Abstract 489: Buoyant targeted microbubble technology provides efficient and superior quality of hematopoietic stem cells isolated from umbilical cord blood
Authors: Shi Guixin , Wu Christina C.N. , Darmadi Sean , Wang Zhiyong , Carson Dennis , Liu Yu-Tsueng , Liu Yu-Tsueng 
Published: September 2020

Purposes: Various separation methods to isolate and enrich hematopoietic stem/progenitor cells (HSPC) have been developed in the past several decades. However, there is still growing interest in clinical trial settings to purify CD34+ cells for ex vivo HSPC expansion and for gene therapy. We have recently developed BUBLES (Buoyancy Enabled Separation), an innovative cell sorting technology using the lipid shell microbubble that is self-molding to external forces. In conjunction with a gas core, it is a very gentle material for cell isolation. This patented technology has the potential to overcome some key hurdles of cell therapy in humans, including targeted multi-parametric cell isolation in bulk for clinical applications. In this study, we aimed to demonstrate that the BUBLES technology is able to efficiently enrich CD34+ HSPC from umbilical cord blood (UCB) and preserve the unique characteristics of stem cells by both in vitro cell assays and in vivo animal study.

Methods: Human mononuclear cells (MNCs) were first isolated from fresh UCB samples using a Ficoll-based density gradient method. Subsequently, MNCs were divided to two groups for side-by-side comparisons of CD34+ HSPC isolation using either BUBLES system or a current marketed magnetic nanobead-based CD34+ isolation kit, EasySep. Flow cytometry (FACS) assays were carried out to characterize the isolated HSPC and determine the efficiency of cell enrichment. To assess the quality and functionality of isolated HSPC, in vitro assays, including CD34+ cell expansion with StemSpan™ and colony formation experiments, were performed. Furthermore, isolated CD34+ HSPC were transplanted to immunodeficient NSG-S mice via tail vein and periodic blood screens were conducted to evaluate the engraftment efficiency and differentiation outcomes.

Results: FACS analysis of the isolated human HSPC (n = 6) revealed that the average purity and recovery rate of CD34+ cells using the microbubble-based BUBLES technology were 89.8% ± 3.6% and 51.0% ± 13.9%, respectively, whereas 49.5% ± 19.6% and 72.7% ± 16.9% were observed with EasySep. The BUBLES-isolated CD34+ HSPC were expanded 32-43 fold and 301-1188 fold, higher than those isolated by EasySep at day 7 and day 14, respectively (n=2). An average of 50 colony-forming units per 100 CD34+ cells were observed in the BUBLES group, compared to 31 units in the ES group (n=4). Moreover, as early as 5 weeks post-transplantation, 1.26%-2.22% of human CD45+ cells were detected in the peripheral blood of transplanted mice with BUBLES-isolated CD34+ cells (n = 2) whereas no engraftment was observed in those with EasySep. Normal B and T cell differentiation was observed in both groups of transplanted mice after 8 weeks.

Conclusion: Based on both in vitro and in vivo results, we demonstrate that BUBLES technology effectively isolates CD34+ HSPC from UCB and preserves the stemness quality.

Citation Format: Guixin Shi, Christina C.N. Wu, Sean Darmadi, Zhiyong Wang, Dennis Carson, Yu-Tsueng Liu, Yu-Tsueng Liu. Buoyant targeted microbubble technology provides efficient and superior quality of hematopoietic stem cells isolated from umbilical cord blood [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 489.