a-microfluidic-platform-for-high-throughput-single-cell-isolation

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

National Health Research Institutes, Taiwan

National Chung Hsing University

In this study, several in vivo and in vitro comparisons were performed to test the possibility of using adipose-derived stem cells (ADSCs), a more convenient cell source than dental pulp stem cells (DPSCs), in tooth regeneration. Using an efficient, non-engineering implantation method, we first demonstrated that both implants of ADSCs and DPSCs were able to grow self-assembled new teeth in adult rabbit extraction sockets with high success rate. The stem cells were necessary because the implants grew no tooth without them. A stepwise comparison showed that the regenerated teeth from these two types of adult stem cells were living with nerves and vascular system and remarkably similar to a normal tooth in many details. Further strictly controlled, side-by-side comparisons between the two types of stem cells also showed that the expression patterns of gene markers and the broad differentiation potentials induced by specific methods in vitro were very similar. Although a few differences were found, they did not affect the tested tooth regeneration in vivo or differentiation in vitro. Furthermore, rabbit ADSCs had a higher growth rate and a better senescence resistance in culture. All these findings suggest that ADSCs, one of the richest adult stem cells in mammals, are very similar and useful as DPSCs for regenerative dentistry.

A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration.

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.

Single-cell enzyme-free dissociation of neurospheres using a microfluidic chip.

In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 μm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

A microfluidic dual-well device for high-throughput single-cell capture and culture.

Neurosphere assay is a common and robust method for identification of neural stem/progenitor cells, but obtaining large numbers of live single cells from dissociated neurospheres is difficult using nonenzymatic methods. Here, we present an enzyme-free method for high-efficiency neurosphere dissociation into single cells using microfluidic device technology. This method allows single cell dissociation of DC115 and KT98 cells with high cell viabilities (80-85 %), single-cell yield (91-95 %), and recovery (75-93 %).

Article	Year
A comparison between adipose tissue and dental pulp as sources of MSCs for tooth regeneration. Biomaterials\| PubMed ID: 21696818	2011
Single-cell enzyme-free dissociation of neurospheres using a microfluidic chip. Analytical chemistry\| PubMed ID: 24228937	2013
A microfluidic dual-well device for high-throughput single-cell capture and culture. Lab on a chip\| PubMed ID: 26060987	2015
Enzyme-Free Dissociation of Neurospheres by a Microfluidic Chip-Based Method. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27044047	2016

Hao-Chen Chang

Biography

JoVE Journal Publications

Other Publications