Maximal Cord Blood Recovery and CD34⁺ Progenitor Cell Collection Using Machine Pulsatile Perfusion of Placentas.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells (HSC) because of greater availability, less stringent HLA matching requirements, and lower incidence and severity of GVHD. Currently, UCB transplant procedures in adults are limited by low collection volumes of total nucleated cells and CD34⁺ HSC. Approaches to ex vivo expand long-term engraftable HSC have been widely unsuccessful. Recent studies have clearly demonstrated that infusion of a greater number of cells UCB cells enhances the rate of engraftment and lowers the risk of transplantation-related mortality. Machine pulsatile perfusion (MPP) has been successfully used to select cadaveric renal allografts for transplantation, to isolate human islets from pancreata and shown to be a useful cardiac preservation technique in canine heart transplant studies. In this study, the feasibility of using machine pulsatile perfusion to collect human UCB total nucleated cells and CD34⁺ HSCs was evaluated using placentas designated for research purposes. Immediately following delivery UCB (65 ± 15 mL, n=5) was first collected by needle aspiration from the umbilical cord vein in accordance with standard procedures then followed by MPP (~500 ml) of the placental arteries within 2–3 hours of delivery. Clinically total nucleated cells count (TNC), CD34⁺ cell numbers and myeloid, erythroid and multipotent CFU progenitor cell content of UCB units are used as predictive measurements of hematopoietic/engraftment potential. Low-density cells (<1.077 g/mL) were isolated by density centrifugation. The median number of viable low density cells obtained was 488 × 10⁶ (range, 240–652 × 10⁶), and 1541 × 10⁶ (range, 888–1800 × 10⁶) for UCB and MPP collections, respectively. MMP low density cell preparations contained significantly more mature segmented neutrophils with a low percentage (<0.1%) of sheared-off vessel wall endothelial cells. Both UCB and MPP low density cells collections showed similar number of assayable CFU-GEMM, CFU-GM, CFU-M, and CFU-G progenitor cells. In contrast, MMP collected cells contained 2–3 times more erythroid BFU-E colonies than UCB collections. Equivalent numbers of CD34⁺ HSC were enumerated by FACS analysis and subsequently isolated by positive immunomagnetic MACS selection from MPP and UCB collections. Likewise, the progenitor cell content (CFU-GEMM, CFU-GM, CFU-M, CFU-G and BFU-e) of the isolated CD34⁺ cell populations derived from each cell collection were very similar. These results demonstrate that pulsatile perfusion can be performed easily after traditional UCB collection procedures. This technique effectively recovers on average twice as many TNC and multilineage CD34⁺ HSC cells when compared to traditional UCB collection procedures. Altogether these results are particular promising since increased numbers of UCB HSC available for infusion should result in accelerated hematopoietic recovery. Moreover, the demonstrated enhanced HSC cell yield together with the simplicity of collection could potentially widen the clinical applicability of UCB transplants in adults.