Cryopreserved Leukopaks Maintain Cell Viability and Functionality: A Solution for Cell Therapy Logistics

Introduction

Human cells are critical raw materials for research and manufacturing of cell therapy products. Fresh products maintain good viability and functionality for the first 24 hours of transport. However, beyond 48 hours, the number of viable cells decreases dramatically. When considering global transportation and the cell therapy supply chain, the need for a practical shipping option is essential. Hence, we set out to evaluate cryopreserved leukopaks (LP) frozen on day 0 compared to fresh leukopaks at 24 hours. For this comparison, we evenly split leukopaks in half. Half was immediately cryopreserved using a control rate freezing process. The other half was held at room temperature for 24 hours, simulating shipment. We isolated CD3 and CD14 cells from both leukopaks and put them into functional assays. Total lymphocyte (CD45) counts between Day 1 fresh LPs and post-thaw cryopreserved LPs were not significantly different using a paired student t-test (2.26 billion and 2.19 billion average cells per time point, p= 0.73). The number of CD3 cells in Day 1 and post-thaw LPs were not statistically different (0.93 billion and 0.92 billion average cells per time point, p=0.81). The number of CD14 cells in Day 1 LPs and post-thaw LPs were not significantly different (0.38 billion and 0.40 billion average cells per time point, p=0.61). Viability of the cells, measured by flow cytometry, was greater than 99% in all groups. Dendritic cell generation was similar between the CD14 cells isolated from both the Day 1 (32.7 million) and post-thaw LPs (37.8 million), p= 0.75. Dendritic cell purity was greater than 99% in both groups. CD3 cells isolated from Day 1 and post-thaw LPs showed similar high proliferation function. Starting counts were 30 million and the proliferation amounted to 42 million and 39 million respectively, p=0.61. Leukopaks from healthy donors, cryopreserved per the HemaCare collection and processing model, maintain viability and functionality of target cells. This presents a scalable option for emerging autologous and allogeneic cell therapies that require apheresis shipments from HemaCare to cell therapy processing facilities around the world.

Materials and Methods

  1. Fresh LP were stained for cell count and cell distribution, then split into two equal halves. One half was incubated at room temperature (2-8°C) for 24 hours (Day 1 LP) to simulate shipping, the other half was cryopreserved in CryoStor® CS10 (BioLife Solutions, Bothell, WA) the same day as leukaphersis draw.
  2. CD45 white blood cell and mononuclear cell counts and distribution were measure by flow cytometry on Day 0, Day 1 LPs, and the post-thaw of cryopreserved LPs.
  3. CD3 and CD14 cells were isolated using positive immunomagnetic selection from the Day 1 and post-thaw leukopaks using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany).
  4. T-cell proliferation was observed and compared by monitoring carboxyfluorescein succinimidyl ester (CFSE) division between Day 1 and post-thaw cryopreserved LPs.
  5. The differentiation ability of CD14 cells into dendritic cells (DC) from Day 1 LPs and post-thaw LPs was measured by flow cytometry. CD14 monocytes were cultured for 7 days, then the number and purity of dendritic cells were measured using flow cytometry.

Results

Post Thaw Cell Counts

The data below shows cell counts taken at day 0, day 1, and post thaw.

 

 

 

Viability

 

Post Culturing Cell Counts

 

 

Purity of Dendritic Cells

 

Proliferation of CFSE Labeled CD3 T Cells

 

Discussion

In conclusion, leukopaks from healthy donors cryopreserved following the HemaCare collection and processing protocols maintain purity, viability and functionality of target cells. This presents a scalable option for emerging autologous and allogeneic cell therapies for the global research community that requires sustainable apheresis shipments from a global provider of primary hematopoietic cells.