell banking book.
  3. Centrifuge cells at 150g for 5 min. Pour off the supernatant and resuspend cells in 10ml of appropriate media in a 1 x 25cm2 tissue culture flask. If no CO2 incubator is used, gas with 5% CO2 in air, and incubate at 35 to 38°C as appropriate.
  4. Subculture cells when confluent. Check microscopically for characteristic growth properties (healthy cells) and microbial contamination, i.e. rods, cocci or yeast cells.
  5. If the cell line develops microbial contamination discard the flask and repeat steps 1-4, omitting step 2. If the cell line is contaminated a second time, discard the complete freeze run and document this on the relevant database.
  6. If the cell line develops uncharacteristic growth properties, inform the responsible and put a hold on the freeze run in question until the problem has been investigated.
  7. If the cell line is a master stock prepare a 1 x 75cm2 flask of cells for isoenzyme analysis (according to AHC/1998/3/3.1/2.2).
  8. If the cell line is a hybridoma prepare a 1 x 25cm2 flask of cells for antibody testing (according to AHC/1998/3/3.5/2).
  9. At the end of approx. 7 days cells are tested for mycoplasma and bacteria and fungi (AHC/1998/3/3.3/2.1).

Acceptance Criteria

Cell lines can fail prior to 'in-house' mycoplasma testing for the following reasons:

  • Viability less than 80% viable, or
  • Viability less an 2 x 106 viable cells/ampoule, or
  • Fail to become confluent within 5 days after resuscitation

If any of these criteria fail, it is up to the discretion of the Head of Quality Control to accept or reject the cell line or cell line batch.

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

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