Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Abstract

Background aims. Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. Methods. We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. Results. The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^– CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells). Conclusions. This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.

Key Words::

• good manufacturing practice
• organ transplantation
• vertebral bone marrow

Acknowledgments

The authors wish to thank Dr Thomas E. Starzl (University of Pittsburgh) for his leadership in this project. We also wish to acknowledge Drs Gerald Brandacher and Benson Pulikkottil for their participation and expertise in the operating room and laboratory, and Dr Noriko Murase (University of Pittsburgh) and Dr Camillo Ricordi (Diabetes Research Institute, Miami, FL) for sharing their experience in the preparation of cadaveric BM.

This work was funded in part by the Armed Forces Institute for Regenerative Medicine and the Orthopedic Trauma Research Program and the University of Pittsburgh Medical Center.

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