The cell diameters are 12 m in (c), 20 m in (d), 30 m in (e), and 40 m in (f) (scale bar: 50 m)
September 29, 2021
The cell diameters are 12 m in (c), 20 m in (d), 30 m in (e), and 40 m in (f) (scale bar: 50 m). Solitary cell sphere formation rate and sphere size comparison To validate the high-throughput single-cell-derived sphere assay, four different cell lines, including SUM-159, SUM-149, MCF-7, and T47D, were loaded and cultured in the device for 14 days. to isolate single-cells in each well for sphere tradition.11C13 However, without a robotic system, this method is labor rigorous and limited in throughput because the capture rate is limited by Poisson distribution (10C30%). Fluorescence-activated cell sorting (FACS) can automate the single-cell dispensing process and accomplish higher single-cell seeding rate; however, high shear stress during the sorting can potentially affect cell viability and influence the results.14 Given the low throughput of conventional methods, people can barely quantify the sphere formation rate; therefore, it is difficult to investigate the cellular heterogeneity within rare CSC populations. The controversy of the correlation between malignancy cell size and stemness is definitely one example. In recent publications, some experts reported evidence showing that smaller malignancy cell size is definitely associated with malignancy stem-like cell activity,15C17 whereas additional experts reported higher mitochondria mass and improved cell size correlate with malignancy stemness and chemo-resistance.18C20 To study the heterogeneity in the CSC populations, there is an unmet need of an high-throughput approach for rare cell studies. Microfluidic tradition systems emerge to be a powerful method for RU 24969 hemisuccinate single-cell studies.21 Combined with a non-adherent tradition substrate, single-cell capture chips were developed for single-cell-derived sphere assay.22,23 However, previous works using hydrodynamic capture techniques required extended meander channels to achieve a high cell capture rate (>80%), which constrained the number of wells per area.23C28 The long term time for imaging over a large area limits the assay throughput and could potentially affect cell viability if an environmental chamber is not used during image capture under a microscope. For additional capture methods, droplet systems can achieve high-throughput analysis by encapsulating solitary cells in aqueous droplets.29 However, droplet approaches are limited by short assay time due to the difficulty in media exchange. Clonal sphere assay would typically require 14 days for tradition and thus cannot be implemented by droplet systems. Micro-well systems are another simple yet effective tool to isolate solitary cells for clonal tradition.30,31 However, most micro-well systems rely on random seeding with a low cell capture rate around 10C30%. Techniques using dielectrophoresis pressure or dual-wells can facilitate higher capture rates, but they either require sophisticated active control or show size-dependent capture.32,33 High density and high capture rate can be realized by a filter array structure system.34,35 However, in these works, capture sites were connected in series, resulting in high flow resistance and low flow rate, given that flow rate (2 L h?1), a high concentration of cells and a long loading time are required. In that case, cell aggregation and clogging may inevitably happen for several malignancy cell lines. In addition, without automatic cell analysis, it requires manual inspection to read out data from microscope images, which is demanding when hundreds or thousands of single-cells are analyzed. To address these challenges, we developed a high-throughput single-cell capture device utilizing highly-parallelized constructions for single-cell-derived tumorsphere studies. The highly scalable fluidic structure enables reliable solitary cell capture from 800 wells per chip to up to 12 800 wells per chip. The capture plan can RU 24969 hemisuccinate reliably sample a representative cell populace from the bulk. With an automatic analysis system, assay results from thousands of cells and spheres can be analyzed after microscopic imaging. The high-throughput tradition system with automatic analysis enables the analysis of heterogeneity within the CSC populations to study malignancy stemness and cell size correlation and single-cell-derived sphere growth dynamics. Experimental Device design and fabrication The high-throughput device was built using a PDMS (polydimethylsiloxane) piece with microfluidic patterns bonded to another blank PDMS piece or a glass slip. PDMS was patterned by standard smooth CD72 lithography. The SU-8 mold utilized for soft-lithography was created by a 3-coating photolithography process RU 24969 hemisuccinate with 10 m, 40 m, and 100 m solid SU-8 (Microchem) following a manufacturers protocol. The pattern was designed using a computer-aided design software (AutoCAD 2015, Autodesk?), and the masks.