Hydrogel nanovial technology for single-cell sorting based on extracellular vesicle production

摘要 分泌性细胞外小泡(EVS)是一种新兴的治疗方法，在临床上具有巨大的潜力。 疾病的治疗。然而，这些细胞来源的研究和生产仍然面临着严峻的挑战 治疗学。异质细胞群体，如间充质干细胞(MSCs)，产生不同的 EVS的数量和类型以及其生物发生的未知机制阻碍了Uniform的缩放 用于临床翻译的电动汽车产品。基于肠道病毒分泌率的分类可以使遗传研究成为可能 导致高电动汽车生物形成的基础。被转基因以分泌生物制品的生产细胞，如 基于抗体的疗法，传统上是根据它们的分泌率进行分类，以分离产生 大量的抗体，以及类似的方法来分类和选择高分泌群体将是 有利于获得高质量的肠道病毒分泌细胞。然而，快速分选单个细胞的方法 目前还不能获得分泌高水平的EV，也不能批量评估细胞间的可变性， 使得以更高的速率或具有更均匀的内容物来分离产生EV的细胞是不可行的。 我们建议在粒子平台上建立一个实验室，在单细胞水平上根据EV分泌对MSCs进行分选 使用标准的荧光激活细胞分类器(FAC)。我们的方法利用含有空洞的水凝胶 微颗粒，或纳米小瓶，允许MSCs种植和黏附，随后均匀形成 通过简单的移液步骤将分泌的电动汽车限制在纳米瓶周围的液滴，以捕获颗粒上的电动汽车。 可以对捕获的EV进行染色，并根据EV的分泌水平对相应的分泌细胞进行分类 使用商用的FACS机。我们建议建立一种基于纳米管的免疫分析方法来检测分泌物 并在对分泌最高水平EVS的人MSCs进行分选时进行评估。我们将确定 MSCs选择用于这种高分泌表型的时间段是稳定的。我们的技术可以帮助 以肠病毒为基础的治疗的临床研究，定义在 高费率，并将进一步扩大定义基于电动汽车的产品的蜂窝基础的能力。改进 电动汽车的生产可以使基于MSC的EV(MSC-EV)治疗在一次治疗中显示出初步疗效 目前有10项正在进行的试验，其中包括两项针对新冠肺炎的临床试验。

SUMMARY Secreted extracellular vesicles (EVs) are an emerging therapeutic category with significant potential in the treatment of disease. However, critical challenges remain with the study and production of these cell-derived therapeutics. Heterogeneous populations of cells, like mesenchymal stem cells (MSCs) that produce different amounts and types of EVs along with unknown mechanisms of their biogenesis hamper the scaling of uniform EV products for clinical translation. Sorting based on EV-secretion rate can enable the study of genetic underpinnings that lead to high EV biogenesis. Producer cells that are transfected to secrete biologics, such as antibody-based therapeutics, are traditionally sorted based on their secretion rate to isolate clones that produce large quantities of antibody, and similar approaches to sort and select highly-secreting populations will be advantageous for obtaining high quality EV-secreting cells. However, methods to rapidly sort out single cells secreting high levels of EVs are not currently available and cell-to-cell variability cannot be assessed in bulk, such that the separation of cells that produce EVs at higher rates or with more uniform contents is not feasible. We propose to develop a lab on a particle platform to sort MSCs based on EV secretion at the single-cell level using standard fluorescence activated cell sorters (FACS). Our approach leverages cavity-containing hydrogel microparticles, or nanovials, that allow seeding and adhesion of MSCs followed by the uniform formation of droplets around the nanovials with simple pipetting steps to confine secreted EVs for capture on the particles. Captured EVs can be stained and the corresponding secreting cells sorted based on the level of EVs secreted using commercial FACS machines. We propose to develop the on-nanovial immunoassay to detect secreted EVs and evaluate it in sorting of human MSCs that secrete the highest levels of EVs. We will identify the timeframe over which MSCs selected for this high secretion phenotype is stable. Our technology can aid in clinical studies for EV-based therapeutics, defining populations of cells that produce uniform EV therapeutics at high rates, and will further expand the ability to define the cellular underpinnings of EV-based products. Improved EV production can enable MSC derived EV-based (MSC-EV) treatments which have shown initial efficacy in one clinical trial for chronic kidney disease, and there are currently 10 on-going trials including two for COVID-19.