The SmartBio System for the Improved Preservation of Human Hematopoietic Stem Cells

摘要 冷冻保存（CP）的最终目标是开发一种生物库过程， 细胞活力和功能水平与未经过保存的姐妹群体相同。 大多数方法用于临床水平的造血干细胞（hHSC）及相关干细胞 然而，细胞由于解冻后坏死和凋亡而导致较差的恢复，从而产生较差的细胞 输注到患者体内的产品。鉴于临床上超过一半或更多的干细胞产品 今天的试验，包括hHSC，在10%DMSO中冷冻保存，这是高毒性的，许多研究 研究了提高CP以及替代冷冻保护剂（CPA）（如糖）的方法 醇以避免DMSO对hHSC分化的已知毒性和影响。然而，迄今为止， 消除DMSO的hHSC CP工艺已经商业化开发。CPSI生物技术公司假设 必须实现多个里程碑，以产生最佳的hHSC CP工艺， 干细胞患者的最佳结果，同时最大限度地减少或消除对DMSO的需求。第一、 必须开发一种独特的高通量冷冻装置， 被定义和执行。第二，DMSO必须用可以防止形成的试剂代替。 以及CP过程中致命冰的再结晶。第三，必须有一个独特的干燥解冻系统， 开发，将消除潜在的样品污染，服从文件， 重要是，可以提供hHSC匹配的解冻特征，其可以用多种不同的 生物库小瓶和袋子最后，解冻后回收调节试剂和输血培养基 必须开发可以防止解冻后延迟发生的细胞死亡并改善 移植期间的hHSC。本第二阶段计划旨在解决以下每个里程碑 SA 1a-开发StemFreeze -一种独特的高通量自动冷冻系统， 系统比当前系统具有更多的操作员和容器灵活性， 可以开发冻结配置文件; SA 1b-开发StemThaw -SmartThaw系统的衍生产品 由第一阶段的母项目支持，该项目将明确设计用于优化解冻 hHSC和其他干细胞的过程导致解冻后活力和功能的改善; SA 2a- 开发StemCP -一种含有冰再循环抑制剂（IRI）的无毒CPA冷冻鸡尾酒， 替代DMSO，DMSO可以添加到选择的任何载体培养基中以改善hHSC在细胞中的CP。 不存在DMSO; SA 2b-配制StemRevive和StemInfusion -解冻后的首个同类产品 调节剂和稀释培养基设计用于改善解冻后细胞凋亡/坏死增加 干细胞输注疗效和SA 3-评估干细胞特异性SmartBio平台， 体外和体内HSC的CP。该多组分系统将共同包含干细胞 CPSI SmartBio平台下的特定产品线。总之，CPSI假设，解决所有 这五项挑战将简化生物库流程，并使hHSC CP科学更加接近 最终的里程碑是获得相当于非冷冻hHSC的冷冻保存产品。 这一成就将改善接受干细胞移植的患者的预后。

Abstract The ultimate goal of cryopreservation (CP) is to develop a biobanking process that results in a post-thaw level of cell viability and function identical to a sister population that has not undergone preservation. Most methods used for the clinical level CP of human hematopoietic stem cells (hHSCs) and related stem cells, however, result in poor recovery due to post-thaw necrosis and apoptosis yielding an inferior cell product which is infused into patients. Given that over half or more of all stem cell products in clinical trials today, including hHSCs, are cryopreserved in 10% DMSO which is highly toxic, many studies have investigated ways to improve CP as well as alternative cryoprotective agents (CPAs) such as sugar alcohols to avoid the known toxicity and impact of DMSO on hHSC differentiation. Yet, to date no new hHSC CP process that eliminates DMSO has been developed commercially. CPSI Biotech hypothesizes that multiple milestones must be achieved to generate the best possible hHSC CP process to yield optimal outcome for the stem cell patient while minimizing or eliminating the requirement for DMSO. First, a unique high throughput freezing device must be developed so that hHSC-specific freezing profiles can be defined and executed. Second, DMSO must be replaced with agents that can prevent the formation and re-crystallization of lethal ice during the CP process. Third, a unique dry thaw system must be developed that will eliminate potential sample contamination, be amenable to documentation and most importantly can provide for hHSC-matched thaw profiles that can be achieved with a diverse array of biobanking vials and bags. Finally, a post-thaw recovery conditioning reagent and transfusion medium must be developed that can prevent post-thaw delayed onset cell death and improve engraftment of hHSC during transplant. This Phase II program is designed to address each of these milestones under the following specific aims: SA1a – Develop StemFreeze – a unique high throughput automated freezing system that has more operator and container flexibility than current systems such that hHSC-matched freezing profiles can be developed; SA1b - Develop StemThaw – a derivative of the SmartThaw system supported by the parent Phase I project that will be expressly designed for optimizing the thawing process of hHSCs and other stem cells resulting in improved post-thaw viability and function; SA2a - Develop StemCP - a non-toxic CPA cryo-cocktail containing Ice Recrystallization Inhibitors (IRIs) to replace DMSO which can be added to any carrier medium of choice to improve CP of hHSCs in the absence of DMSO; SA2b - Formulate StemRevive and StemInfusion – a first in class post-thaw conditioning reagent and dilution media designed to ameliorate post-thaw apoptosis/necrosis increasing stem cell transfusion efficacy and SA3 – Evaluate the stem cell specific SmartBio platform for optimized CP of HSCs in vitro and in vivo. Collectively this multicomponent system will comprise a stem cell specific product line under CPSI’s SmartBio platform. In summary, CPSI hypothesizes that addressing all five challenges together will streamline the biobanking process and move the hHSC CP sciences closer to the ultimate milestone of achieving a cryopreserved product that is equivalent to non-frozen hHSCs. This achievement will result in improved outcome for patients receiving stem cell transplants.