Graft-versus-host disease (GVHD) is a frequent and severe complication following hematopoietic cell transplantation. Natural CD4+25+ regulatory T cells (nTregs) have proven highly effective in preventing GVHD and autoimmunity in murine models. Yet, clinical application of nTregs has been severely hampered by their low frequency and unfavorable ex vivo expansion properties. Previously, we demonstrated that umbilical cord blood (UCB) nTregs could be purified and expanded in vitro using GMP reagents; however, the initial number of nTregs in UCB units is limited, and average yield after expansion was only 1×109 nTregs. Therefore, we asked whether yield could be increased by using peripheral blood (PB), which contains far larger quantities of nTregs. PB nTregs were purified under GMP conditions and expanded 80-fold to yield 19×109 cells using anti-CD3 antibody loaded, cell-based artificial antigen presenting cells (aAPCs) that expressed the high affinity Fc receptor and CD86. A single re-stimulation increased expansion to ~3,000-fold and yield to >600×109 cells, while maintaining FoxP3 expression and suppressor function. nTreg expansion was ~50 million-fold when flow-sort purified nTregs were re-stimulated four times with aAPCs. Indeed, cryopreserved donor nTregs re-stimulated four times significantly reduced GVHD lethality induced by the infusion of human T cells into immune deficient mice. The capability to efficiently produce donor cell banks of functional nTregs could transform the treatment of GVHD and autoimmunity by providing an off-the-shelf, cost-effective, and proven cellular therapy.
Our data indicate that the thawing and washing results in a substantial loss of cells, with TNC loss approaching 20 percent when compared with PF counts; the wash step was responsible for nearly half of the cell loss. The reduced PT viability was expected. Elapse of time PW resulted in further loss of NCs but no detectable significant changes in CD34+ cell content and viability and/or CFU.
Clinical-scale production of NK cells is efficient and can be performed under GMPs. The purified NK cell product results in high NK cell purity with minimal contamination by T cells, monocytes, and B cells, but it requires more time for processing and results in a lower NK cell recovery when compared to NK cell enrichment with CD3 cell depletion alone. Additional laboratory studies and results from clinical trials will identify the best source and type of NK cell product.
Microbial contamination of hematopoietic stem cell products is a rare but potentially fatal complication of hematopoietic stem cell transplantation. We report the incidence of contaminated products and describe the clinical outcomes for 35 patients at the University of Minnesota who received contaminated products from January 1990 to December 2004. In total, 2935 products were infused for 2863 transplants during this time, 36 of which 36 (1.2%) were contaminated. Coagulase negative Staphylococcus was the predominant species isolated on culture of the hematopoietic stem cell products. Patients received prophylactic antibiotics before infusion of the contaminated product based on the organism identified from culture and antibiotic sensitivities, if known. After transplantation, blood cultures from 2 patients grew the same pathogen as in the infused contaminated product, including 1 patient who had blood cultures positive for Pseudomonas cepacia. All patients who received contaminated products had benign post-transplantation courses except for the patient with Pseudomonas bacteremia, who ultimately died from complications. These results suggest that, although rare, microbial contamination of stem cell products does occur and there must be ongoing efforts by physicians and laboratory personnel to minimize the risk for introduction of contaminants. Prophylactic antibiotics are useful for certain contaminants; however, caution must be exercised when gram-negative contaminated products are administered.
Background
Current methods of MSC cryopreservation result in variable post thaw recovery and phenotypic changes due to freezing. The objective of this investigation is to determine the influence of ex-vivo cell expansion on phenotype of MSCs and the response of resulting phenotypes to freezing and thawing.
Methods
Human bone marrow aspirate was purchased from Lonza (Walkersville, MD). MSCs were isolated, and cells were assessed for total count, viability, apoptosis, and senescence over 6 passages (8–10 doublings/passage) in ex vivo culture. One half of cells harvested at each passage were re-plated for continued culture, and the other half were frozen at 1°C/min in a controlled rate freezer. Frozen samples were stored in liquid nitrogen, thawed, and reassessed for total cell count, viability, and senescence immediately and 48 hours post thaw.
Results
Viability did not differ significantly between samples pre freeze or post thaw. Senescence increased over time in pre freeze culture, and was significantly higher in one sample that experienced growth arrest both pre freeze and post thaw. Freezing resulted in similar initial post thaw recovery in all samples, but 48 hour post thaw growth arrest was observed in the sample with high senescence only.
Conclusion
High freeze senescence appears to correlate with poor post thaw function in MSC samples, but additional studies are necessary to obtain a sample size large enough to quantify results.
Background-Rapid release testing reduces the waiting period for administration of time-sensitive cell therapy products. Current assay systems are labor intensive and time consuming. The Endosafe ® Portable Test System (PTS™) is a chromogenic Limulus Amebocyte Lysate (LAL) portable endotoxin detection system which provides quantitative results in approximately 15 minutes. To evaluate Endosafe ® performance, specifically with cell therapy products, side-by-side testing of traditional LAL systems, and the Endosafe ® system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National
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