Figure 1.
Device schematic and system setup.
(A) The microfluidic sorting device is shown with the location of the inlets and outlets. Inset represents a zoom-in section of the post array with FLUENT simulation of the fluid streamlines. An illustration of expected CMs purity from each outlet is shown at the bottom. (B) An image of the system setup with color dyes illustrating stable hydrodynamic focusing. The blue dye is focused by the red dyes into the center of the chamber and exits only from outlet #2. A 20 mm filter unit is placed upstream from the sorting chamber to eliminate cell clumps.
Table 1.
Design Parameters of the Post Array.
Figure 2.
(A) Size distribution of primary cardiac cell mixture from digested heart tissue of neonatal rat (1–2 days old) determined with Coulter Counter. Cell counts were normalized to the total number of cells counted. n = 3 (B) Fraction of total cell input collected by each outlet of the device. n = 3. (B, inset) Comparison between the total number of cells collected and the total number of input cells. (C) Cell size distribution from each outlet of the device. Cell counts were normalized to the total number of cells counted. n = 3 (D) CMs purity before and after sorting analysed by Flow Cytometry. There is a significant difference between sorted cell population from Outlet 4 and cell population before sorting, p = 0.01. n = 3 (E) Representative flow cytometry plots. Blue dots represent Troponin T (+) cells. Red dots represent Troponin T (−) cells. PI staining is used to identify nuclei. n = 3.
Figure 3.
Viability and function of sorted cardiomyocytes.
(A) Cell viability was measured by staining with trypan blue to confirm cell membrane integrity after sorting. The number of trypan blue negative cells was counted against the total number of cells to get the fraction of live cells. The control represents cell viability before sorting. (B) Excitation threshold and maximum capture rate measurement for cardiac patches engineered from enriched CMs. (C) Sorted CMs and (D) CMs collected after pre-plating were cultured on abraded surfaces to demonstrate cell alignment. Fluorescence image of Troponin T staining shows cell alignment and elongation in the direction of the grooves. Vimentin Staining labels the nonmyocyte population (DAPI(blue), Troponin T(green), and Vimentin (red)). Cross striations indicated by arrow heads can also be observed. Black arrow indicate the direction of the grooves. (E) Sorted CMs and (F) CMs collected after pre-plating were cultured on 3-D scaffold. Confocal images shows the CMs population (Troponin T, green) and non-myocyte population (vimentin, red)(E, F) Confocal images of sorted CMs in 3-D scaffold to show cell junction. (DAPI(blue), Troponin T(green), Connexin 43(red)).