Figure 1. Recovery of Viable Cells Is Higher When Leukopaks Are Stored at Fridge Temperature Compared to Room Temperature

Stability of fresh leukopaks (LP) was evaluated after storage for up to 5 days after collection, at a refrigerated temperature (“FT”; 2 - 8°C) or at room temperature (RT; 15 - 25°C). Full leukopaks were collected from three (3) unique donors, then divided into ten (10) equally sized fractions and stored at FT or RT (n = 5 at each temp, per donor). Over the following 5 days, one (1) 1/10th leukopak fraction was processed from each condition daily, to identify changes in cellular composition and functionality that may have occurred during storage. Viable cell yield (A) and percent cell viability (B) were determined daily, following red blood cell (RBC) lysis of leukopak fractions. By day 5 after collection, leukopak fractions stored at RT showed a 68% loss of viable cells and an accompanying 54% decrease in cell viability. Conversely, those stored at FT showed a much less pronounced 19% decrease of viable cells and just 5% decrease in viability over the 5 days. All data points represent average ± standard deviation values from leukopak fractions of 3 unique donors.

Figure 2. Refrigeration of Leukopaks Ameliorates Time-Dependent Loss of Mature Blood Cells

Recovery of mature blood cells from leukopaks stored at RT or FT was calculated from the viable total cell recovery combined with lineage frequency assessment by flow cytometry. For all mature cell types examined, recovery decreased at a faster rate when leukopaks were stored at RT compared to FT. A pronounced loss of T and NK cells was particularly evident over the 5 days in leukopaks stored at RT. Less pronounced losses of B cells, monocytes and granulocytes were also observed, and these losses were also ameliorated by FT storage. All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.

Figure 3. Mean Percentages of Cell Subpopulations in a Leukopak

Representative chart showing the average frequencies of major immune subsets in leukopak products, as measured by flow cytometry. Values shown are mean percentages of total viable leukocytes present in a leukopak (n ≥ 30).

Figure 4. T Cell Expansion and Activation Ability Is Better Preserved When Leukopaks are Stored at Fridge Temperature

Using EasySep™ Human T Cell Isolation Kit, T cells were isolated from 1 leukopak fraction of each storage condition daily for 5 days, and 1 x 10^6 isolated cells were cultured in ImmunoCult™-XF T Cell Expansion Medium supplemented with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator and 10 ng/mL IL-2 for 10 days with assessment of CD25 activation marker expression on day 3 of culture. (A) Representative flow cytometry data from leukopaks stored 1 day at FT, showing that both CD4+ and CD8+ cells are CD25-negative at the start of culture (day 0), and upregulate CD25 expression by day 3 of culture. (B) Cellular expansion and corresponding cell yield over 7 days of culture decreases in correlation with storage duration of leukopak fractions. Leukopaks that were stored for 1 - 2 days at either RT or FT had high expansion potential yielding 1.5 - 2 x 10^7 cells, and this 15 - 20-fold expansion potential is maintained in T cells from leukopaks stored at FT for up to 5 days. In contrast, little or no expansion is observed in T cell cultures from leukopaks stored at RT for 3 or more days, indicating a loss of proliferative capacity. Moreover, T cells show a gradual reduction in their ability to become activated by ImmunoCult™ T Cell activator, as shown by a reduction in day 3 CD25 expression in gated CD4+ (bottom left) or CD8+ (bottom right) cells over time, and the effect is most pronounced with storage at RT for 3 or more days. All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.

Figure 5. Refrigeration of Leukopaks Preserves Monocyte-to-Macrophage Differentiation Efficiency for Up to 5 Days

Monocytes were isolated (using EasySep™ Human Monocyte Isolation Kit) from 1 leukopak fraction of each storage condition daily for 5 days, and 1 x 10^6 isolated cells were cultured in ImmunoCult™-SF Macrophage Medium supplemented with 50 ng/mL Human Recombinant M-CSF for a further 6 days. (A) Representative flow cytometry plot from leukopaks stored 1 day at FT, showing maintenance of CD14 and upregulation of CD68 expression over the 5 day differentiation to M0 macrophages. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into M0 macrophages, those stored at RT for over 3 days failed to differentiate, as shown by low percentages of CD14+CD68+ cells. Moreover, monocytes harvested from Day 5 leukopaks stored at RT failed to thrive in the 6-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.

Figure 6. Refrigeration of Leukopaks Preserves Monocyte-to-Dendritic Cell Differentiation Ability for Up to 5 Days

Monocytes were isolated (using EasySep™ Human Monocyte Isolation Kit) from 1 leukopak fraction of each storage condition daily for 5 days, and 1 x 10^6 isolated cells were cultured in ImmunoCult™-ACF Dendritic Cell (DC) Medium supplemented with ImmunoCult™-ACF Dendritic Cell Differentiation Supplement. (A) Representative flow cytometry plot from leukopaks stored 1 day at FT, showing efficient downregulation of CD14 and upregulation of CD11c over 6 days of culture. (B) While monocytes isolated from FT-stored leukopaks efficiently differentiated into DCs, those stored at RT for over 3 days show reduced DC differentiation ability and CD14-CD11c+ cell output. Moreover, monocytes harvested from day 5 RT-stored leukopaks failed to thrive in the 5-day culture, resulting in very few viable cells recovered (not shown). All data points represent average ± standard deviation values from leukopak fractions of n = 3 unique donors.