Sorting Single Stem Cells Adhered to Microhydrogel Carriers based on Growth Factor Secretion using FACS

Mesenchymal stem cells (MSCs) are a promising therapeutic for currently intractable diseases, mainly due to their production of paracrine factors. A barrier to translation is the inherent heterogeneity in secretory function of MSCs, which varies based on initial tissue source and cell expansion conditions. Sorting MSCs based on their secretions would enable production of better-defined therapeutics that link function to efficacy. Current platforms for single-cell isolation based on secreted products, such as droplet-based encapsulation or microwells, are incompatible with adherent cells or low-throughput.
We present fluorescent activated cell sorting (FACS)-compatible microhydrogels (“nanovials”) with a spherical cavity, which can carry adhered single MSCs and capture secreted molecules, such as vascular endothelial growth factor (VEGF). These nanovials can enrich single MSCs or MSC populations with high VEGF secretion, and investigate microenvironment effects on VEGF secretion in a single-cell format.
We engineered these nanovials to feature an inner coating of gelatin using a novel aqueous two-phase composition of gelatin and PEG-acrylate, which enables cell adhesion exclusively inside the cavity. Additionally, the amine groups on gelatin can be functionalized post-fabrication with NHS-biotin to allow for conjugation of secretion capture antibodies selectively inside the cavity. Due to localized secretion capture antibodies in proximity to the secreting cell, secretions can be captured without emulsifying nanovials in oil so that MSCs can maintain cell-to-cell communication as well as nutrient exchange. There is minimal off-target signal, with cell-loaded nanovials having an average VEGF detection antibody intensity 2.6 or 3.6x higher than that of empty nanovials after 12- and 24-hour secretion, respectively (p < 0.001).
We improved cell loading in nanovials, with >25% recovery of cells in nanovials as single cells, which is beneficial for precious therapeutic cell samples. By decreasing the size of our nanovials from previously reported 85 µm diameter to 55 µm diameter, we can better avoid cell multiplets, with >85% of cell loaded nanovials containing a single cell. These smaller nanovials are also compatible with commercial FACS systems, such as the Sony SH800.

Using these new formulations, we demonstrate sorting of primary adherent cells (MSCs) based on the quantity of secreted growth factor using FACS for the first time. MSCs are known to secrete growth factors at a rate 5 to 50-fold less than the previously used producer cell lines, and the ability to detect lower levels of cell secretion expands the repertoire of cell types and secreted factors compatible with nanovials. We have also demonstrated that we can expand single cell colonies from nanovials sorted into individual wells, with a colony formation efficiency of 35%. Going forward, nanovials can be used to evaluate the maintenance of high-VEGF secretion phenotype after sorting and correlate secretion to other cell phenotypes such as surface markers or biophysical properties.