Purpose

To describe the preparation of a Working Cell Bank

 Safety

See SP 09-001 for lab safety considerations for the cell culture lab.

 Equipment

1. Laminar Flow Hood
2. Freezers, -70^oC or Rate-Controlled Freezer
3. Liquid Nitrogen Freezer

Materials

1. Cryovials, 1.8 ml (Nunc or equivalent)
2. Cryovial rack (Nunc or equivalent)
3. Sterile Centrifuge tubes, 50 ml (VWR # 21008-146 or equivalent)
4. Fetal Bovine Serum, heat inactivated (BioWhittaker # 14-503F or equivalent)
5. Sterile DMSO (Sigma # D2650 or equivalent)
6. Sterile Pipets of appropriate sizes
7. Ice
8. Permanent Marking Pen
9. Lab Coat
10. Latex Gloves
11. 70 % alcohol or equivalent
12. Trypan Blue, 0.4% (GIBCO # 630-5250AG or equivalent)
13. Hemocytometer
14. Nunc Freezing Container (# 5100-0001)
15. Freezer Log

Procedure

1. The Master Cell Bank must be tested for sterility and viability before the Working Cell Bank is prepared/
2. The Working Cell Bank consists of 50 vials of cells at 5 x 10⁶ cells/vial.
3. The day before freezing, refeed the cells with fresh medium, or add additional medium to suspension cultures to ensure that they are in log phase of growth.
4. Chill the Freezing medium on ice.
5. Label with cryotubes with the cell line name, the cells/vial, and the date.  Include on the label that the vials are the Working Cell Bank.  Place the vials in the cryotube rack and place the rack on ice.
6. Prepare a single cell suspension, use trypsin for adherent cell line.
7. Take an aliquot for a cell count.  Count the cells using a hemocytometer, according to the current revision of SP 05-009.  Determine the viability using trypan blue stain, according to the current revision of SP 09-005. NOTE:  The cells must have greater than 90% viability before proceeding.  If the cells are less than 90% viability, do not freeze.
8. Transfer the cell suspension to centrifuge tubes.
9. Centrifuge at 1000 rpm for 5 minutes, 2-8^oC.
10. Siphon off all the medium.
11. Slowly add chilled freezing medium to yield 5 x 10⁶ cells/ml.  Resuspend the cells in the freezing medium by gently pipetting.
12. Place the tube on ice.  Dispense the cells, 1 ml/vial, into the labeled cryovials.  Recap vials tightly.  Complete all vials.
13. Place the vials into the Nunc Freezing Container.  Replace the lid.
14. Place the Freezing Container in the -70⁰C Freezer overnight.
15. Transfer the vials to the vapor phase of the liquid nitrogen freezer.
16. Record the cell line information and location in the Freezer Log.