High Efficiency Microfluidic Purification of Stem Cells to Improve Transplants

描述(申请人提供)：造血干细胞移植(HSPC)是许多恶性和非恶性疾病的既定治疗方法。HSPC来源于3种来源：G-CSF动员的成人外周血(PBSC)、骨髓(BM)和脐带血(UCB)。因为红细胞增加了移植患者的有害副作用的风险和冷冻保存的成本，他们 必须从收获的HSPC组织中耗尽。脐带血移植的主要问题是小容量(100-300毫升)脐带血中的HSPC总数较少。这会导致延迟植入或植入失败的高风险(随之而来的是高死亡率、发病率和成本)，特别是在较大的儿童或成人患者中。目前的技术，包括密度梯度离心法和差速沉淀法，会导致红细胞不完全耗尽，在加工过程中可能会平均损失25%的白细胞。由于移植的成功率和速度取决于每受体体重的白细胞和HSPC的数量，因此开发新的细胞分离方法以确保从采集的脐带血中获得高纯度、有活力的白细胞和HSPC是必要的。这个STTR项目的商业目标是通过销售一种高效和强大的处理系统来改善干细胞库和移植，该处理系统能够出色地回收存活的白细胞和HSPC，重点是将脐带血作为一个直接目标，但涉及微流控细胞分离技术，该技术可以在未来进行修改，以进一步纯化HSPC和其他类型的干细胞。我们将利用微流体确定性横向位移(DLD)，其中细胞通过微流体系统的路径基于大小，并且是确定性的，即绝对确定的，不受随机过程的影响。这一STTR项目的关键创新是使用DLD来去除脐带血中的红细胞用于造血移植；这是一种新的临床用途，使这一应用具有商业新颖性和临床益处。该研究战略利用了GPB Science LLC的微机械系统专家、马里兰大学(Civin实验室)的造血细胞生物学和细胞处理专家以及普林斯顿大学(Sturm-Austin实验室)的微流体专家之间的合作。在具体目标1中，该团队将优化从脐带血中去除红细胞和回收白细胞的效果，解决可能阻止微流体流动的细胞聚集事件，并将微流体吞吐量提高到临床相关体积。在具体目标2中，该系统将被改装用于无菌临床应用，以便在随后的第二阶段项目中，这种大容量微流控血液分离系统可以用于评估体外和体内的HSPC，以确定其功能在红细胞耗尽后是否被保存。这项技术还将扩展到用于PBSC和BM收获。其价值主张是明确的：GPB技术应该提供更多更高质量的移植移植物(即更多的移植物和更多的HSPC)，这将导致更大的移植成功。 公共卫生相关性：在冷冻保存之前，迫切需要一种快速、有效的方法从动员G-CSF的外周血(PBSC)、骨髓(BM)，特别是脐带血(UCB)中去除红细胞并回收白细胞。不完全去除移植物中的红细胞增加了造血干细胞移植中不良副作用的风险，而存活的白细胞和CD34+细胞的恢复不良降低了移植的成功率，并限制了可治疗的患者数量。开发一种新的、高效的系统来去除红细胞和纯化白细胞将提高脐带血和其他移植移植物的质量，从而显著改善患者的预后。

DESCRIPTION (provided by applicant): Hematopoietic stem-progenitor cell (HSPC) transplantation is an established therapy for many malignant and non-malignant diseases. HSPCs are harvested clinically from 3 sources: G-CSF mobilized adult peripheral blood (PBSC), bone marrow (BM), and umbilical cord blood (UCB). Because erythrocytes increase both the risk of harmful side effects in transplant patients and the cost of cryopreservation, they must be depleted from the harvested HSPC tissues. The major problem in UCB transplantation is the low total number of HSPCs in the small volume (100-300 ml) of UCB units. This leads to high risk for delayed engraftment or engraftment failure (with attendant high mortality, morbidity and costs), especially in larger children or adult patients. Current techniques, including density gradient centrifugation and differential sedimentation, result in incomplete erythrocyte depletion and may lose 25% leukocytes (on average) during processing. Since success and speed of engraftment depend on the numbers of leukocytes and HSPCs per recipient body weight, it is essential to develop new cell separation methods to ensure high yields of pure, viable leukocytes and HSPCs from harvested UCB. The commercial goal of this STTR project is to improve stem cell banking and transplantation by marketing an efficient and robust processing system that results in superior recoveries of viable leukocytes and HSPCs, focusing on UCB as an immediate objective but involving a microfluidic cell separation technology that can be modified in the future to further purify both HSPCs and other stem cell types. We will take advantage of microfluidic deterministic lateral displacement (DLD), in which the paths cells take through the microfluidic system is based on size and is deterministic, i.e. absolutely determined, not subject to random processes. The key innovation of this STTR project is the use of DLD to deplete erythrocytes from UCB for hematopoietic transplant; this is a new clinical use, making this application commercially novel as well as clinically beneficial. The research strategy leverages a collaboration among micromechanical systems experts at GPB Scientific LLC; hematopoietic cell biology and cell processing experts at University of Maryland (Civin lab); and microfluidics experts at Princeton University (Sturm-Austin lab). In Specific Aim 1, the team will optimize the efficacy of erythrocyte depletion and leukocyte recovery from UCB, solving cell clumping events which can block microfluidic flow and increasing microfluidic throughput to clinically relevant volumes. In Specific Aim 2, the system will be modified for aseptic clinical us such that in the subsequent Phase II project, this high- volume microfluidic blood separation system can be used to assess HSPCs in vitro and in vivo to determine if their functionalities are conserved after erythrocyte depletion. The technology will also be extended for use with PBSC and BM harvests. The value proposition is clear: the GPB technology should deliver greater numbers of higher quality transplant grafts (i.e. more grafts with more HSPCs) that will lead to greater transplant success. PUBLIC HEALTH RELEVANCE: There is a critical unmet need for rapid, efficient methods to deplete erythrocytes and recover leukocytes from G-CSF mobilized peripheral blood (PBSC), bone marrow (BM), and especially umbilical cord blood (UCB), prior to cryopreservation. Incomplete erythrocyte removal from transplant grafts increases the risk of harmful side effects in hematopoietic stem cell transplants, while poor recovery of viable leukocytes and CD34+ cells reduces engraftment success and limits the treatable patient population. Development of a novel, highly efficient system to remove erythrocytes and purify leukocytes would raise the quality of UCB and other transplant grafts, thereby significantly improving patient outcomes.