Shape, shrink, spheroid: a DIY high-throughput spheroid generation device

3D spheroids, which closely replicate three-dimensional cell-cell and cell-extracellular matrix interactions, offer superior predictive capabilities compared to conventional 2D monolayer cultures, positioning them as forward-looking platforms in drug testing, cancer biology, and regenerative medicine. However, high-throughput generation of uniform sized spheroids is still a technological challenge. In one hand, the use of conventional ultra-low attachment (ULA) multiwell plates for this purpose is labor intensive and complex. On the other hand, the use of microfabricated facilities demands cutting-edge infrastructure such as clean room, photolithography, and microfluidic setup which are often unavailable for the resource constrained laboratories. In this study, we addressed these problems by developing a low-cost do-it-yourself (DIY), polydimethylsiloxane (PDMS) and agarose-based spheroid generation device, capable of producing and maintaining hundreds of spheroids with minimal user intervention. We have demonstrated two variants based on their size, termed here as S1 and S2 devices which fit into 6-well and 12-well plates, and can generate 600 and 1200 uniform-sized spheroids respectively. We validated our device with various cell lines including primary and cancerous cell lines. We further demonstrated the drug testing capabilities of the device by estimating the IC₅₀value of the anticancer drug Temozolomide on U87-MG. The value was comparable with the same obtained from the spheroids generated using conventional ULA plates. Additional attachment of a perfusion system made the device suitable for long-term spheroid culture without much user intervention. Furthermore, the devices can also be used for the production of spheroids with gradually changing diameters in a controlled manner, resembling a size gradient. This feature is useful for checking the effect of drugs on different-sized spheroids and for co-culturing spheroids with varying cell densities, mimicking the disease architecture. We have co-cultured two types of the placental trophoblast cells, i.e. extravillous trophoblast (HTR-8) and syncytiotrophoblast (BeWo) with varying densities. In summary, this paper demonstrates a unique DIY method for a high-throughput uniform-sized spheroid generation at a fraction of cost which can be deployed to resource-constrained labs.