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Background & Aim Patients with primary immune-deficiency (PID) may develop pulmonary complications and at that stage most are ineligible for either lung transplant (LTX) or bone marrow transplant (BMT) alone due to futility. We are conducting a first in human prospective trial, ClinicalTrials.gov: NCT01852370 where PID patients in lung failure -past the opportunity for curative BMT alone- may receive a 2 of 6 HLA antigen-matched cadaveric BOLT and minimum 8 weeks alter cryopreserved BMT from the same deceased UNOS donor. We hypothesized: 1) hematopoietic stem cells prepared from organ donor vertebral bodies (VB) would yield greater CD34+ cell dose compared to iliac crest aspirates-in part due to limited harvest time to avoid delay in the activity of organ recovery teams; 2) cryopreserved CD3/CD19 depleted bone marrow could generate durable hematopoiesis and donor derived immunity. Here we present the first 5 consecutive cases vertebral bone marrow (VBM) processing experience at Children's Hospital of Pittsburgh and subsequent CD3+/CD19+ cell depletion on the CliniMACS® device. With a threshold viability of 70% prior to cryopreservation the CD3/CD19-depleted product must contain 1 × 10E+6/kg CD34+ cells and 1 × 10E+5/kg CD3+ T cells to achieve durable engraftment and reduce the risk of GVHD respectively.® Methods, Results & Conclusion For details of pre/post processing, see Table. Since early 2017 we introduced chest/abdominal cavity wash with 1:1 saline-betadine mix and scrubbing T11-L4VB logs with chlora-prep that has eliminated bacterial contamination for the last 3 products. The first 2 were infused without adverse events following IRB/FDA approved antibiotic prophylaxis. Initially, processing of the VBs was arduous, using a scalpel and wire brush to clean the logs, then dissecting into small pieces with a mallet and osteotome. Since early 2017 a Stryker RemB saw has been used. A physical task that had taken 2 technologists 3 hours to complete has become almost effortless and one tech is freed up. The target pre-cryo CD34+ dose and viabilities have met target at all times, in fact CD34+ recovery has increased >70%, see Table. The entire process (dissection, crushing, filtering, CD3/CD19 depletion and cryopreservation) can be completed in 12 hours with 3 technologists. In summary, we have developed a complex sequence of procedures that even exceeded our target of 2E+06 CD34/kg pre-cryo dose (mean: 4.49E+06) leading to prompt and multilineage engraftment in 3 of the first 4 analyzable patients.

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 fi rst and second rounds of tumbling, respectively. Cells were pooled and fi ltered (500, 200 μ m) 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 fl ow 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.

(2001). Good manufacturing practices (GMP) in a hospital environment: the ideal and the real. Cytotherapy: Vol. 3, No. 2, pp. 63-65.

PBPC or BM is increasingly being harvested in remission for possible use in the event of relapse. Although the value of this approach has not been demonstrated, the long-term storage of progenitor cell components has become commonplace in many facilities.We used multi-parameter flow cytometry to determine the viability of 11 long-term cryopreserved BM components (mean = 11.8 years) in liquid phase nitrogen. The components, prepared for autotransplantation but deaccessioned after confirming patient death, were carefully thawed, washed, and assayed immediately. The flow cytometry assay was performed according to the ISHAGE protocol, modified by the addition of 7AAD for analysis of progenitor viability (CD45+ CD34+ 7AAD-) and total leukocyte viability (CD45+ 7AAD-). In addition, total viability was assessed by fluorescence microscopy using acridine orange dye exclusion; granulocyte-monocyte colony-forming units (CFU-GM) were measured after 14 days culture.Leukocyte viability by flow cytometry and fluorescence microscopy agreed well (r2 = 0.55, slope = 0.83, P0.0005 by linear regression). CFU-GM did not correlate with CD34% or any of the viability parameters. Compared with short-term stored (mean = 33 days) PBPC assayed at infusion, long-term stored BM had a comparable percentage of CD34+ cells, comparable CFU-GM activity, increased CD34 viability, but decreased total cell viability, the latter most likely due to an increased proportion of differentiated myeloid cells.The results indicate that BM products can be cryopreserved for more than a decade without apparent loss of progenitor activity, as measured by these laboratory surrogates. This agrees with clinical anecdotes describing successful engraftment with long-term stored BM, and argues that expiration dates cannot be set for cryopreserved hematopoietic stem-cell components stored in liquid phase nitrogen.