Introduction

When ordering human or animal blood cells for research, many scientists subscribe to the adage fresh is best. But just how viable and functional are those “fresh” PBMC you ordered after sitting out of the body for 24 hours or more? The term freshness is often used when referring to the quality of donor blood samples. However, freshness is not the measure with which you should judge sample quality. Instead, focus on the measures that matter to the outcomes of your experiments: viability and functionality.

We set out to put this freshness myth to rest using human PBMC from our lab. We received PBMC from two donors just hours after collection and evenly divided each sample into two groups. One group sat at room temperature for 24 hours to simulate one-day shipping conditions before being isolated and cryopreserved. The other group was isolated and cryopreserved within 3 hours of arrival to our lab. We measured post-thaw cell viability and recall response of our cryopreserved samples using flow cytometry and a recall antigen assay testing cytokine secretion.

Methods

  1. We received fresh PBMC from two normal donors the same day as collection. Each sample was split into two halves. One half sat at room temperature (20–25°C) for 24 hours (to simulate same-day shipping conditions) before being isolated and cryopreserved. The other half was isolated and cryopreserved the same day as collection using standard methods.
  2. Viability was measured for all cell types in each batch using flow cytometry.
  3. The proportion of cell types present (T cells, B cells, monocytes and NK cells) was determined by flow cytometric analysis of cells stained with fluorescently tagged antibodies from Thermo Fisher Scientific.
  4. All batches were tested for cytokine secretion using our recall antigen assay.

Results

  • Viability of Cells is Higher when Frozen the Day of Collection

Cells that were isolated and cryopreserved the same day as collection were more viable after thaw than cells that were isolated and cryopreserved 24 hours after collection. Cells frozen the same day as collection were 47% more viable (Donor 369) and 94% more viable (Donor 328), respectively. This decrease in viability is greater than we have observed with leukopaks received after overnight shipping.

We performed the comparison with a third donor and left a portion of cells in the original collection bag for delayed isolation. This batch had higher viability after thaw, comparable to the cells preserved the same day as collection, which may be due to better gas exchange in the collection bag compared to the cells stored in tubes overnight.

Post-Thaw Cell Viability chart
Figure 1. Post-thaw viability of cells isolated and cryopreserved the same day as collection and 24 hours after collection.

  • Freshness Impacts Viability Differently Across Cell Types

When it comes to viability, we found that delayed cryopreservation had varied impacts on different cell types. We analyzed the cell types present in each batch by flow cytometry after thawing. Dead cells were excluded from the analysis. We noticed a decrease in the proportion of T cells and NK cells when the samples were cryopreserved 24 hours after collection instead of the same day. The viability of B cells and monocytes were not significantly impacted by delayed cryopreservation.

Donor 328 graph
Figure 2. Proportion of cells in the PBMC samples from Donor 328 isolated and cryopreserved the day of collection and the day after collection, respectively.
Donor 369 graph
Figure 3. Proportion of cells in the PBMC samples from Donor 369 isolated and cryopreserved the day of collection and the day after collection, respectively.

  • Recall Response is Stronger for Cells Cryopreserved Soon After Collection

We tested both cell batches using our recall antigen assay to see how cytokine secretion was affected by time of cryopreservation relative to collection. Before performing the assay, we equalized the number of viable cells in each batch. Even though equal numbers of viable cells were used, the recall response was much weaker in cells isolated after 24 hours compared to cells cryopreserved soon after collection.

INF-gamma Production Stimulated by Tetanus Toxoid graph
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.
INF-gamma Following PHA Stimulation graph
Figure 5. PHA was used to stimulate T cells, showing that there were live T cells in both samples, though the amount of IFNγ produced by the cells isolated after 24 hours was much lower.
TNF-alpha Following LPS Stimulation graph
Figure 6. Monocytes respond to LPS by producing TNFα. While still present and viable in the samples, the ability of the monocytes to produce TNFα was reduced.

Conclusion

The sooner blood cells are frozen after collection, the more viable and functional the cells will be upon thaw. When purchasing cells for research, it’s better to buy cells that were isolated and cryopreserved the same day as collection. Cells that were isolated and cryopreserved 24 hours after collection or later will have lower viability, lower T cell and NK cell counts, and weaker recall responses.