Cell Separation System for Fully Automated Clinical Scale Separation of CD34+ Cells from Leukapheresis Products

In vitro CD34 enrichment of stem cell grafts is widely used for depleting T cells from the graft. A high ex vivoT cell depletion enables effective prevention of Graft-Versus Host Disease (GVHD) – a major complication after allogeneic stem cell transplantation. Until now, the clinical scale enrichment of CD34 positive cells is a complex procedure involving numerous manual steps.

We have developed a fully automated clinical scale process within a closed sterile system to purify CD34⁺cells from human leukapheresis products. In this context, cell washing, erythrocyte reduction, cell labeling and the conditions for immunomagnetic separation were optimized.  

To determine the process performance, CD34⁺cells were separated from human leukapheresis products with an initial volume of 120 mL to 211 mL (n=5). We performed colony-forming unit (CFU) assays, which allowed us to evaluate the differentiation potential of the enriched cells.

The total processing time was <4 h. The number of enriched CD34⁺ cells was 5.4x10⁶ (range: 2.6x10⁶ to 9.8x10⁶). The average yield was 73% and the average viability of the separated CD34⁺ cells achieved 76% (range: 65.0% to 85.0%). The depletion of CD34 negative cells was >99.9%. CFU assays performed after the fully automated enrichment process showed that the CD34⁺cell fraction contained primitive and multipotent progenitor cells, such as CFU-GEMM and CFU-GM.

The cell separation system described provides a safe and easy way to purify CD34⁺ cells from leukapheresis within <4 h without any intermediate manual steps. The cell preparation in a closed sterile system facilitates a fast and robust enrichment of CD34⁺ cells. The cells are eluted in a volume of approximately 80 mL and can be used directly for further applications.