1071-B - Automated Long-term 3D Cell-Based Toxicity Studies Using a Novel Flowchip System
Monday, January 27, 2020
There is an increasing interest in using three-dimensional (3D) cell structures for modeling tumors, organs, and tissue to accelerate translation research. Significant progress has been made in formation of such structures to recapitulate the in vivo environment but performing complex assays with them can be challenging. For example, manual treatment, staining, and processing of spheroids and organoids is typically labor-intensive and prone to disruption or loss of samples. Here we report on use of a novel microfluidic-based system to perform automated assays with spheroids, organoids, and microtissues. It is ideal for applications for long-term toxicity, oncology therapeutics, single organoid secretion, and metabolite sampling. The flowchip has sample chambers connected to multiple reservoirs to enable assay steps such as media and buffer exchange, supernatant sampling, and in situ lysing. A key aspect to the device is a protective chamber that holds 3D cell models and allows reagent exchanges from adjoining reservoirs to be performed without disrupting the structures. The chambers and reservoirs are arranged in a convenient multiwell plate format (384-well spacings) and provide up to 32 tests per plate. Multiple reagent exchanges are possible with the current flowchip allowing complex assay protocols to be performed. After spheroids and reagents are loaded, the plate is placed in a Pu·MA System and fluid movements are done automatically through microfluidic channels connected to the sample chambers. Assay protocols are pre-loaded into the system and experiments are done using an intuitive touch-screen interface. The whole system can be placed in an incubator to run assays at 37°C and 5% CO2. The system architecture and use of pneumatics to move fluids provides gas exchange to the sample chambers. The capabilities of the system were demonstrated by assaying spheroid toxicity over a 3 to 5 day period. Spheroids were formed with HCT116 colon cancer cells in low-adhesion U-bottom 384-well plates and transferred into Pu·MA System flowchips. Media or “media + compound” were dispensed into flowchip reservoirs. The flowchip plate was loaded into a Pu·MA System located in an incubator and a 48-hour incubation protocol was run. Media in the spheroid sample chambers were exchanged at the beginning of the protocol and at 24 hours. The spheroids were analyzed by ImageXpress Micro Confocal Imaging System for Live/Dead cells and by luminescence using a SpectraMax iD5 Plate Reader for ATP activity. Assay performance was assessed using established anti-cancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different read-outs. This new format offers potential for fully automated 3D cell-basd assays that mimic in vivo conditions, perform multi-dosing protocols and multiple media exchanges, handle spheroids and microtissues gently, and allow a wide range of assay detection modalities.