reproducible-manufacturing-spot-as-high-throughput-scaffold-based

Reproducible Manufacturing of SPOT as a High-throughput Scaffold-based Culture Platform

Patient-derived organoids (PDOs) are becoming increasingly used in the field of cancer research to model tumors. They combine the benefits of in vivo and ...

University of Toronto

Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.

Computationally guided high-throughput design of self-assembling drug nanoparticles.

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

Patient-derived organoids have emerged as a useful tool to model tumour heterogeneity. Scaling these complex culture models while enabling stratified analysis of different cellular sub-populations, however, remains a challenge. One strategy to enable higher throughput organoid cultures is the scaffold-supported platform for organoid-based tissues (SPOT). SPOT allows the generation of flat, thin, and dimensionally-defined microtissues in both 96- and 384-well plate footprints that are compatible with longitudinal image-based readouts. SPOT is currently manufactured manually, however, limiting scalability. In this study, an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler is optimized and comparable within- and between-sample variation to standard manual manufacturing is shown. Further, a liquid handler-supported cell extraction protocol to support single-cell-based end-point analysis using high-throughput flow cytometry and multiplexed cytometry by time of flight is developed. As a proof-of-value demonstration, 3D complex tissues containing different proportions of tumour and stromal cells are generated to probe the reciprocal impact of co-culture. It is also demonstrated that primary patient-derived organoids can be incorporated into the pipeline to capture patient-level tumour heterogeneity. It is envisioned that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets.

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

Triggerable coatings, such as pH-responsive polymethacrylate copolymers, can be used to protect the active pharmaceutical ingredients contained within oral solid dosage forms from the acidic gastric environment and to facilitate drug delivery directly to the intestine. However, gastrointestinal pH can be highly variable, which can reduce delivery efficiency when using pH-responsive drug delivery technologies. We hypothesized that biomaterials susceptible to proteolysis could be used in combination with other triggerable polymers to develop novel enteric coatings. Bioinformatic analysis suggested that silk fibroin is selectively degradable by enzymes in the small intestine, including chymotrypsin, but resilient to gastric pepsin. Based on the analysis, we developed a silk fibroin-polymethacrylate copolymer coating for oral dosage forms. In vitro and in vivo studies demonstrated that capsules coated with this novel silk fibroin formulation enable pancreatin-dependent drug release. We believe that this novel formulation and extensions thereof have the potential to produce more effective and personalized oral drug delivery systems for vulnerable populations including patients that have impaired and highly variable intestinal physiology.

Article	Year
Computationally guided high-throughput design of self-assembling drug nanoparticles. Nature nanotechnology\| PubMed ID: 33767382	2021
An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues. Biomaterials\| PubMed ID: 36343611	2022
An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays. Advanced healthcare materials\| PubMed ID: 37086259	2023
Silk Fibroin-Based Coatings for Pancreatin-Dependent Drug Delivery. Journal of pharmaceutical sciences\| PubMed ID: 37690778	2024

Ruonan Cao

Biography

JoVE Journal Publications

Other Publications