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

Vijay S. Gorantla, Stefan Schneeberger, Linda R. Moore, Vera S. Donnenberg, Ludovic Zimmerlin, W. P.Andrew Lee, Albert D. Donnenberg

Research output: Contribution to journalArticle

19 Scopus citations

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 L4T8 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 × 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+ CD117dim 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.

Original languageEnglish (US)
Pages (from-to)104-113
Number of pages10
JournalCytotherapy
Volume14
Issue number1
DOIs
StatePublished - Jan 2012
Externally publishedYes

Keywords

  • good manufacturing practice
  • organ transplantation
  • vertebral bone marrow

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Genetics(clinical)
  • Cell Biology
  • Cancer Research
  • Transplantation

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