Abstract
Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.
Highlights
There is substantial evidence that many cancers are heterogeneous and hierarchically organized, and that at the apex of this hierarchy are cells that display stem cell properties
We report a scalable single-cell suspension culture chip with 1,024 micro-chambers with non-adherent surface coating, which can provide robust single-cell isolation, tracking, and continuous media perfusion to avoid any difficulty in cell seeding and media exchange
We investigated the relationship between sphere formation and expression of genes related to cell ‘stemness’
Summary
There is substantial evidence that many cancers are heterogeneous and hierarchically organized, and that at the apex of this hierarchy are cells that display stem cell properties These cancer stem-like cells (CSCs) drive tumor growth and metastasis and contribute to treatment resistance[1,2,3,4,5,6,7]. First utilized for the identification of neural stem cells, in vitro sphere formation assays have been suggested as a marker free methodology for culture and identification of stem-like cells in breast and other cancers[13,14,15]. There are a number of microfluidic approaches for performing sphere assays on-chip, including hanging droplet methods[23,24,25], micro-rotation flow[26], and micro-well[27] These platforms are generally not clonal, and they do not exclude the possibility of cellular aggregation. Despite its advantages over many conventional suspension culture coatings and devices, the patterned surface requires expensive deep reactive ion etching and complicates optical imaging
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