Homo sapiens (Peripheral mononuclear cells (PBMC))

(Xeno-free, Chemically Defined PBMC Cryopreservation)

Intro/Description:

The article this protocol was published in sought to characterize a xeno-free and fully chemically defined cryopreservation medium for use with PBMC. The authors found that the new medium had a comparable cryopreservation efficiency after short-term storage and after 6 months. Longer durations were not tested. Viabilities of over 97% and recoveries over 84% were obtained.

It may be worth noting that the authors of this study are from the institute that, at the time of publication, had already licensed this cryopreservation media to Fischer Procryotect for commercial sale.

Materials:

• Lymphocyte separation medium (PAA, Cölbe)
• PBS (Gibco, Karlsruhe)
• Pharm Lyse (BD, Heidelberg)
• Distilled water (B. Braun, Melsungen)
• FBS (PAA, Cölbe)
• IBMT-Medium w/ 10% DMSO (Fischer Procryotect, Zurich)
• Cryovials
• Freezing container
• IMDM medium (Gibco, Karlsruhe)
• L-glutamine (Gibco, Karlsruhe)

Protocol:

1. Isolate peripheral blood mononuclear cells (PBMC) by density gradient centrifugation over lymphocyte separation medium.
2. Collect the buffy coat layer and wash with PBS.
3. Lyse contaminating red blood cells by incubating 2 × 108 cells in 20ml of 1/10 diluted Pharm Lyse in distilled water for 30 min in darkness.
4. Stop the reaction by adding 30 ml of PBS with 1% pretested FBS.
5. Resuspend the PBMC at a concentration of 11.5 × 106 cells/ml in IBMT-Medium w/ 10% DMSO.
6. Transfer 1 ml aliquots of cell suspension into pre-cooled (-20°C) cryovials in a cell freezing container.
7. Freeze the PBMC at a rate of -1°C/min and store overnight at -80°C.
8. Transfer the samples to the vapor phase of a liquid nitrogen tank for long-term storage.
9. To thaw, directly transfer the cryovials from the liquid nitrogen tank to a 30°C water bath and leave until almost no ice remains.
10. Slowly add 1 ml of the thawing medium (IMDM medium containing L-glutamine, 25 mM HEPES buffer, and 3.024 g/l sodium bicarbonate, supplemented with 10% pretested, heat-inactivated FBS and 1 mML-glutamine) to the PBMC suspension.
11. Transfer the PBMC suspension to a 50 ml polypropylene tube containing 9 ml pre-warmed thawing medium.
12. Centrifuge at 400 x g for 5 min.
13. Resuspend the PBMC pellet in 10 ml thawing medium.
14. Place in a cell incubator (5% CO2, 37 °C) overnight with a loose cap.

References:

Schulz JC, Germann A, Kemp-Kamke B, Mazzotta A, von Briesen H, Zimmermann H. Towards a xeno-free and fully chemically defined cryopreservation medium for maintaining viability, recovery, and antigen-specific functionality of PBMC during long-term storage. J Immunol Methods. 2012 Aug 31;382(1-2):24-31.

Added by: Carlton Hoyt on July 25, 2012

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