Label-Free, High-Throughput Purification of Satellite Cells Using Microfluidic Inertial Separation

Skeletal muscle satellite cells have tremendous therapeutic potential in cell therapy or skeletal muscle tissue engineering. Obtaining a sufficiently pure satellite cell population, however, presents a significant challenge. We hypothesized that size differences between satellite cells and fibroblasts, two primary cell types obtained from skeletal muscle dissociation, would allow for label-free, inertial separation in a microfluidic device, termed a Labyrinth, and that these purified satellite cells could be used to engineer skeletal muscle. Throughout tissue fabrication, Labyrinth-purified cells were compared with unsorted controls to assess the efficiency of this novel sorting process and to examine potential improvements in myogenic proliferation, differentiation, and tissue function. Immediately after dissociation and Labyrinth sorting, cells were immunostained to identify myogenic cells and fibroblast progenitors. Remaining cells were cultured for 14 days to form a confluent monolayer that was induced to delaminate and was captured as a 3D skeletal muscle construct. During monolayer development, myogenic proliferation (BrdU assay on Day 4), differentiation and myotube fusion index (alpha-actinin on Day 11), and myotube structural development (light microscopy on Day 14) were assessed. Isometric tetanic force production was measured in 3D constructs on Day 16. Immediately following sorting, unsorted cells exhibited a myogenic purity of 39.9% +/- 3.99%, and this purity was enriched approximately two-fold to 75.5% +/- 1.59% by microfluidic separation. The BrdU assay on Day 4 similarly showed significantly enhanced myogenic proliferation: in unsorted controls 47.0% +/- 2.77% of proliferating cells were myogenic, in comparison to 61.7% +/- 2.55% following purification. Myogenic differentiation and fusion, assessed by fusion index quantification, showed improvement from 82.7% +/- 3.74% in control to 92.3% +/- 2.04% in the purified cell population. Myotube density in unsorted controls, 18.6 +/- 3.26 myotubes/mm(2), was significantly enriched in the purified cell population to 33.9 +/- 3.74 myotubes/mm(2). Constructs fabricated from Labyrinth-purified cells also produced significantly greater tetanic forces (143.6 +/- 16.9 mu N) than unsorted controls (70.7 +/- 8.03 mu N). These results demonstrate the promise of microfluidic sorting in purifying isolated satellite cells. This unique technology could assist researchers in translating the regenerative potential of satellite cells to cell therapies and engineered tissues.