I have been searching the literature regarding PBMCs isolation, manipulation and conservation. Regarding freezing medium composition, I have found that there is a number of reactants that can be used in different proportions, some of them being: RPMI, FBS, FCS, human AB serum, HSA, glutamine, dextran-40 and an ever-changing proportion of DMSO (7%, 10%, 20%...).
I was wondering about the pros and cons of different combinations, if any, from someone who has experience in this topic.
Thank you in advance,
Helena
Hi Helena,
At our laboratory we collect human PBMC samples using 90% FBS + 10% DMSO combination. After isolating PBMC with Ficoll-hypaque gradient, the cells are washed with RPMI containing penicillin/streptomycin twice. Freezing medium (90% FBS + 10% DMSO) is prepared before use and kept on ice. Then the cells are resuspended with the freezing medium to achieve the final cell concentration of 3 million cells/ml. The cells are then preserved in -80c freezer overnight before transferring to liquid nitrogen tank.
The cells viability is about 90% with this method and it works well with our samples. I've never tried varying DMSO concentration before so I'm not sure how it will affect the cells. The major drawback of the method is that it consumes so much FBS.
I hope that helps
Anunya
At the biobanque of Picardie we use the following medium to cryopreserve PBMC
10%DMSO+ 10%FBS + 80%RPMI. We have 90 to 95% viability.
This protocol is used by the major biobanks
Good a luck
Thank you Abderrahmane! I see that the most important component of the final medium is the 10% of DMSO.
Hola Helena:
Yo utilizo 10% de DMSO en Suero Fetal Bovino, como Anunya Opasawachai, sin embargo, los lavados los realizo con DMEM y la concentración que conservo es en torno a 10 millones por militro. También tengo una alta viabilidad (90%). Sin embargo, mi modelo de estudio no es humano sino pinnípedos.
Mi antigua maestra de cultivo celular afirmaba que la glicerina al 10% puede sustituir al DMSO al 10% como protector de las células con los mismos resultados y es menos tóxica que éste cuando descongelas las células. He conservado líneas celulares con glicerina (T3 y otras que ahora no recuerdo) pero, con PBMCs, no lo he probado. Si las conservas con DMSO, eso sí, debes lavarlas rápidamente una vez que las descongelas, si no, pueden intoxicarse y morir.
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