Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.
Author(s): Zhang J, Yin Z, Liang Z, Bai Y, Zhang T, Yang J, Li X, Xue L
Publication: J Transl Int Med, 2024, Vol. 12, Page 51-63
PubMed ID: 38525442 PubMed Review Paper? No
Purpose of Paper
This paper compared the population, viability, activation, proliferation, and gene expression between fresh peripheral blood mononuclear cells (PBMCs) and matched cryopreserved PBMCs that were stored for 6 or 12 months and between cryopreserved ascites mononuclear cells from ovarian cancer patients that were freeze-thaw cycled twice versus once after 12 months of storage.
Conclusion of Paper
Relative to fresh PBMCs, cryopreserved PBMCs had lower viability, fewer cells, fewer monocytes, and fewer lymphocytes; however, because of the proportionally larger decrease in monocytes versus lymphocytes after cryopreservation, the percentage of monocytes increased with cryopreservation while the percentage of lymphocytes decreased. Cell population subsets were differentially affected by cryopreservation, but the effects were most apparent for CD4+ T-cells. While the percentage of T-cells (CD4+ or CD8+) secreting IFN-ɣ was not affected by cryopreservation, cryopreserved PBMCs had a higher percentage of T-cells (CD4+ or CD8+) secreting granzyme B+ and a lower percentage secreting IL-2. CD4+ and CD8+ T-cell proliferation was lower in cryopreserved cells and cell permeability (% 7-AAD+) was higher, particularly in CD4+ T-cells. Cryopreservation increased CD4+ but not CD8+ T-cell mitochondrial reactive oxygen species (ROS), and in cryopreserved CD4+ T-cells, there was a corresponding upregulation of genes involved in the “response to oxygen-containing compounds” and “cellular response to oxidative stress” relative to fresh CD4+ T-cells. In comparison to CD4+ T-cells, CD8+ cell sequencing had enrichment of genes involved in negative regulation of oxidative stress-induced neuron death and cell redox homeostasis. The authors conclude that elevated ROS after cryopreservation results in CD4+ T-cell death. There were no differences observed in cell number or viability between PBMCs cryopreserved for 6 months versus 12 months, but changes in the population of some cell types were affected by storage duration and ROS increased with storage of CD4+ cells.
Ascites monocytes that were refrozen and thawed an additional time after the initial 12 months of storage had fewer total cells, monocytes, and lymphocytes and displayed changes in monocyte subtype percentages. Ascites monocytes that were freeze-thaw cycled twice had a lower percentage of T-cells (CD4+ or CD8+) secreting IFN-ɣ or IL-2 and higher CD4+ cytoplasmic ROS compared to cells that were freeze-thaw cycled once.
Studies
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Study Purpose
This study compared the population, viability, activation, proliferation, and gene expression of fresh peripheral blood mononuclear cells (PBMCs) with matched cryopreserved PBMCs that were stored for 6 or 12 months. Peripheral blood was collected from 21 healthy volunteers into EDTA tubes. Blood was centrifuged at 2000 g for 10 min at 4°C before separation of mononuclear cells by Ficoll density gradient centrifugation at 400 g for 25 min. Cells were washed in phosphate-buffered saline (PBS), counted, and then cryopreserved in a solution consisting of 10% dimethylsulfoxide (DMSO) and 90% fetal bovine serum (FBS) at -80°C. After 24 h, cryopreserved PBMCs were transferred to liquid nitrogen and stored for 0, 6, and 12 months before thawing in a 37°C water bath where they were shaken to speed up the thawing process. Once thawed, cells were aspirated into tubes containing pre-warmed RPMI media, centrifuged at 500 g for 5 min, and then washed and resuspended in PBS. Cells were stained with antibodies for cell surface markers and analyzed by fluorescence-activated cell sorting (FACS) using a CytoFLEX S flow cytometer. To evaluate T-cell functionality, lymphocytes were cultured with a cell activation cocktail, stained with surface marker antibodies, fixed, permeabilized, and stained with the Biolegend Staining Buffer Kit and then analyzed by flow cytometry. PBMC proliferation was evaluated by Carboxyfluorescein diacetate succinimdyl ester (CFSE) staining using a flow cytometer. ROS was measured by incubating PBMCs with H2DCFDA (cytoplasmic) and MitoSOX (mitochondrial) dye; visualization was by flow cytometry. Single-cell whole transcriptome analysis was performed on BD Rhapsody WTA Amplification libraries using an Illumina NovaSeq platform. PBMC cell classification of the sequencing data was performed with Azimuth and differentially expressed genes were identified using the Seurat function in FindMarkerS.
Summary of Findings:
Relative to fresh PBMCs, cryopreserved PBMCs had lower viability (80-85%, P<0.001), fewer cells (30-40%, P<0.001), fewer monocytes (P<0.001), and fewer lymphocytes (P<0.0001); however, because of the proportionally larger decrease in monocytes versus lymphocytes after cryopreservation, the percentage of monocytes increased with cryopreservation (P<0.001) and the percentage of lymphocytes decreased (P<0.0001). Cell number and viability did not differ between PBMCs that were cryopreserved for 6 months versus 12 months. Further analysis revealed that after cryopreservation of PBMCs, there were higher percentages of classical monocytes (CD14+CD16-), B-cells, natural killer T (NKT) cells, and CD4+ T-cells but lower percentages of CD8+ T-cells and CD45+ leukocytes that were CD3+ T cells. Further analysis of T-cell subsets identified a slight decrease in the proportion of naïve T cells (CD45RA+CCR7+) and an increase in terminally differentiated effector memory CD8+ T-cells (CD45RA-CCR7-) after cryopreservation, with larger differences found when they were stored for 12 months. Analysis of NK cell subsets found no clear differences with cryopreservation or storage. The percentage of T-cells (CD4+ or CD8+) secreting IFN-ɣ was not affected by cryopreservation or storage; however, compared to fresh PBMCs, cryopreserved PBMC had a higher percentage of T-cells (CD4+ or CD8+) secreting granzyme B+ (P<0.01 and P<0.05, respectively) and a lower percentage secreting IL-2 (P<0.01 and P<0.001, respectively). The proliferation of CD4+ and CD8+ cryopreserved T-cells was lower than in fresh T-cells (P<0.01, both), but cell permeability (% 7-AAD+) increased with cryopreservation and was higher in CD4+ than CD8+ T-cells (P<0.0001). Mitochondrial ROS increased in CD4+ T-cells with cryopreservation (P<0.01) and in cells stored for 12 months, but no increase in mitochondrial ROS was observed following cryopreservation of CD8+ T-cells. Correspondingly, pathways analysis of genes found to be upregulated in cryopreserved relative to fresh CD4+ T cells identified the “response to oxygen-containing compounds (GO: 1901701)” and “cellular response to oxidative stress (GO: 0034599)”, but these pathways were not upregulated in cryopreserved relative to fresh CD8+ T-cells. When treated with H2O2, CD4+ cells displayed greater sensitivity than CD8+ T-cells. Further, CD8+ cell sequencing showed enrichment of genes involved in negative regulation of oxidative stress-induced neuron death and cell redox homeostasis relative to CD4+ T-cells. The authors conclude that elevated ROS after cryopreservation results in CD4+ T-cell death.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry RNA Next generation sequencing Protein Flow cytometry Small molecule Flow cytometry Morphology Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Storage Storage duration 0 months
6 months
12 months
-
Study Purpose
This study compared the population, viability, activation, proliferation, and gene expression of cryopreserved ascites mononuclear cells that were stored for 12 months with those stored for 12 months then thawed and refrozen for an additional week; ascites mononuclear cells were isolated from ovarian cancer patients. A fresh specimen of ascites mononuclear cells was also analyzed to determine cell population proportions. Ascites was collected from eight patients with ovarian cancer (further details not provided) and centrifuged at 2000 g for 10 min at 4°C before separation of mononuclear cells by Ficoll density gradient centrifugation at 400 g for 25 min. Cells were washed in phosphate-buffered saline (PBS), counted, and then cryopreserved in a solution consisting of 10% dimethylsulfoxide (DMSO) and 90% fetal bovine serum (FBS) at -80°C. After 24 h, cryopreserved mononuclear cells were transferred to liquid nitrogen, stored for 12 months, thawed, refrozen, and thawed 1 week later. Cells were thawed in a 37°C water bath where they were shaken to speed up the thawing process. Once thawed cells were aspirated into tubes containing pre-warmed RPMI media, they were centrifuged at 500 g for 5 min and then washed and resuspended in PBS. Cells were stained with antibodies for cell surface markers and analyzed by FACS using a CytoFLEX S flow cytometer. To evaluate T-cell functionality, lymphocytes were cultured with a cell activation cocktail, stained with surface marker antibodies, fixed, permeabilized, and stained with the Biolegend Staining Buffer Kit and then analyzed by flow cytometry. PBMC proliferation was evaluated by Carboxyfluorescein diacetate succinimdyl ester (CFSE) staining using a flow cytometer. ROS was measured by incubation of PBMCs with H2DCFDA (cytoplasmic) and MitoSOX (mitochondrial) dye and was visualized by flow cytometry. Single-cell whole transcriptome analysis was performed on BD Rhapsody WTA Amplification libraries using an Illumina NovaSeq platform. PBMC cell classification of the sequencing data was performed with Azimuth and differentially expressed genes were identified using the Seurat function in FindMarkerS.
Summary of Findings:
Relative to ascites monocytes that were stored for 12 months and thawed once, those stored for an additional week and thawed again had fewer total cells, monocytes, and lymphocytes (P<0.05, all), but the percentage of monocytes or lymphocytes were unaffected. Further analysis revealed that ascites monocytes thawed twice had lower percentages of monocytes classified as intermediate (CD14+CD16+), non-classical (CD16+CD14-), or Myeloid-derived suppressor cells (MDSC, CD45+HLA-DR-CD11b+CD33+), and a lower percentage of CD3+ T-cells than those thawed once, but the percentages of CD8+ T cells, NK cells, NKT cells, and B cells were higher in ascites monocytes that were thawed twice than those thawed once. Analysis of T-cell populations revealed a decrease in the percentage of naïve T cells following cryopreservation. A decrease in the percentage of naïve T cells and an increase in central memory and terminally differentiated effector memory CD8+ T cells was observed after the second freeze-thaw event. The percentage of T-cells (CD4+ or CD8+) secreting IFN-ɣ or IL-2 was lower in ascites monocytes that were thawed twice rather than once (P<0.05, all). Cell permeability (%7-AAD+) increased with cryopreservation of CD4+ (P<0.05) but not CD8+ T-cells. The cytoplasmic ROS of CD4+ T-cells, but not CD8+ T-cells, increased with the second freeze-thaw event.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry RNA Next generation sequencing Small molecule Flow cytometry Morphology Flow cytometry Protein Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Type of fixation/preservation Frozen
None (fresh)
Storage Freeze/thaw cycling 1 cycle
2 cycles
0 cycles
Storage Storage duration 0 months
12 months
12 months and 1 week