Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy

Abstract

Background and Aims. With the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34⁺ selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices. Methods and Results. We developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10⁶ CD34⁺ cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34⁺ bone marrow cells revealed a significant proportion of CD45⁻ CD34⁺ cells. Conclusions. Intra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45⁻ cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.

Key Words::

• Baxter Isolex
• bone marrow
• CD34 selection
• flow cytometry
• Hetastarch

Acknowledgments

This project was supported by Production Assistance for Cellular Therapy (PACT) under contract numbers N01-HB-37165 and R01-HL-085819 from the National Heart, Lung, and Blood Institute.

The authors would like to acknowledge the technical assistance provided by Heather Stanczak and Eileen Koch for cell separation and enumeration, Deborah Livingston, Baxter Oncology, for assisting with the use of the Isolex device, Dr Amit Patel for collecting the bone marrow and injecting the product, G. Phillip Zorich for assisting with bone marrow harvests, and Dr James P. Bradley for providing bone marrow aspirates from orthopedic patients.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.