Ex vivo expansion of haematopoietic stem/progenitor cells for pooled cord blood transplantation

Abstract

Introduction: Umbilical cord blood transplantation (UCBT) has become an established therapy for patients with various cancers and disorders of the blood and immune system. However, low numbers of haematopoietic stem and progenitor cells (HSPC) has limited this application in adult recipients. As such, studies are underway to explore various expansion strategies to increase HSPC. We proposed to expand one cord blood (CB) to be transplanted at day 0, with another unexpanded unit to be transplanted at day 1. Methods: Frozen cord blood units were thawed and enriched for CD133+ cells using the CliniMACS prior to ex vivo expansion with cytokines in suspension culture. Processes were refined through several rounds of optimisation in thaw and selection steps. Results: Initial cell recovery rate from thawing and washing steps was 34.80±1.20% (n=3). After increasing the centrifuge speed and reducing the washing steps, cell recovery rate improved to 77.25±4.73% (n=8). CD34+ percentages increased 13.41±4.08% to 49.33±11.47% after optimisation included increase in protein concentration and discontinuation of use of DNAse in selection buffer. Consequently, the total nucleated cell (TNC) expansion increased to as much as 99.20±2.79 fold. CD34+CD45+ cells, CD33+CD45+ cells and CFU-GM were expanded 3.63±1.90 fold, 504.50±146.02 fold and 14.78±7.23 fold respectively. Conclusion: The current protocol produced 100x increase of total nucleated cells, without loss of CD34+CD45+ HSPC, but with an efficient expansion of myeloid progenitor cells to enhance post-transplant haematopoietic reconstitution. This current protocol has been incorporated in our translational clinical platform for ex vivo cord blood expansion.