Bioprocess Development for the Large Scale Production of Extracellular Vesicles From Adult Stem Cells

干细胞在人体内几乎所有的组织中都以非常小的数量存在。例如，间充质干细胞（MSC）是一种可以在骨髓和脂肪中发现的成体干细胞。人们对MSC有着巨大的兴趣，因为它们植入受损组织通常会导致某种程度的功能恢复。尽管MSC的广泛临床应用存在重大障碍（例如监管问题，成本），但已经进行了数百项临床试验来研究MSC如何用于治疗心脏病，中风和I型糖尿病等疾病。现在有证据表明，MSC植入后观察到的许多积极结果是由于它们分泌的产物。特别感兴趣的是细胞外囊泡（EV），由MSC分泌的包含由生物活性分子组成的货物的小球形结构。已经观察到EV从产生它们的细胞迁移并将它们的货物递送到靶细胞，导致靶细胞执行所需的动作，例如启动修复过程。因此，MSC衍生的EV（MSC-EV）具有作为有效治疗剂在临床上施用的潜力，这是一种与MSC植入相比具有更少障碍的方法。此外，现在已知电动汽车是自然产生的，从我们胚胎的时候到我们死亡的时候。它们被认为是细胞之间复杂的通信系统，并涉及组织发育，功能和衰老的过程。因此，研究人员可以使用EV来更多地了解健康个体的这些过程，并了解这些过程的变化如何导致出生缺陷，组织退化和疾病。电动汽车领域目前处于起步阶段，并准备爆炸性增长。阻碍进展的一个巨大挑战是缺乏研究人员可以使用的定义电动汽车。由于它们不能轻易地直接从组织中获得，研究人员收集了由MSC在体外生长的血管中产生的EV，这些血管含有由鼓励分离的MSC分裂和分泌EV的成分组成的液体培养基。标准的大规模EV生产方法尚不存在，并且目前从培养基中收集EV的方法费力、耗时且回收率低。我们有一个既定的生物制造研究计划，在该计划中，我们以前已经开发出强大的技术，以在称为生物反应器的大型受控容器中扩大干细胞生产。在这里，我们建议扩展我们的计划，并在未来5年内，（i）设计标准方法，用于在生物反应器中大规模生产定义的功能性MSC-EV种群，以及（ii）设计一种能够从培养基中快速收集EV的新型设备。这项工作的成果将是创造支持电动汽车生产、研究和应用开发的新技术，以及培训有资格在这一迅速崛起的新领域工作的人员。

Stem cells exist in very small numbers in almost all tissues within the body. For example, mesenchymal stem cells (MSCs) are a type of adult stem cell that can be found in bone marrow and fat. There is huge interest in MSCs because their implantation into a damaged tissue often leads to some degree of functional recovery. Despite there being significant barriers (e.g. regulatory issues, cost) to the widespread clinical use of MSCs, hundreds of clinical trials have been undertaken to investigate how MSCs can be used to treat medical conditions such as heart disease, stroke and type I diabetes. Evidence now suggests that many of the positive outcomes observed after MSC implantation are due to the products they secrete. Of particular interest are extracellular vesicles (EVs), small spherical structures secreted by MSCs that contain cargo composed of bioactive molecules. EVs have been observed to migrate from the cell where they were produced and deliver their cargo to target cells, causing the target cells to perform a desired action, such as initiating a repair process. Thus, MSC derived EVs (MSC-EVs) have the potential to be administered clinically as effective therapeutic agents, an approach with fewer barriers compared to MSC implantation. Moreover, EVs are now known to be naturally produced in all of us, from the time we are embryos to the time that we die. They are believed to serve as a complex communication system between cells and are implicated in the processes of tissue development, function and aging. Thus, researchers can use EVs to learn more about these processes in healthy individuals and understand how changes in these processes can lead to birth defects, tissue degeneration and disease. The field of EVs is currently in its infancy and primed for explosive growth. A huge challenge hampering progress is a lack of defined EVs with which researchers can work. Because they can't be easily obtained directly from tissues, researchers collect EVs made by MSCs that are grown outside the body in vessels which contain a liquid medium made up of ingredients that encourage isolated MSCs to divide and secrete EVs. Standard large-scale EV production methods do not yet exist, and current methods to collect EVs from the medium are laborious, time consuming and have low recoveries. We have an established biomanufacturing research program in which we have previously developed robust technologies to scale-up stem cell production in large, controlled vessels called bioreactors. Here we propose to extend our program, and over the next 5 years, (i) engineer standard methods for the large-scale production of defined, functional MSC-EV populations in bioreactors, and (ii) design a novel device capable of rapidly collecting EVs from medium. Outcomes of this work will be the creation of novel technologies that support EV production, study and application development, and the training of individuals qualified to work in this rapidly emerging new field.