An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues

An Automation Workflow for High‐Throughput Manufacturing and Analysis of Scaffold‐Supported 3D Tissue Arrays

“…The hydrogel blend used for PPTO.46 cells comprised 25% Matrigel and 75% 3 mg mL −1 collagen hydrogel, as reported previously. [58] As described previously, [22] 70 μL of sterile PBS was added to each interwell space in the 96-well plate to prevent evaporation while seeding before use, and then the plate was chilled for 30 min to ensure no clumping of cell-gel solution during seeding. For manual seeding, a single-channel micropipette (Gilson) was used to deposit 5 μL of cell-gel into the center of each 96-SPOT well by carefully dispensing the gel above the paper surface and gently touching the tip of the pipette to the paper.…”

“…A custom ImageJ script (adapted from refs. [22,58]) was written to measure the MGV of the GFP signal of 100 randomly selected squares to obtain the standard deviation within one well. Standard deviations were used to calculate the coefficient of variation associated with each well using the following:…”

“…We therefore set out to optimize a digestion sequence to enable the recovery of single cells for downstream end-point analysis. Previous work [22,23,44] using paper scaffolds infiltrated with 5 μL of cell-gel showed successful gel digestion and cell retrieval using 1 mL of a solution containing collagenase, protease and DNAase enzymes incubated at 37 °C for 45 min in a thermomixer, and followed by vigorous pipetting. We applied 200 μL of the same digestion solution with adapted protocols (i.e., no need for a plate shaker during incubation) but focused on optimizing pipetting sequences to be compatible with 96-SPOT digestion.…”

“…Some examples have been reported where automation is beginning to be incorporated into workflows using these complex culture models, primarily with a focus on automation of 3D microtissue/tumour fabrication and/or dispensing drugs to perform a drug response assay. [4,6,8,15,[27][28][29][30][31][32][33] For example, engineered microtissues/tumours have been fabricated automatically using bioprinting techniques to control the deposition of cell-laden bioinks, such as extrusion-based bioprinting, [29,30] stereolithography, [31] acoustic bioprinting, [32] magnetic force field, [8] contact-capillary wicking to infiltrate cellgel into existing scaffolds, [22,34] and FRESH bioprinting using sacrificial support materials. [33] However, the remaining manual downstream processes, such as maintenance, screening, and high-content analysis, still significantly limit the throughput of these culture platforms and potentially introduce unwanted experimental variation.…”

“…Further, the thick geometry of the plug distributes cells into multiple im-age planes often necessitating the use of confocal microscopy to decipher different cell populations. Microtissues/tumours manufacturing approaches that involve geometric templating of the hydrogel matrix to ensure a flat and thin gel geometry can significantly improve the capacity to perform imaging and obtain single-cell resolution data [13,22] however to date, these systems have not been automated with a view toward scaling throughput.…”