Here is one that works well for us:
1. Purify cells on Ficoll-Paque and wash cells well twice in RPMI.
2. Make a cytospin of the purified cell product and have it stained in
hematology. This will help in looking at the cells later after thawing and
comparing cell numbers, morphology etc.
3. Place the tube holding the remaining cells in an ice bath and add the
following chilled reagents in the indicated order:
1. 3.75 ml RPMI
2. 0.75 ml Pooled Human Serum
3. 0.50 ml DMSO
4. Add the DMSO to the sample dropwise while mixing by agitation. Rapid
addition of the DMSO will denature the pooled human serum additive. Keep
samples and freezing reagent on ice the entire time.
5. Aliquot one ml of this cell mixture into each of 5 clean and properly
labeled cryotubes.
6. Place the cryotubes at -80 degrees C freezer.
7. After 24 hrs., they can be transferred to a liquid nitrogen freezer if
desired.
Thawing Procedure:
1. Remove desired samples and thaw quickly in warm tap water (not hot!)
2. Once thawed, immediately wash 3X with RPMI to remove the DMSO from the
cells.
3. Reconstitute to desired volume in RPMI.
Technical notes:
1. Once the cells have been removed from the ficoll tube they should remain on
ice until the final transfer to the freezer. This is particularly important
when adding the freezing mixture to the cells.
2. When adding the DMSO to the cells, the tube should remain in the ice bath
and mixed frequently to prevent denaturation of the pooled human serum
additive.
3. When thawing the cells, it is desirable to get the DMSO off them as soon as
possible. It has been reported that DMSO can be toxic to living cells when
exposed at room temperature.
Good luck.
Haywood Pyle
King Faisal Specialist Hospital and Research Center, MBC#3, Box 3354, Riyadh
11211 Saudi Arabia
tel/fax: 966-1-464-7272 ext. 32860
e:pyle@kfshrc.edu.sa
_______________________________________________________________________________
Subject: Cryopreservation of leukemia/lymphoma/normal cells
From: <hbpulido@ix.netcom.com (Hector Bauson Pulido Jr.) > at INTERNET-MAIL
Date: 30/11/97 05:40
Hello everyone!
I am interested in cryopreserving cells for both research and quality
control purposes. Does anyone know a good method to preserve cells
with minimal cell loss and high viability after thawing? Also, what
equipment should one have for this procedure?
Thanks in advance,
Hector Pulido
HBPulido@ix.netcom.com
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