Introduction: Several methods have been developed to produce cell spheroids in the laboratory; including rocking method, agarose silicone moulds, and micro-well plates. However, most of available ready-made silicone moulds and micro-well plates are considered as expensive options. We here describe a simple low-cost method to produce agarose micro-wells using commercially available Japanese floral frogs.

Methods: 2500 µL of sterile 1.5% w/v agarose were placed per well in 12-well plates and commercially available stainless steel Japanese floral frogs (autoclaved) were placed in the well-plates upside down to produce the micro-grooves inside the agarose. After gelation, the floral frogs were removed, and the micro-wells were used for cell seeding (Figure 1). 20,000 HepG2 cells/well were used for demonstration.

Results: HepG2 spheroids were produced on day 3-5 after cell seeding and were retrieved through gentle pipetting. The MTT assay will be performed in comparison to spheroids made in ready-made spheroids microplates and 2D culture and the results will be presented at the conference.

Conclusion: Using Japanese floral frogs is a simple and low-cost method to produce spheroids. The testing of cell viability and proliferation in the new model is still ongoing.