Figure 1.
Label-free isolation of adrenal cortical progenitor cells.
(a) The photograph of the microfluidic device used for progenitor cell isolation and (b–d) schematic showing the inertial focusing of living cell clumps in microscale flow. (b) Solution containing randomly distributed heterogeneous tissue digest is injected at the inlet of the device and (c) flowing cells experience two lateral forces, namely wall effect lift, FWL, and shear-gradient lift force, FSL, as they travel through the straight focusing region, and these forces induce lateral migration of cells and focus them at different locations base on the size (i.e., larger cells were closer to the channel center, and smaller cells were closer to the channel walls). (d) Differentially focused flowing cells can be directed and collected at designated outlets based on apparent-diameter variation. All schematics represent the top view of the microfluidic device.
Figure 2.
Label-free separation of murine adrenal cortical progenitor cells using deformability activated cell sorting technique.
Fluorescent images of cells collected at (a) outlet 1, (b) outlet 2, and (c) outlet 3. Cells were stained post-collection with DAPI (blue) and Nile Red (green) to identify intracellular lipid droplets and nuclei, respectively. There was a significantly lower number of cells expressing high levels of lipid droplet contents (Nile Red bright, somatic adrenal cortical cells) found in the fraction collected from outlet 1, and the population of Nile Red bright cells was the highest in the fraction collected from outlet 3. Scale bar is 50 µm.
Figure 3.
Relative gene expression levels of Sf1, Cyp11b1, and Cyp11b2 of all adrenal cells collected at each fraction of outlets.
Each adrenal specific gene expression level was normalized to that of pooled neonatal adrenal glands. The levels of Sf1 were similar (P = 0.9) for all conditions, whereas Cyp11b1 and Cyp11b2 were differentially expressed for fractions collected at different outlets. The expression of zonal-specific genes (Cyp11b1 and Cyp11b2) was significantly lower in the fraction of outlet 1 than that for the rest of samples. Red asterisks indicates P<0.05 compared with NR bright cells from outlet 3. Error bar represents the standard deviation of measurements from two animals.
Figure 4.
Processed cells remain highly viable.
(a) Colorimetric viability test showed that cells flowed through the device remain highly viable similar to control cells, which have not been processed. (b) Collective bright field images of 10 day cultured cells. Various cell types with different morphologies were observed. Scale bar is 100 µm.