Shipping of therapeutic somatic cell products.

BACKGROUND AIMS: Shipment of therapeutic somatic cells between a current good manufacturing practice (cGMP) facility and a clinic or between different cGMP facilities requires validated standard operating procedures (SOP). Under National Heart Lung & Blood Institute (NHLBI) sponsorship, the Production Assistance for Cellular Therapies (PACT) group conducted a validation study for the shipping SOP it has created, including shipments of cryopreserved somatic cells, fresh peripheral blood specimens and apheresis products. METHODS: Comparisons of pre- and post-shipped cells and cell products at the three participating facilities included measurements of viability, phenotypic profiles and cellular functions. The data were analyzed at the University of Pittsburgh Biostatistics Facility. RESULTS: No consistent shipping effects on cell viability, phenotype or functions were detected for cryopreserved and shipped peripheral blood mononuclear cells (PBMC), monocytes, immature dendritic cells (iDC), NK-92 or cytotoxic T cells (CTL). Cryopreserved mesenchymal stromal cells (MSC) had a significantly decreased viability after shipment, but this effect was in part because of inter-laboratory variability in the viable cell counts. Shipments of fresh peripheral blood and apheresis products for the generation of CTL and dendritic cells (DC), respectively, had no significant effects on cell product quality. MSC were successfully generated from fresh bone marrow samples shipped overnight. CONCLUSIONS: This validation study provides a useful set of data for guiding shipments of therapeutic somatic cells in multi-institutional clinical trials.

Intra-operative preparation of autologous bone marrow-derived CD34-enriched cellular products for cardiac therapy.

BACKGROUND AND AIMS: With the advent of regenerative therapy, there is renewed interest in the use of bone marrow as a source of adult stem and progenitor cells, including cell subsets prepared by immunomagnetic selection. Cell selection must be rapid, efficient and performed according to current good manufacturing practices. In this report we present a methodology for intra-operative preparation of CD34(+) selected autologous bone marrow for autologous use in patients receiving coronary artery bypass grafts or left ventricular assist devices. METHODS AND RESULTS: We developed a rapid erythrocyte depletion method using hydroxyethyl starch and low-speed centrifugation to prepare large-scale (mean 359 mL) bone marrow aspirates for separation on a Baxter Isolex 300i immunomagnetic cell separation device. CD34 recovery after erythrocyte depletion was 68.3 ± 20.2%, with an average depletion of 91.2 ± 2.8% and an average CD34 content of 0.58 ± 0.27%. After separation, CD34 purity was 64.1 ± 17.2%, with 44.3 ± 26.1% recovery and an average dose of 5.0 ± 2.7 × 10(6) CD34(+) cells/product. In uncomplicated cases CD34-enriched cellular products could be accessioned, prepared, tested for release and administered within 6 h. Further analysis of CD34(+) bone marrow cells revealed a significant proportion of CD45(-) CD34(+) cells. CONCLUSIONS: Intra-operative immunomagnetic separation of CD34-enriched bone marrow is feasible using rapid low-speed Hetastarch sedimentation for erythrocyte depletion. The resulting CD34-enriched product contains CD45(-) cells that may represent non-hematopoietic or very early hematopoietic stem cells that participate in tissue regeneration.

Effects of storage and leukoreduction on lymphocytes and Epstein-Barr virus genomes in platelet concentrates.

BACKGROUND: Epstein-Barr virus (EBV) persists in infected B lymphocytes in blood donors. Lymphocytes are viable during platelet (PLT) storage. The effects of storage and leukoreduction on lymphocytes and EBV genomes are evaluated. STUDY DESIGN AND METHODS: Forty nonleukoreduced PLT concentrates were stored at 20 to 24°C for up to 7 days. EBV genomes in B cells were quantified on Days 1 and 5. Viable white blood cells (WBCs) and T and B cells were quantified in 10 of 40 units on Days 1, 3, 5, and 7 of storage. For the leukoreduction study, four pools of PLTs were leukoreduced within 24 hours of collection. B cells from before leukoreduction and all peripheral blood mononuclear cells from after leukoreduction were assayed for EBV. RESULTS: Viable WBCs and T cells were stable whereas viable B cells were reduced to 71% of the Day 1 level by Day 5. A total of 31 of 37 (83.8%) units were EBV positive. Although EBV genomes remained stable in most units, 12 of 37 units demonstrated a median of 5.1 (range 2- to 134)-fold increase in EBV genomes per 105 B cells on Day 5. For the leukoreduction study, EBV genomes were detected in four of four pools before leukoreduction with a median of 3.8 (range, 0.2-93.6) EBV genomes per 105 B cells. EBV genomes were not detected in any of the postleukoreduction specimens. CONCLUSIONS: Seventy percent of B lymphocytes are viable on Day 5 of PLT storage. Although the mean number of EBV genomes remained stable, a subset of units had increased EBV genomes during storage. Leukoreduction removed polymerase chain reaction-detectable EBV genomes from PLT pools.