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

干细胞的数量非常少，存在于人体几乎所有组织中。例如，间充质干细胞（MSCs）是一种可以在骨髓和脂肪中发现的成体干细胞。人们对间充质干细胞非常感兴趣，因为将其植入受损组织中通常会导致一定程度的功能恢复。尽管在临床上广泛使用间充质干细胞存在重大障碍（例如监管问题、成本），但已经开展了数百项临床试验，以研究如何使用间充质干细胞治疗心脏病、中风和I型糖尿病等疾病。现在有证据表明，骨髓间充质干细胞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（EVs），这是一种由间充质干细胞分泌的小球形结构，含有由生物活性分子组成的货物。已经观察到电动汽车从产生它们的细胞迁移，并将它们的货物运送到目标细胞，导致目标细胞执行期望的动作，例如启动修复过程。因此，MSC衍生的ev （MSC- ev）有潜力作为有效的治疗药物在临床上使用，与MSC植入相比，这种方法的障碍更少。此外，我们现在知道，从胚胎到死亡，我们所有人都会自然产生电动汽车。它们被认为是细胞间复杂的通讯系统，与组织发育、功能和衰老过程有关。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、组织变性和疾病。电动汽车领域目前处于起步阶段，即将迎来爆发式增长。阻碍进展的一个巨大挑战是缺乏研究人员可以使用的明确的电动汽车。因为它们不容易直接从组织中获得，研究人员收集了由骨髓间充质干细胞制造的内皮细胞，这些内皮细胞生长在体外的血管中，血管中含有一种液体培养基，这种液体培养基由促使分离的骨髓间充质干细胞分裂和分泌内皮细胞的成分组成。目前还没有标准的大规模电动汽车生产方法，目前从培养基中收集电动汽车的方法费力、耗时且回收率低。我们有一个成熟的生物制造研究项目，在这个项目中，我们之前已经开发出了强大的技术，可以在被称为生物反应器的大型受控容器中扩大干细胞的生产。在此，我们建议扩展我们的计划，并在未来5年内，(i)设计用于在生物反应器中大规模生产定义的功能性MSC-EV种群的标准方法，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，并培训有资格在这一迅速崛起的新领域工作的个人。

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.