title: Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting.
abstract: OBJECTIVE
  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 × 10(10) total cells, 6.2 ± 2.2 × 10(8) 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.
authors: Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
authors: Vijay S
authors: VS
authors: Gorantla
authors: Stefan
authors: S
authors: Schneeberger
authors: Linda R
authors: LR
authors: Moore
authors: Vera S
authors: VS
authors: Donnenberg
authors: Ludovic
authors: L
authors: Zimmerlin
authors: W P Andrew
authors: WP
authors: Lee
authors: Albert D
authors: AD
authors: Donnenberg
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology; D002102:Cadaver; D002469:Cell Separation; D002470:Cell Survival; D002478:Cells, Cultured; D046528:Chimerism; D005240:Feasibility Studies; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007108:Immune Tolerance; D045164:Mesenchymal Stem Cell Transplantation; D016377:Organ Transplantation; D044968:Regenerative Medicine / Q000379:methods / Q000639:trends; D013131:Spine / Q000166:cytology*; D014019:Tissue Donors