Novel microfluidic platform isolates single cells from tissue faster and more thoroughly for single cell diagnostics and primary culture models.. Novel microfluidic platform isolates single cells from tissue faster and more thoroughly for single cell diagnostics and primary culture models.

Tissues are composed of highly heterogeneous mixtures of cell subtypes, and this diversity is increasingly being characterized using high-throughput single cell analysis methods. However, these efforts are hindered by the fact that tissues must first be dissociated into single cell suspensions that are viable and still accurately represent phenotypes from the original tissue. Current methods for breaking down tissues are inefficient, labor-intensive, subject to high variability, and potentially biased towards cell subtypes that are easier to release. Here, we present a microfluidic platform consisting of three different tissue processing technologies that can perform the complete tissue to single cell workflow, including digestion, disaggregation, and filtration. First, we developed a new microfluidic digestion device that can be loaded with minced tissue specimens quickly and easily, and then use the combination of proteolytic enzyme activity and fluid shear forces to accelerate tissue breakdown. Next, we integrated dissociation and filter technologies into a single device, which enhanced single cell numbers and fully prepared the sample for single cell analysis. The final multi-device platform was then evaluated using a diverse array of tissue types that exhibited a wide range of properties. For murine kidney and mammary tumor, we found that microfluidic processing produced 2.5-fold more single, viable cells. Single cell RNA sequencing (scRNA-seq) further revealed that device processing enriched for endothelial cells, fibroblasts, and basal epithelium, and did not increase stress responses. For murine liver and heart, which are softer tissues containing fragile cell types, processing time could be reduced to 15 min, and even as short as 1 min. We also demonstrated that periodic recovery at defined time intervals produced substantially more hepatocytes and cardiomyocytes than continuous operation, most likely by preventing damage to fragile cell types. In future work, we will seek to integrate additional operations such as upstream tissue preparation and downstream microfluidic cell sorting and detection to create powerful point-of-care single cell diagnostic platforms. Overall design: Six (6) samples were included. Three (3) from murine kidney tissue and three (3) from mammary tumor tissue samples. For each tissue type, there were two samples that were processed using the device platform and collected at 15 and 60 min intervals, and we also evaluated the 60 min control.

组织由高度异质性的细胞亚型混合物构成，此类细胞多样性正日益通过高通量单细胞分析手段得以解析。然而，此类研究仍受限于一项核心前提：需先将组织解离为活的单细胞悬液，且该悬液需能准确反映原组织的细胞表型。当前常用的组织解离方法效率低下、操作耗时费力、重复性差，且可能对易于解离释放的细胞亚型存在偏好性偏差。

本研究提出一种集成三种组织处理技术的微流控平台，可完成从组织到单细胞的全流程操作，涵盖酶解、解离与过滤三个环节。其一，本研究开发了一款新型微流控酶解装置，可快速便捷地装载切碎的组织标本，并结合蛋白水解酶活性与流体剪切力加速组织解离。其二，本研究将解离与过滤技术集成于单一装置中，可提升单细胞产出量，并为后续单细胞分析完成充分的样本制备。随后，本研究针对一系列具有多样特性的不同组织类型，对该多装置集成平台进行了性能评估。

针对小鼠肾脏与乳腺肿瘤组织，研究发现微流控处理可获得2.5倍数量的活单细胞。单细胞RNA测序（single cell RNA sequencing, scRNA-seq）进一步分析显示，该装置处理可富集内皮细胞、成纤维细胞与基底上皮细胞，且不会增强细胞应激反应。针对质地较软且含有脆弱细胞类型的小鼠肝脏与心脏组织，该平台的处理时间可缩短至15分钟，甚至仅需1分钟。本研究还证实，相较于持续操作，在指定时间间隔内进行阶段性收获，可获得更多的肝细胞与心肌细胞，这一效果大概率是通过避免对脆弱细胞造成损伤实现的。

在未来的研究中，本团队将致力于集成更多操作环节，例如上游组织制备与下游微流控细胞分选及检测技术，以构建高性能的即时检测单细胞诊断平台。

实验整体设计：本研究共纳入6份样本，其中3份来自小鼠肾脏组织，另外3份来自乳腺肿瘤组织。针对每一类组织，均有2份样本通过该装置平台进行处理，分别于15分钟与60分钟时间点收集样本，同时还设置了60分钟时长的对照组进行评估。