CRYOPRESERVATION OF CELL CULTURES (Appendix X)

Generally research use only:

1. When a new bottle of cells to be frozen arrives, transfer 30 mL aliquots of cells into 125 cm² flasks (about 3 flasks).

2. Incubate at 36^oC until a complete monolayer is obtained (approximately 4 - 5 days).

3. Follow steps 1 - 5 in Appendix IX (Trypsinization and maintenance of monolayer cell cultures).

4. Transfer the trypsinized monolayer to a sterile 15 mL centrifuge tube.

5. Centrifuge at 2000 rpm (700 x g) for 10 minutes.

6. Discard the supernate and resuspend the cells in 10 mL cell freezing medium.

7. Distribute 1.0 mL volume into freezing vials labelled with cell line information.

8. Place the vials into a "Mr. Frosty" freezing container and hold in -70^oC freezer for 2 - 3 hours.

9. Record in the computer Freezer Program details of the cell lines stored. Use study "CEL".

10. Transfer the vials to liquid nitrogen freezer.

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Reference

Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infection. A.P.H.A. 1989. Sixth Edition. Pg. 72.

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CRYOPRESERVATION OF VIRUS ISOLATES (Appendix XII)

1. Record in the computer Freezer Program details of the isolate stored.

a. Select "A. Clinical Records".

b. "Add new entries?": enter "Yes".

c. Enter lab number, ensure patient data is correct, enter date frozen, enter study (ie "VIR"), ensure specimen type is correct.

d. Enter name of isolate to be frozen once.

e. Note the freezer locations in which the frozen isolates are to be placed.

- Freezer "G" & "J" refer to Liquid Nitrogen freezers.

- Shelf no. refers to the canister number.

- Box no. refers to the cane number. Each cane holds 5 freezer vials.

 

2. Discard maintenance medium on monolayer to be frozen.

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3. Add 1.0 mL viral freezing medium (see Appendix VII) to positive cells. Scrape monolayer and mix well with freezing medium.

 

4. Distribute into freezing vial labelled with LIS label.

 

5. Store the vial immediately in liquid nitrogen freezer in the location specified by the freezer program.

 

FREEZING CAP SPECIMENS AND ISOLATES:

1. After planting CAP specimens, add 1 mL of viral freezing medium to the sample and transfer to a freezing vial. Enter into the freezer program as described above using "VQC" for study. Enter "CAPSAM" as isolate #1.

 

2. Freeze one vial of CAP viral isolates after identification using steps 2 - 5 described above. The frozen isolate will be isolate #2 and is entered using the code for the name of the virus. (The original sample and frozen isolate may be on different canes, this is acceptable).

 

Reference

Laboratory Diagnosis of Infectious Diseases. 1988 Volume II. Pg. 268.

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