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

基本信息

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
项目类别：
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

项目摘要

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.

概括分泌型细胞外囊泡（EV）是一种新兴的治疗类别，在治疗领域具有巨大潜力治疗疾病。然而，这些细胞衍生的药物的研究和生产仍然面临着严峻的挑战。疗法。异质细胞群，例如产生不同细胞的间充质干细胞 (MSC) EV 的数量和类型以及其生物发生的未知机制阻碍了统一的规模化用于临床翻译的 EV 产品。基于 EV 分泌率的分类可以使遗传研究成为可能导致高 EV 生物发生的基础。被转染以分泌生物制剂的生产细胞，例如基于抗体的疗法，传统上根据其分泌率进行分类，以分离产生的克隆大量的抗体，以及类似的方法来分类和选择高分泌群体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目前无法分泌高水平的 EV，并且无法批量评估细胞间的变异性，因此，以更高的速率或更均匀的含量生产 EV 的细胞的分离是不可行的。我们建议在颗粒平台上开发一个实验室，根据单细胞水平的 EV 分泌对 MSC 进行分类使用标准荧光激活细胞分选仪（FACS）。我们的方法利用含腔水凝胶微粒或纳米瓶，允许 MSC 播种和粘附，然后均匀形成通过简单的移液步骤，在纳米瓶周围形成液滴，以限制分泌的 EV 捕获在颗粒上。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业 FACS 机器。我们建议开发纳米免疫测定法来检测分泌的 EV 并在分选分泌最高水平 EV 的人类 MSC 时对其进行评估。我们将确定为这种高分泌表型选择的 MSC 保持稳定的时间范围。我们的技术可以帮助基于 EV 的疗法的临床研究，定义了产生统一 EV 疗法的细胞群高速率，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 生产可以实现基于 MSC 衍生的 EV（MSC-EV）治疗，该治疗已在一种治疗中显示出初步疗效慢性肾脏病的临床试验，目前有 10 项正在进行的试验，其中包括两项针对 COVID-19 的试验。