Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Effect of Warming Process on the Survival of Cryopreserved Human Peripheral Blood Mononuclear Cells.

Author(s): Xu Y, Zou Q, Gao F, Wang D, Xue S, Lin H, Guo H, He X, Yang H, Gao D

Publication: Biopreserv Biobank, 2021, Vol. , Page

PubMed ID: 34061624 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to investigate the effect of thawing temperature of frozen PBMCs on cell viability, subpopulation proportions, and cytokine production. White cells were isolated by Ficoll-Paque density gradient centrifugation from venous blood of healthy volunteers (collection details not provided), suspended in freezing medium at a concentration of 1 x 107 cells/mL, aliquoted into cryovials (~1 mL), immediately placed into a rate-controlled freezing container, stored overnight at -80°C, and then transferred into a liquid nitrogen freezer until analysis (4-6 weeks). PBMC suspensions were thawed in a stirred water bath at 37°C, 42°C, or 65°C until ice crystals disappeared. To remove DMSO, cells were diluted in pre-warmed culture media and incubated overnight at 37°C. Cell viability and lymphocyte proliferation were assessed using an automated cell counter. PBMC subpopulations were determined by staining lymphocyte surface antigens (CD3, CD4, CD8, CD16, CD56, CD19, and CD14) and flow cytometric analysis. PBMCs were incubated for 6 days in the presence of phytohemagglutinin (PHA) and cytokine production was measured using ELISA kits for IFN-γ and IL-2.

   Summary of Findings:

   Thaw time for PBMC suspensions decreased by 20% and 40% when thawed in the 42°C and 65°C water baths compared to the 37°C water bath, respectively. Further, cell viability increased when specimens were thawed in 42°C and 65°C water baths compared to 37°C (P<0.05 and P<0.01, respectively). While cell recovery was comparable between specimens thawed in 42°C and 37°C water baths (76.06% ± 3.6% and 77.5% ± 4.37%, respectively), PBMC recovery was significantly higher when specimens were thawed in water baths at 65°C rather than at 42°C (81.1% ± 2.23% versus 76.06% ± 3.6%, P=0.04) or at 37°C (81.1% ± 2.23% versus 77.5% ± 4.37%, P=0.0661). Thawing temperature had no effect on proportions of PBMC subpopulations (T cells, B cells, NK cells, and monocytes). PHA-stimulated cells produced significantly more IL-2 and IFN-γ in PBMCs thawed in water baths at 42°C or 65°C than those thawed in water baths at 37°C (P<0.05, all).

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Coulter counter
   Cell count/volume	Flow cytometry
   Protein	ELISA
   Cell count/volume	Lymphocyte proliferation assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Thaw temperature/condition	37°C water bath 42°C water bath 65°C water bath

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