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

总结 分泌的细胞外囊泡（EV）是一种新兴的治疗类别，在治疗中具有显著的潜力。 疾病的治疗。然而，关键的挑战仍然与研究和生产这些细胞衍生的 治疗学异质细胞群，如间充质干细胞（MSC），产生不同的细胞分化。 电动汽车的数量和类型沿着与未知的机制，他们的生物成因阻碍了规模的统一 用于临床翻译的EV产品。基于EV分泌速率的分选可以使得能够研究遗传性的细胞毒性。 导致高EV生物发生的基础。被转染以分泌生物制剂的生产细胞，例如 基于抗体的治疗剂，传统上基于它们的分泌速率进行分选，以分离产生 大量的抗体，以及类似的方法来分选和选择高分泌群体将是 有利于获得高质量的EV分泌细胞。然而，快速分选单细胞的方法 分泌高水平EV的细胞目前不可用并且不能大量评估细胞间的变异性， 使得以更高速率或以更均匀的含量产生EV的细胞的分离是不可行的。 我们建议在粒子平台上建立一个实验室，在单细胞水平上基于EV分泌对MSC进行分选 使用标准荧光激活细胞分选仪（FACS）。我们的方法利用了含有空腔的水凝胶 微颗粒或纳米管，其允许MSC的接种和粘附，随后均匀形成微颗粒或纳米管。 通过简单的移液步骤将微滴移到纳米管周围，以限制分泌的EV用于捕获在颗粒上。 可以对捕获的EV进行染色，并基于分泌的EV水平分选相应的分泌细胞 使用商业化的流式细胞仪。我们建议开发纳米管免疫分析来检测分泌型 并在分泌最高水平EV的人MSC的分选中对其进行评估。我们将确定 选择用于这种高分泌表型的MSC是稳定的。我们的技术可以帮助 基于EV的治疗剂的临床研究，定义了产生均匀EV治疗剂的细胞群， 高速率，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 EV产生可以使MSC衍生的基于EV的治疗（MSC-EV）成为可能，所述治疗已经在一个实施方案中显示出初始功效。 目前有10项正在进行的试验，包括两项针对COVID-19的试验。

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.