当使用PBMC用于实验时，大部分科学家都倾向于使用Fresh PBMC。但是当您订购的“Fresh” PBMC离体24h或更久时间后是否对于PBMC的活性和功能有影响呢？
Materials and Methods
密度梯度离心（Ficoll是蔗糖的多聚体，呈中性，平均分子量为400,000，未超出正常生理性渗透压,也不穿过生物膜）blood samples are meticulously layered over Ficoll and centrifugedfor 45 minutes at 400g. The PBS medium layer is aspirated off, and the PBMClayer then is carefully pipetted into a fresh tube and washed three times withPBS.
CellViability and Recovery
冷冻：–1 °C/minute with cryopreservationmedium containing 10% dimethyl sulfoxide (DMSO) and 90% fetal bovine serum(FBS).
LeukocyteCell Subset Frequencies
FACS, the cell subset frequencies wereanalyzed before and after cryopreservation for each isolation condition.
Cells that were isolated and cryopreservedthe same day as collection were more viable after thaw than cells that wereisolated and cryopreserved 24 hours after collection. Cells frozen the same dayas collection were 47% more viable (Donor 369) and 94% more viable (Donor 328),respectively.
This decrease in viability is greater thanwe have observed with Leukopaks received after overnight shipping. We performedthe comparison with a third donor and left a portion of cells in the originalcollection bag for delayed isolation. This batch had higher viability afterthaw, comparable to the cells preserved the same day as collection, which maybe due to better gas exchange in the collection bag compared to the cellsstored in tubes overnight.
Figure 1. Post-thaw viability of cells isolated and cryopreserved thesame day as collection and 24 hours after collection.
When it comes to subset, we found thatdelayed cryopreservation had varied impacts on different cell types. Weanalyzed the cell types present in each batch by FACS after thawing. Dead cellswere excluded from the analysis.
We noticed a decrease in the proportion ofT cells and NK cells when the samples were cryopreserved 24 hours aftercollection instead of the same day. The viability of B cells and monocytes werenot significantly impacted by delayed cryopreservation.
Figure 2. Proportion of cells in the PBMC samples from Donor 328isolated and cryopreserved the day of collection and the day after collection,respectively.
Figure 3. Proportion of cells in the PBMC samples from Donor 369isolated and cryopreserved the day of collection and the day after collection,respectively.
We tested both cell batches using ourrecall antigen assay to see how cytokine secretion was affected by time ofcryopreservation relative to collection. Before performing the assay, weequalized the number of viable cells in each batch.
Even though equal numbers of viable cellswere used, the recall response was much weaker in cells isolated after 24 hourscompared to cells cryopreserved soon after collection.
Figure 4. The response to tetanus toxoid is primarily a T cell response.There was no significant IFNγ produced by the cells isolated after 24 hours.
Figure 5. PHA was used to stimulate T cells, showing that there werelive T cells in both samples, though the amount of IFNγ produced by the cellsisolated after 24 hours was much lower.
Figure 6. Monocytesrespond to LPS by producing TNFα. While still present and viable in thesamples, the ability of the monocytes to produce TNFα was reduced.
Isolating PBMCs from blood stored for 24hours at room temperature with either method led to significantly moregranulocyte contamination. It has been well documented that granulocytes becomeactivated over prolonged storage, which affects their buoyancy profile andcompromises the efficiency of separation by density-gradient isolation.
Figure 7: Leukocyte subset frequencies are compared, calculated as apercentage of CD45+cells of PBMCs isolated by Ficoll and SepMate methods asmeasured by flow cytometry.
1.Maximizing PMBC Recovery and Viability: A Method to Optimize andStreamline Peripheral Blood Mononuclear Cell Isolation, Cryopreservation, andThawing https://bioprocessintl.com/manufacturing/cell-therapies/maximizing-peripheral-blood-mononuclear-cells-recovery-and-viability-a-method-to-optimize-and-streamline-peripheral-blood-mononuclear-cell-isolation-cryopreservation-and-thawing/
2.McKenna KC, et al. Delayed Processing of Blood Increases theFrequency of Activated CD11b+ CD15+ Granulocytes Which Inhibit T Cell Function.J. Immunol. Meth. 341, 2009: 68–75.
3.DeRose R. Granulocyte ContaminationDramatically Inhibits Spot Formation in AIDS Virus–Specific ELISpot Assays:Analysis and Strategies to Ameliorate. J. Immunol. Meth. 297, 2005:177–186.
4.How Fresh Are Your PBMC by Astrobio
Peripheral Blood Mononuclear Cells, Frozen, 15 million/vial
Peripheral Blood Mononuclear Cells, Frozen, 25 million/vial
Peripheral Blood Mononuclear Cells, Frozen, 50 million/vial
Peripheral Blood Mononuclear Cells, Frozen, 100 million/vial
Peripheral Blood CD3+ Pan T Cells, Frozen, 20 million/vial
Peripheral Blood CD3+ Pan T Cells, Frozen, 30 million/vial
Peripheral Blood CD3+ Pan T Cells, Frozen, 40 million/vial
Peripheral Blood CD4+ Helper T Cells, Frozen, 10 million/vial
Peripheral Blood CD4+ Helper T Cells, Frozen, 15 million/vial
Peripheral Blood CD4+CD25+ Regulatory T cells, Frozen, 2 million/vial
Peripheral Blood CD4+CD45RO+ Memory T Cells, Frozen, 5 million/vial
Peripheral Blood CD4+CD45RA+ Naïve Helper T Cells, Frozen, 5 million/vial
Peripheral Blood CD8+ Cytotoxic T Cells, Frozen, 10 million/vial
Peripheral Blood CD8+CD45RA+ Naïve Cytotoxic T Cells, Frozen, 5 million/vial
Peripheral Blood CD56+ Natural Killer Cells, Frozen, 5 million/vial
Peripheral Blood CD14+ Monocytes, Frozen, 40 million/vial
Peripheral Blood Monocyte-Derived Macrophages, Frozen, 1.5 million/vial
Peripheral Blood Monocyte-Derived Immature Dendritic Cells, Frozen, 1.5 million/vial
Peripheral Blood CD19+ B Cells, Frozen, 10 million/vial
Peripheral Blood CD19+ CD27+ Memory B Cells, Frozen, 0.5 million/vial
Peripheral Blood CD19+ CD27+ Memory B Cells, Frozen, 1 million/vial
Peripheral Blood CD19+ CD27+ Memory B Cells, Frozen, 2 million/vial
Peripheral Blood CD19+CD27- Naïve B Cells, Frozen, 1 million/vial
Peripheral Blood CD19+CD27- Naïve B Cells, Frozen, 2 million/vial
Peripheral Blood Eosinophils, Frozen, 1 million/vial
Peripheral Blood Basophils, Frozen, 0.3 million/vial