Cryo-preservation of Cell lines.

 

PROCEDURE:

  1. Ensure the Mr. Frosty (fisher, cat. 15-350-50) is pre-chilled to -20 C.
  2. Label each cryo-vial with the following information:

                                                              i.      Cellline

                                                            ii.      Population doubling (PD) #

                                                          iii.      Approximate Cell # or dish/flask size

                                                          iv.      Freeze date

                                                            v.      Operator Initials

  1. Trypsinize and collect cells in centrifuge tubes
  2. Centrifuge cells at 700-900 rpm (~ 500g) for 2 minutes to pellet.
  3. Aspirate medium and replace with 1 mL of cold Freezing media. Re-suspend pellet into a homogeneous cell suspension.
  4. Transfer 1mL of cell suspension to each labeled cryovial
  5. Put the cryovials in a -20C Mr. Frosty and place in a -80C freezer.
  6. After 40-50 minutes, transfer vials to liquid nitrogen storage.
  7. All vials frozen must be recorded in the freezer logbooks, and/or in the respective computer database(s) as early as possible.

 

Extra Notes:

5e6 cells per mL is a good quantity for freezing cells. The cells that were shipped to your lab were frozen in culture media plus 5%DMSO. A few people have also used 10% DMSO successfully.

 

I’ve attached our general protocol for freezing cells. The Mr. Frosty takes the cells down a degree a minute, available from FISHER, Cat; 15-350-50

 

Thawing Cryo-preserved cells

PROCEDURE:

  1. Remove cryovial from cryostorage tank and put in -20C Mr. Frosty or on ice for transfer to water bath.
  2. Thaw the vial by gentle shaking in a 37C water bath or by holding in hand for approximately 2 minutes.
  3. Observe and remove the vial from the water bath when only a small piece, approximately 3mm, of ice remains.
  4. Mist vial with 70% isopropanol, then transfer in the Biological Safety Cabinet (BSC).

All subsequent steps should be done in a Biological Safety cabinet (BSC)

  1. Transfer the content of the tube to a sterile 15 or 50 mL conical tube.
  2. Rinse the cryovial with 1mL media and add it to the conical tube containing the cells.
  3. Centrifuge at 700-900rpm, (~500g) for 2 minutes. Return tube to the BSC>
  4. Remove supernatant and resuspend the cell pellet in culture medium.
  5. Unless specific bulk lots have been made, prepare some cells for expansion culture to replace thawed vial and the remained for experimental use.
  6. Record thaw date and initials in the appropriate cell bank freezer logbook.
  7. Replace the thawed vial with a new vial of the same cell line as early as possible, i.e within 3-5 population doublings.
  8. To avoid cross contamination, DO NOT have vials and tubes of different cell lines open simultaneously. Great care must be taken to clean the BSC and gloves in between handling of different cell lines.