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

Abstract

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid- containing microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platform's usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

An automation workflow for high-throughput manufacturing and analysis of scaffold-supported 3D tissue arrays

“…To this end, we characterized three scaffold candidates (II, III and IV) and compared them with the original paper scaffold (I) used in 96/384-SPOT [22]. The three scaffold candidates were preliminarily selected from a larger pool of candidates based on thickness (similar thickness as scaffold I), material types (cellulose scaffolds are preferred), and material size compatibility with well-plate dimensions.…”

“…The 96/384-SPOT plates were fabricated as previously described [22]. Cellulose scaffolds were partially infiltrated with a poly(methyl methacrylate) (PMMA, 120KDa, Sigma-Aldrich, USA) solution in acetone (Sigma-Aldrich, USA) to block the inter-well regions using the AxiDraw V3 (Evil Mad Scientists, USA).…”

“…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 cell-gel 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.…”

“…The growth of tumour organoids relies on the presence of 3D bio-matrix, such as Matrigel™, which has poor structural integrity that can result in uncontrolled culture deformation when using high cell densities, during long-term culture, or even during the frequent washing steps necessary for analysis [13]. A variety of strategies have emerged to better preserve the fragile 3D cultures including models incorporating a solid substrate such as elastomer-based microfluidic devices [14][15][16] and scaffold-supported platforms [13,[17][18][19][20][21][22]. By improving the structural integrity and control over culture assembly, some of these models have also enabled increased culture complexity and relevance such as the incorporation of multiple cell types including cancerassociated fibroblasts [13,23], mesenchymal stromal cells [24,25] and endothelial cells [26].…”

“…Further the thick geometry of the plug distributes cells into multiple image plane often necessitating the use of confocal microscopy to decipher different cell populations. Microtissues/tumours manufacturing approaches that involve micromolding 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 towards scaling throughput.…”