Isolating single cells in a neurosphere assay using inertial microfluidics

Abstract

Sphere forming assays are routinely used for in vitro propagation and differentiation of stem cells. Because the stem cell clusters can become heterogeneous and polyclonal, they must first be dissociated into a single cell suspension for further clonal analysis or differentiation studies. The dissociated population is marred by the presence of doublets, triplets and semi-cleaved/intact clusters which makes identification and further analysis of differentiation pathways difficult. In this work, we use inertial microfluidics to separate the single cells and clusters in a population of chemically dissociated neurospheres. In contrast to previous microfluidic sorting technologies which operated at high flow rates, we implement the spiral microfluidic channel in a novel focusing regime that occurs at lower flow rates. In this regime, the curvature-induced Dean’s force focuses the smaller, single cells towards the inner wall and the larger clusters towards the center. We further demonstrate that sorting in this low flow rate (and hence low shear stress) regime yields a high percentage (> 90%) of viable cells and preserves multipotency by differentiating the sorted neural stem cell population into neurons and astrocytes. The modularity of the device allows easy integration with other lab-on-a-chip devices for upstream mechanical dissociation and downstream high-throughput clonal analysis, localized electroporation and sampling. Although demonstrated in the case of the neurosphere assay, the method is equally applicable to other sphere forming assays.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

“…Since both forces are functions of the particle size, the particles of different size occupy distinct lateral positions near the channel wall and exhibit different degrees of focusing, allowing size-based separation. [56][57][58][59][60] The smaller blood cells, including red blood cells and leukocytes, migrate along the Dean vortices toward the inner wall and then back to the outer wall again, while the larger CTCs experience additional strong inertial lift forces and focus along the microchannel inner wall. The advantage of the hydrodynamic method is that it offers a quick, simple and label-free way to isolate CTCs.…”

Micro- and Nanoscale Technologies for Delivery into Adherent Cells

“…To understand complex intracellular input-output relationships and to develop mathematical descriptions of cellular behavior, it is essential to have tools for multiplexed cell manipulation and analyses, e.g., by adding cell sorting [91] and multiplexing [92] schemes to the cell access and analysis module (Figure 3), that can be performed with throughput that is statistically significant and practical with respect to research time per data point (1000 cells have been suggested as a reasonable goal for statistical relevance using single-cell technologies [2, 93]). In addition, systems biology analyses of the temporal data are needed to systematically process large sets of data from multiplexed cell analysis to elucidate the cellular behavior or mechanisms being studied [82, 83].…”

“…Example of envisioned integrated micro/nanofluidic platform for transfection, sampling, biomolecular detection, sorting, and on-chip cell culture [3, 15, 35, 88, 91, 95–97]. Through temporal analysis, intracellular processes leading to mechanistic understanding through systems biology is possible [82, 83].…”

“…Through temporal analysis, intracellular processes leading to mechanistic understanding through systems biology is possible [82, 83]. (Reproduced from [88] and [91], [35] and [97], [96], and [83] with permission of The Royal Society of Chemistry, American Chemical Society, The American Association for the Advancement of Science, and the National Academy of Sciences, respectively)…”

Novel fluid shear‐based dissociation device for improved single cell dissociation of spheroids and cell aggregates

“…There is one report that adapted these methods that was originally designed for tissue dissociation in order to dissociate spheroids into single cells . Other attempts focused on using microfluidic chip devices for spheroid disscoation . However, the utility of these devices is limited, as they do not allow for the large‐scale processing of spheroids.…”