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

基于细胞外囊泡产生的单细胞分选水凝胶纳米瓶技术

• 批准号: 10193200
• 负责人: Dino Di Carlo
• 金额: $ 17.61万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10193200
• 关键词: Adhesions; Agitation; Alpha Particles; Antibodies; Antibody Therapy; Binding; Biocompatible Materials; Biogenesis; Biological; CD81 gene; COVID-19; Categories; Cell Separation; Cell Therapy; Cells; Cellular Structures; Characteristics; Chemistry; Chinese Hamster Ovary Cell; Chronic Kidney Failure; Clinical Research; Clinical Trials; Conditioned Culture Media; Disease; Dose; Emulsions; Encapsulated; Engineering; Fluorescence; Fluorescence Microscopy; Foundations; Genetic study; Heterogeneity; Human; Hydrogels; Immunoassay; Incubated; Investigation; Label; Lead; Lipids; Mechanics; Membrane Proteins; Mesenchymal Stem Cells; Messenger RNA; Methods; MicroRNAs; Microfluidic Microchips; Molecular Profiling; Oils; Phenotype; Population; Population Heterogeneity; Pre-Clinical Model; Production; Proteins; Signal Transduction; Sorting - Cell Movement; Stains; Surface; Techniques; Technology; Therapeutic; Water; anti-IgG; base; cell type; clinical translation; cost; extracellular vesicles; fluorescence activated cell sorter device; immunoregulation; improved; intercellular communication; novel; particle; protein biomarkers; regenerative; response; single cell technology

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.

概括 分泌的细胞外囊泡（EV）是新兴的治疗类别 疾病的治疗。但是，这些细胞衍生的研究和生产仍然存在着关键的挑战 疗法。细胞的异质种群，例如产生不同的间质干细胞（MSC） 电动汽车的数量和类型以及其生物发生的未知机制阻碍了均匀的尺度 EV产品用于临床翻译。基于EV分泌率的分选可以使遗传研究 导致高EV生物发生的基础。转染给分泌生物制剂的生产者细胞，例如 基于抗体的疗法，传统上是根据其分泌率分离出产生的克隆的分类速率 大量的抗体以及类似的分类和选择高度分泌种群的方法 有利于获得高质量分泌的细胞。但是，快速整理单个单元的方法 当前无法分泌高水平的电动汽车，细胞到细胞的可变性不能批量评估， 因此，以较高速率或更均匀的含量产生EV的细胞分离是不可行的。 我们建议在粒子平台上开发一个实验室，以基于单细胞级别的EV分泌对MSC进行分类 使用标准荧光激活的细胞分角（FACS）。我们的方法利用含腔的水凝胶 微粒或纳米维型，允许MSC的播种和粘附，然后均匀形成 用简单的移液步骤将纳米维型的液滴限制在颗粒上捕获的分泌的电动汽车。 捕获的电动汽车可以染色，并根据分泌的电动汽车的水平进行染色，并分类相应的分泌细胞 使用商业FACS机器。我们建议开发纳维亚免疫测定法以检测分泌 电动汽车和评估人类MSC的分类，该MSC分泌最高水平的电动汽车。我们将确定 为此高分泌表型选择的MSC稳定的时间范围是稳定的。我们的技术可以帮助 基于EV的疗法的临床研究，定义了在AT A上产生均匀EV治疗的细胞种群 高率，并将进一步扩大定义基于EV产品的细胞基础的能力。改进 EV生产可以实现MSC衍生的基于EV（MSC-EV）的处理 慢性肾脏疾病的临床试验，目前正在进行10项正在进行的试验，其中包括两项Covid-19。