Selective Photomechanical Detachment and Retrieval of Divided Sister Cells from Enclosed Microfluidics for Downstream Analyses

Abstract

Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells, a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photomechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube-polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using the presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.

Keywords: carbon nanotube; cell detachment; cell retrieval; microfluidics; photoacoustics; single-cell transcriptome analysis.

Fig. 1.

Schematic diagram of single-cell detachment setup. (a) The cells were cultured in the microfluidic chamber coated with CNT-PDMS composite. A short pulse laser is used to detach the target cell. (b) the single-cell hydrodynamical capture scheme in the microfluidic chamber.

Fig. 2.

Selective single cell detachemnt by optical generation of shear forces on the CNT-PDMS film: (a) Scanning electron microscope (SEM) image of the CNT grown on quartz substrate (scale bar: 5 μm), (b) SEM image of CNTs (separate sample) after spin coating with PDMS (scale bar: 5 μm). (c-e) An example of laser detachment of a MDA-MB-231 cell: (c) before detachment, (d) immediately after pulsed laser irradiation, and (e) cell detached and flowed away from the original culture location (scale bar: 50 μm). Note that the cell is highly viable preserving its membrane without biochemical modification observed in the trypsinization (f-h) An example of partial detachment of a MDA-MB-231 cell: (f) before detachment, (g) after laser irradiation onto the left extension of the cell, and (h) the partially detached cell only having the physical contact on the right end (scale bar: 50 μm).

Fig. 3.

Cell retrieval and viability validation. (a-c) Three different flow schemes of the microfluidic device: (a) cell loading into the chambers for culturing and monitoring, (b) retrieving cells in the even rows, and (c) retrieving cells in the odd rows. (d-g) The recovery of a MDA-MB-231 cell: (d) before detachment, (e) right after detachment, (f) the retrieved cell, (g) proliferation after 4 days (scale bar: 100 μm). (h-k) Scanning electron microscope (SEM) images of laser detached and trypsinized cells: (h) the laser detached MDA-MB-231 cell (scale bar: 5 μm) and (i) its enlarged view (scale bar: 500 nm), (j) the trypsinized MDA-MB-231 cell (scale bar: 3 μm) and (k) its enlarged view (scale bar: 500 nm).

Fig. 4.

Single cell gene-expression data of trypsinized and laser detached T47D cells utilizing Fluidigm C1/Biomark HD for multiplexed gene expression analysis. (a) Principal component analysis (PCA) plot of single cell expression analysis for 20 trypsinized (green) and 20 laser detached (red) cells. Each dot represents a cell. (b) Heatmap hierarchical clustering of single cell expression analysis for 20 trypsinized (green triangle) and 20 laser detached (red circle) cells. In the heatmap, the red color indicates high gene expression, and the blue color indicates low gene expression. Two types of cells are mixed together, indicating no significant alteration in the gene expression when laser detachment was used, as compared to trypsinization. (c) The violin plots of gene expression for 20 trypsinized cells (green) and 20 laser detached cells (red). 96-gene expression of single cells was analyzed. The vertical axis indicates relative expression levels in log2 scale, and the horizontal axis indicates the distribution of cell population. The cells detached by both methods maintain typical T47D cell expression with no significant variation.

Fig. 5.

Comparison of asymmetrically and symmetrically divided sister cells using single cell transcriptome analysis. (a) A single Notch+ T47D cell was captured in a chamber. (b) After 3 days, the Notch+ cell asymmetrically divided to two cells: i.e. Notch+ (green) and Notch- (non-green). (c) The Notch+ cell was selectively detached and retrieved first, leaving the Notch- cell alone in the chamber. (d) The Notch- cell was retrieved, leaving no cells. (e) Principal component analysis (PCA) plot of 2 pairs of asymmetrically divided sister cells (red), 2 pairs of symmetrically divided Notch+ sister cells (blue), and 2 pairs of symmetrically divided Notch- sister cells (blue). Each dot represents a cell. Comparison of the distances between the cell pairs indicates that the asymmetric division causes significant alteration in the gene expression profiles between two sister cells. (f) Heatmap hierarchical clustering of the asymmetrically and symmetrically divided cells (red - high gene expression, blue - low gene expression). The symmetrically divided cells are more likely to cluster together. (g) The pair-wise difference in the gene expression of 12 cells. The gene expression difference is defined as the average difference in cycle threshold (Ct) of all 96 genes. The blue color indicates low difference (closer expression profile), and the red color indicates high difference (more different expression profile). The results suggest that symmetric division generates two similar sister cells, while asymmetric division generates distinctive sister cells. (h) Identification of genes altered in symmetric and asymmetric division. Each dot represents a gene(horizontal axis - the average difference in Ct of symmetrically divided cell pairs, vertical axis - the average difference in Ct of asymmetrically divided cell pairs). The genes were categorized into 4 types.