Abstract
The dissociation of tissue and cell aggregates into single cells is of high interest for single cell analysis studies, primary cultures, tissue engineering, and regenerative medicine. However, current methods are slow, poorly controlled, variable, and can introduce artifacts. We previously developed a microfluidic device that contains two separate dissociation modules, a branching channel array and nylon mesh filters, which was used as a polishing step after tissue processing with a microfluidic digestion device. Here, we employed the integrated disaggregation and filtration (IDF) device as a standalone method with both cell aggregates and traditionally digested tissue to perform a well-controlled and detailed study into the effect of mechanical forces on dissociation, including modulation of flow rate, device pass number, and even the mechanism. Using a strongly cohesive cell aggregate model, we found that single cell recovery was highest using flow rates exceeding 40 ml/min and multiple passes through the filter module, either with or without the channel module. For minced and digested kidney tissue, recovery of diverse cell types was maximal using multiple passes through the channel module and only a single pass through the filter module. Notably, we found that epithelial cell recovery from the optimized IDF device alone exceeded our previous efforts, and this result was maintained after reducing digestion time to 20 min. However, endothelial cells and leukocytes still required extended digestion time for maximal recover. These findings highlight the significance of parameter optimization to achieve the highest cell yield and viability based on tissue sample size, extracellular matrix content, and strength of cell-cell interactions.
Highlights
Dissociation of aggregated particulates is a fundamental process in diverse scientific fields including polymer suspensions (Petka et al, 1998), microbeads/nanoparticles (Mafuné et al, 2001), and various cellular constructs in the life sciences (Lindvall and Kokaia, 2006)
The goal of this study is to perform a detailed examination of the different dissociation mechanisms offered by the branching channel array and filtration modules, as a function of different operational conditions and cell aggregate types
Shortening digestion time without compromising cell yield is of critical importance to limit the time that enzymes are in contact with cells, as well as stress response pathways that can interfere with transcriptomic analysis (Adam et al, 2017; van den Brink et al, 2017; O’Flanagan et al, 2019), as we have recently shown using the full microfluidic platform including the digestion device (Lombardo et al, 2021)
Summary
Dissociation of aggregated particulates is a fundamental process in diverse scientific fields including polymer suspensions (Petka et al, 1998), microbeads/nanoparticles (Mafuné et al, 2001), and various cellular constructs in the life sciences (Lindvall and Kokaia, 2006). Traditional diagnostic methods provide information about biological traits that have been averaged over an entire population of cells, which masks cellto-cell variability and the presence of rare cell populations (Tung et al, 2017). This necessitates the analysis of individual cells, which can be evaluated globally to better understand normal tissue function and diseased states such as cancer (Heath et al, 2016). Towards this goal, single cells must be liberated from tissues efficiently without changing viability or activation state (Nguyen et al, 2018). Continued development and refinement of rapid and efficient methods for processing tissues and cell aggregates into single cells is a major area of need in the biotechnology and medical arenas
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