Gel-Free Single-Cell Culture Arrays on a Microfluidic Chip for Highly Efficient Expansion and Recovery of Colon Cancer Stem Cells

The microgel single-cell culture approach we developed to expand tumor stem cells (TSCs) is associated with limited TSC production, which can be attributable to cell viability loss in microgel formation and tumorsphere expansion limitation caused by hydrogel stiffness. In this work, we developed a gel-free single-cell culture array on a microfluidic chip to overcome these issues. The microfluidic chip used in the study has a 16,000 hydrophilic microchamber array, which can capture ∼2000 single cells at a time. After cell capturing, the cell culture chambers were enclosed by forming a chitosan layer through interactions between chitosan and alginate, thus preventing cell loss in the gel-free culture. The hydrophilic coating prevented cell adhesion, so only TSCs with anti-apoptosis and self-renewal properties can survive the harsh culture and form tumorspheres. After a 7 day culture, 19.04% of the HCT116 colon cancer cells formed single-cell-derived tumorspheres with an average size of 46.59 ± 10.58 μm. Compared with the microgel single-cell culture, sphere-forming rate and TSC expansion efficiency were significantly improved by using this gel-free single-cell culture array. After cell culture, the chitosan layer could be destabilized easily, thus allowing recovery of the tumorspheres from the microchip by applying a reverse flow. Approximately 13,600 cells could be obtained in a single culture, which can be used for off-chip cell assays. Flow cytometry analysis indicated high proportions of LGR5(+) and SOX2(+) cells within the single-cell-derived tumorspheres. Moreover, the differentiation experiments confirmed the multi-lineage differentiation potential of single-cell-derived tumorspheres. The gel-free single-cell culture offers a label-free approach to obtain sufficient amounts of TSCs, which is valuable for tumor biology research and the development of TSC-specific therapeutic strategies.