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Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations.

Author(s): Ticha O, Moos L, Bekeredjian-Ding I

Publication: J Immunol Methods, 2021, Vol. 495, Page 113081

PubMed ID: 34048717 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared cell viability, B-cell subpopulation proportion, and response to stimulation in fresh PBMC and those stored frozen for up to 12 months. PBMCs were isolated from the buffy coat of 12 healthy blood donors by Pancoll gradient centrifugation. PBMCs were resuspended in fetal calf serum (FCS) and aliquoted. An equal volume of freezing media (20% FCS, 60% RPMI, and 20% DMSO) was added and tubes were mixed by inversion three times. Tubes were placed in a slow-cooling container in a -80°C freezer overnight and then transferred to a single storage box in the -80°C freezer. Monthly, an aliquot from each of the four donors was removed, thawed at 37°C for 1 min, transferred into warming media (50% FCS, 50% HEK medium), and placed in a 37°C incubator for 1 h. Cells were pelleted by centrifugation at 360 x g for 6 min at room temperature and resuspended in Hek media. Cells were counted by trypan blue and transferred to 96 and 48-well plates (200,000/well). The 96-well plates were centrifuged at 360 x g for 6 min at room temperature and cells were resuspended in PBS; pelleted again; stained using LIVE/DEAD Fixable Aqua Dead Cell Stain Kit and antibodies to CD19, CD27, IgM, and CD38; and counted by flow cytometry. Cells from all donors were plated in 48-well plates and stored frozen for <30 days or 10-12 months. Cells in plates were thawed as described above and stimulated with CpG and Tetanus toxoid (TT) adsorbed to alum adjuvant and incubated at 37°C for 5 days. Levels of IgG were quantified by ELISpot.

   Summary of Findings:

   PBMC counts were affected by freezing but also declined with frozen storage duration (P=0.04). Importantly, the counts declined by more than 25% over the year of storage in specimens from three of four donors. However, lymphocyte percentage and viability of lymphocytes remained comparable to fresh specimens over the storage period. The absolute count of B-cells in specimens stored frozen for 12 months was only 42.8% of that in fresh PBMCs. Although the proportion of CD19+ B cells was consistent with fresh specimens over the first two months of storage, the average percentage CD19+ B-cells declined from 7.98% to 5.37% over the 12 months (ANOVA, P<0.0001). The percentage naïve (CD19+ and CD27-) B-cells was stable over the 12 months. The percentage IgM memory (IgM+ and CD27+) and switched memory (IgM- and CD27+) cells fluctuated over the timecourse but there was no clear trend with storage.  Plasmablasts (CD19+ CD27++ and CD38++) constituted a small percentage of the B-cells in the samples, making it too hard to identify any trends, but they remained detectable at all timepoints. PBMCs stored for 10-12 months produced more total IgG in response to stimulation than those stored for 1 month (P=0.012), but there was no difference in anti-tetanus toxoid IgG produced in response to stimulation between cells stored for 1 month and those stored 10-12 months.

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Glycoprotein	ELISA
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	0 months 1 month 2 months 3 months 4 months 5 months 6 months 7 months 8 months 9 months 10 months 11 months 12 months
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)

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