Single-Cell Enzyme-Free Dissociation of Neurospheres Using a Microfluidic Chip

Abstract

Obtaining single dissociated cells from neurospheres is difficult using nonenzymatic methods. In this paper we report the development of a microfluidic-chip-based approach that utilizes flow and microstructures to dissociate neurospheres. We show that this microfluidic-chip-based neurosphere-dissociation method can generate high yields of single cells from dissociated neurospheres of mouse KT98 and DC115 cell models (passage number, 3−8; diameter range, 40−250 μm): 90% and 95%, respectively. The microfluidic-chip-dissociated cells had high viabilities (80−85%) and the ability to regrow into neurospheres, demonstrating the applicability of this device to neurosphere assay applications. In addition, the dissociated cells retained their normal differentiation potentials, as shown by their capabilities to differentiate into three neural lineages (neurons, astroglia, and oligodendrocytes) when cultured in differentiation culture conditions. Since this microfluidic-chip-based method does not require the use of enzymatic reagents, the risk of contamination from exogenous substances could be reduced, making it an attractive tool for a wide range of applications where neurosphere dissociation is needed.

Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method

“…Neurosphere dissociations are commonly performed by using enzymatic methods which can generate high yields of single dissociated cells with reproducible results. However, to avoid protease and collagenolytic activities, which degrade cell membrane proteins [3], mechanical methods including trituration [4], manual cutting with scalpel [5], micro-scissors [6], tissue chopper [7], and microfabricated filter are used [8]. We have developed a microfluidic chip-based mechanical method to dissociate neurospheres [9].…”

Effect of enzymatic and mechanical methods of dissociation on neural progenitor cells derived from induced pluripotent stem cells

“…Investigators using different cell lines, cells from fetal or embryonic tissue, and cells differentiated using other protocols may find other combinations of dissociation methods to be more effective. We did not include mechanical methods of dissociation involving filters, choppers, and/or microfluidic devices that may not be readily available to most laboratories [4, 5, 8]. Finally, we did not evaluate the dissociation methods for their effects on the long-term survivability and maturation of our cells, as this was beyond the scope of our study.…”

“…To avoid this cell loss, multiple practices have been developed to work around having to dissociate these clusters [3-5]. Complete dissociation into a single-cell suspension, however, is necessary for accurate cell counts, which can affect the reproducibility of results, assays involving flow cytometry, and studies of the impact of cell-cell interactions on survival and maturation of cells both in vitro and in vivo [6-8]. …”

Microfluidic Sample Preparation for Single Cell Analysis

“…To achieve strictly single cell dissociation from neurospheres, Lin et al 26 designed a microfluidic cell dissociation chip, termed “µ-CDC”. The single channel device consisted of micro pillars 50 µm wide and 167 µm tall with 20 µm spacing between adjacent pillars (Figure 2B).…”

“…Neurospheres traveling through the micro pillar array are mechanically dissociated into single cells. Reproduced from Lin, C. H.; Lee, D. C.; Chang, H. C.; Chiu, I. M.; Hsu, C. H. Analytical chemistry 2013, 85 (24), 11920-8 (ref 26). Copyright 2013 American Chemical Society.…”