About the automated 3D spheroid culturing
High-content imaging studies using three-dimensional tissue structures involve a series of sequential steps, each of which needs to be well-optimized and matched to achieve maximal output with minimal technical variability. 3D models, in particular multi-cellular spheroids, have gained increasing interest as they have proven to have higher physiological relevance due to their three-dimensional architecture and potential to include all relevant cell types. Further, their scalability and compatibility with plate formats of 96 and more wells make them an ideal model for higher-throughput studies.
Suspended in media as a spherical 200-500 µm diameter compact tissue model, all steps leading up to high-content imaging need to be adapted. These include liquid handling protocols for efficient medium exchange and dosing without tissue loss, staining protocols preserving morphology of the tissue while ensuring dye penetration and selection of imaging compatible culture ware to enable high-resolution multi-color fluorescence imaging.
We have previously demonstrated that using InSphero’s 384-well Akura plates, adherent-free spheroid culturing with multiple medium exchanges and compound dosing is achievable with minimal tissue loss even when more than 90% of the medium is removed in a single pipetting step. This function is enabled by an integrated ledge within the well that protects the microtissue from accidental aspiration.
The well design has been used to implement an automated staining protocol performed directly after the experiment. The protocol includes fixation, permeabilization, blocking and antibody staining of the microtissues with multiple washing steps in between. In total, the protocol has 9 steps and can be run autonomously overnight (15 h). Automation of these steps with long incubation times significantly reduces the time cost of the sample preparation and prevents manual pipetting errors. GF-expressing tumor spheroids have been stained with DAPI for nuclear quantitation and anti-beta integrin for cell surface receptor imaging.
Once stained, the spheroids were imaged using Yokogawa’s CQ1 desktop high-content system. The well plate comprises a 25 µm-thin flat Teflon bottom-foil with the spheroids located in a small 1-mm wide observation window. The thin bottom enables high-quality in-situ imaging with high NA short working distance objectives even for larger 3D spheroids with expanded z-dimensions. The complete acquisition of a complete 384-well plate for 3 fluorescent channels and 7 z-slices takes 93 minutes. We were able to image the GFP signal in the cytosol, DAPI signal in the nucleus and the beta-integrin on the cell membrane.
Download our poster about the automated 3D spheroid culturing
