Multi-size spheroid formation using microfluidic funnels

Abstract

We present a microfluidic platform for automatic multi-size spheroid formation within constant volume hanging droplets (HDs) from a single inlet loading of a constant cell concentration. The platform introduces three technological improvements over the existing spheroid formation platforms: 1) cell seeding control is achieved by enrichment of a cell solution rather than dilution; 2) cell seeding in each HD is fully independent and pre-programmable at the design stage; 3) the fabricated chip operates well using a hydrophobic PDMS surface, ensuring long-term storage possibility for device usage. Pre-programmed cell seeding densities at each HD are achieved using a "microfluidic funnel" layer, which has an array of cone-shaped wells with increasing apex angles acting as a metering unit. The integrated platform is designed to form, treat, stain, and image multi-size spheroids on-chip. Spheroids can be analyzed on-chip or easily transferred to conventional well plates for further processing. Empirically, enrichment factors up to 37× have been demonstrated, resulting in viable spheroids of diameters ranging from 230-420 μm and 280-530 μm for OV90 and TOV112D cell lines, respectively. We envision that microfluidic funnels and single inlet multi-size spheroid (SIMSS) chips will find broad application in 3D biological assays where size-dependent responses are expected, including chemoresponse assays, photodynamic therapy assays, and other assays involving drug transport characterization in drug discovery.

Facile One Step Formation and Screening of Tumor Spheroids Using Droplet‐Microarray Platform

“…The possibility to use only 150 cells per spheroid can be crucial when working with rare and hard to expand cells, such as primary patient derived tumor cells. Spheroids used in research vary in their sizes from 50 to 1000 µm . Even though spheroids smaller than 200 µm usually do not develop necrotic core, it was shown that even very small spheroids, with diameter < 100 µm, have oxygen gradient and develop hypoxia and activation of hypoxia‐responsive element (HRE) already after 2 days of culturing, which confirms their physiological similarities to in vivo tumors.…”

“…Droplet Microarray platform is flexible and allows to grow spheroids of different sizes by varying initial cell number and size of hydrophilic spots. It was shown that spheroids of different sizes might respond differently to drug treatment, therefore, suitable size of spheroids should be chosen depending on application. In this study, our goal was to demonstrate formation and screening of spheroids from low cell numbers to enable large screenings in the 3D format using physiologically relevant rare and difficult to expand cells, such as primary patient‐derived cells and stem cells.…”

On-chip combined radiotherapy and chemotherapy testing on soft-tissue sarcoma spheroids to study cell death using flow cytometry and clonogenic assay

“…First, not all cell lines are able to form and be maintained as spheroids, thereby reducing the number of models. Second, 3D models confer heterogeneity in the concentrations of nutrients, oxygen and lactate within the spheroids, which are dependent on diffusion and therefore spheroid size 24,25 . Thus, consistent control of spheroid size is desired to reduce variability in experimental results.…”

Micropocket hydrogel devices for all-in-one formation, assembly, and analysis of aggregate-based tissues

“…Second, a large number of cells are needed to achieve the high seeding densities required for the uniform spheroid production regime described here. While this is not a concern for cell lines, adapting this platform for limited primary cells will require some modifications, such as a funnel structure to feed a small volume of cell suspension into a single well [30]. Although this would increase device complexity and would require a pipetting step for each spheroid, it would enable production of spheroids from precious cell material.…”

“…Scaled-up methods have been introduced, including forming aggregates in non-adhesive microwells [21] or in stirred vessels [1,[21][22][23], but these stochastic techniques have relatively poor control over aggregate size, which is a critically important parameter for aggregate function. More technically advanced strategies such as the use of microarrayed [3,7,[24][25][26] or microfluidic systems [27][28][29][30][31] have also been developed to provide controlled and addressable culture models, but these are challenging to adopt in standard wet labs as microfluidic systems typically require considerable expertize to fabricate and operate [32].…”