Microfluidic channel-integrated hanging drop array chip operated by pushbuttons for spheroid culture and analysis

Abstract

Although the hanging drop methods have a number of advantages for spheroid culture, they suffer from reagent exchange procedures that depend on tedious and accurate liquid handling by manual pipetting or robotic arms. To simplify these procedures, we developed a method for liquid handling in a hanging drop array (HDA) chip for spheroid culture and analysis by integrating microfluidic channels operated by pushbuttons. Six finger-actuated microfluidic pumping units connected to a 3 × 3 HDA can draw or replenish reagents in an HDA chip without any external equipment. The initial cell seeding, medium exchange, and staining for further analysis can be simply done by pushing the buttons in the programmed order. After the assessment of the reagent exchange ratio of the device, BT474 spheroids of various sizes were cultured in the device for 7 days by exchanging the medium once a day and stained on the same device by exchanging the medium with staining reagents for the LIVE/DEAD assay. Furthermore, the cultured spheroids were embedded into collagen by exchanging the medium with a collagen solution to mimic a cancer metastasis environment.