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.

Cadaveric cardiosphere-derived cells can maintain regenerative capacity and improve the heart function of cardiomyopathy

“…[17][18][19] It has also been shown previously that split-thickness skin grafts could be allografted up to 3 weeks after death if stored at 4 C. 20 More recently, stem and adult cells have been isolated from post mortem brain, spinal cord, and retinal tissue. 3,[21][22][23] These findings showed that, due to the persistence of stem cells, cadaver tissue could be a potential abundant source of these cells. We are the first to report that viable CDCs can be isolated post mortem, in both humans and mice.…”

Clinical Implementation of a Procedure to Prepare Bone Marrow Cells From Cadaveric Vertebral Bodies

“…The clinical transplantation protocol was approved by the University of Pittsburgh Internal Review Board. A total of nine vertebrae (Th8–L4) were harvested according to our published procedure [7]. VBs were rinsed with a saline–betadine solution and double-bagged with 2 l of sterile Custodiol ® preservation solution (Essential Pharmaceuticals, LLC, Newtown, PA, USA).…”

“…Recently, we have expanded the use of this technique to hand and forelimb grafts [6], where the indication for transplantation is quality of life improvement and there is a pressing need to protect the patient from the risks of lifelong immunosuppression. In order to implement the Pittsburgh Protocol in this setting we preformed preclinical experiments to develop and validate a Good Manufacturing Practice (GMP)-compliant method for preparation of a cellular product from cadaveric vertebral bodies [7]. In this article, we describe the implementation of this methodology as a clinical protocol, and publish the full working procedure as Supplementary Material (see online at www.futuremedicine.com/doi/suppl/10.2217/RME.11.89).…”

Surgical and Logistical Aspects of Donor Limb Procurement in Hand and Upper Extremity Transplantation

“…Novel immunomodulation protocols, such as infusion of donor derived bone marrow cells as used by the senior authors, further impact procurement logistics and need to be carefully considered. 12,36,37 Overall, the field of VCA is rapidly evolving, requiring further standardization, guidelines, more consistent reporting, and is ripe with opportunity to advance current practice and knowledge.…”