Fully Automated Clinical-Scale Separation of CD133+ Cells From Bone Marrow Aspirate

There is a growing interest in CD133 antigen expressing stem cell in the field of regenerative medicine such as cardiovascular, peripheral artery, and liver disease. Investigators in particular concentrate on bone marrow-derived stem cells for these applications. Today the clinical scale enrichment of CD133⁺ cells has to be performed as a complex procedure involving numerous manual handling steps.

We have developed a fully automated clinical scale process within a closed sterile system to purify CD133⁺ cells from human bone marrow aspirates. In this context, erythrocyte reduction, generation of autologous plasma, labeling time and the conditions for immunomagnetic separation were optimized.  

To determine the process performance, CD133⁺ cells were separated from human bone marrow aspirates with an initial volume of about 60 mL (n=10). We performed colony-forming unit (CFU) assays, which allowed us to evaluate the differentiation potential of the enriched cells.

The total processing time was reduced from about 4.5 h (previous manual process) to 2.5 h. The number of enriched CD133⁺ cells was 7.9x10⁵ (range: 3.7x10⁵ to 1.9x10⁶). The average yield was 47% and the average viability of the separated CD133⁺ cells achieved 90% (range: 69.9% to 96.9%). The depletion of CD133 negative cells was >99.9%. CFU assays perfomed after the fully automated enrichment process showed that the CD133⁺ 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 CD133⁺ cells from bone marrow aspirates within 2.5 h without any intermediate manual steps. The cell preparation in a closed sterile system facilitates a fast and robust enrichment of CD133⁺ cells. The cells are eluted in a small volume (6 mL) and can be used directly for further applications according to requirements e.g. for use in regenerative medicine.