Human cells are critical raw materials for research and manufacturing of cell therapy products. However, access to freshly procured cells can be limited, creating a crucial need for a suitable alternative to fresh cells that are viable and functional — especially when transporting materials globally.
At tebu-bio, we chose HemaCare to investigate the viability and functionality of lymphocytes, both fresh and cryopreserved, from leukopaks (leukapheresis collections) procured within their FDA registered cGMP donor collection facility.
Fresh vs. Cryopreserved Leukopaks
Fresh leukopaks (LP) from healthy donors were evaluated by HemaCare for cell viability via flow cytometry over the course of seven days. Studies found that viability of LPs in autologous plasma at room temperature was greater than 80% up to 144 hours post collection. However, the cell count decreased steadily over time, leading us to look at how cryopreservation might help to avoid these drawbacks in typical LP transport. The viability of T-cells, monocytes, B-cells, and natural killer (NK) cells from LP were evaluated from the same donor pre- and post-cryopreservation.
Preliminary data for whole LPs shows that post-cryopreserved viability averages 97.5% (+/- 1.2 SEM). The distribution of the CD3+, CD4+, and CD8+ populations were 43.4%, 28.7%, and 12.2%, respectively, within the total LP. Distribution of B-cells, NK cells, and monocytes shows 8.38%, 12.5%, and 20.4%, respectively. T-cell functionality data was also obtained, as this cell type is sensitive to the cryopreservation process.
Methods used for evaluation
- Fresh LP were stained for cell count and cell distribution, then split into two equal halves. One half was incubated at room temperature for 24 hr (Day 1 LP) to simulate shipping, the other half was cryopreserved in CryoStor-CS10 (BioLife Solutions) the same day as drawn.
- Cell count and distribution were quantified on the Day 1 LPs and the post-thaw cryopreserved LPs.
- CD3 and CD14 were isolated from the Day 1 and post-thaw LPs using magnetic beads.
- T-cell proliferation ability was observed and compared by monitoring CFSE division between Day 1 and post-thaw cryopreserved LPs.
- Differentiation ability of CD14 cells into dendritic cells (DC) was observed and compared between Day 1 LPs and post-thaw cyropreserved LPs.
Total cells counts
- Initial total WBC counts between fresh LPs (Day 0), Day1 LPs, and post-thaw cryopreserved LPs were not significantly different (2.64 billion, 2.26 billion and 2.19 billion average cells per time point).
- The number of CD3 in fresh LPs (Day 0), Day 1 LPs, and post-thaw cryopreserved LPs were not significantly different (1.03 billion, 0.93 billion, and 0.92 billion average cells per time point).
- The number of CD14 cells in fresh LPs (Day 0), Day 1 LPs, and post-thaw cryopreserved LPs were not significantly different (0.40 billion, 0.38 billion, and 0.40 billion average cells per time point).