Three-dimensional (3D) cell culture models, including organoids, are expected to increase the quality of compounds in the drug development pipeline, as they are poised to become the most faithful representation of the in vivo environment that can be studied in vitro. If successful, these models could partially replace animal disease models — in which the industry has recognized shortcomings but come to rely upon — thereby increasing the relatability of preclinical research to clinical outcomes.
In order to reach this potential, these complex models must be able to operate in a high-throughput manner. This is driven by a need for efficiency in drug discovery and screening and further motivated by the end goal — providing treatments to patients with the best chance of a positive outcome.
3D Models in Drug Discovery and Screening
3D cell culture has the incredible ability to faithfully model a cell while it’s being cultured ex vivo. 3D culture format varies based on needs and available technology and includes tissue models, spheroids, and organoids. Tissue models with solid synthetic scaffolds mimic a broad range of 3D tissue structures. Spheroid models are simple, widely used multicellular 3D models derived from 2D cell lines that can be generated from a broad range of cell types, such as tumor spheroids, embryoid bodies, hepatospheres, neurospheres, and mammospheres. However, eclipsing both of these models in sophistication and potential are organoids, the most advanced 3D models. Organoids can be generated from pluripotent stem cells (PSCs) and adult stem cells (ASCs). Influenced by growth factors and extracellular matrices (ECMs), organoids support advancements in the study of organogenesis, disease modeling, and subsequent patient-specific therapies.
3D models can play a role in both drug discovery and screening, with the approach and tools tailored to researchers’ needs at each stage. 3D models may be used to screen and discover new therapeutic prospects from molecular libraries or be used to deduce the most effective choice from an array of available therapies.
Organoids are being studied for their predictive ability regarding clinical response. Researchers can look for resistance or sensitivity of the cancer cells to the selected therapeutic regimen.1 Furthermore, these models can be used to investigate which gene transcription changes were correlated with sensitivity and resistance, revealing chemotherapy sensitivity signatures.2
Clinical trials that demonstrate the utility of organoids in personalized medicine are already underway. The Pancreatic Adenocarcinoma Signature Stratification for Treatment (PASS-01) trial in pancreatic cancer randomizes patients to 1 of 2 chemotherapies while establishing patient-derived organoids in parallel with the aim of demonstrating predictive power of patient response.3
“This research collaboration represents a joint deep dive into the scientific opportunities of a clinical trial, which, we are demonstrating, can include following patient responses while predicting them through ex vivo modeling of the patient’s own cancer.”
— Dennis Plenker, Ph.D., Research Investigator (Tuveson lab),
Technical Manager CSHL Organoid Facility, Cold Spring Harbor Laboratory
Bringing 3D Models to a High-Throughput Environment
3D organoid models derived from human tissue are utilized for drug discovery and screening because of their ability to predict clinical human outcomes. They offer incredible potential for the future of personalized medicine. However, to realize this, these models must be able to operate in a high-throughput environment for capacity, efficiency, and practical utility. Translation of 3D cell culture to high throughput is complex, and certain considerations must be accounted for and challenges overcome.
Homogeneity and Reproducibility
One of the most important aspects of any assay is reproducibility. This is a key challenge in bringing organoids into a high-throughput environment. Variability in growth rates, morphologies, and requirements for growth conditions and matrices can hinder the homogeneity and reproducibility of organoid assays, making implementation in high throughput more challenging. Each sample and cell is different, making each assay in many ways unique unto itself.
Culture Materials and Technologies
Cancer treatments are often given in combination, but running comprehensive assays on the optimal number of combination therapies can result in researchers quickly running up against the limitations of their culture materials and technology.
Automation, including creating, removing, and moving the spheroids or organoids into the encapsulation medium can present a technological challenge. One such area automation has been successful in is imaging (batch processing of data). Additionally, equipment can be more costly as methods require a greater degree of sophistication.
Plasticware can contribute to the success or failure of 3D modeling. Signal-to-noise ratio must be carefully optimized when moving from 2D to 3D.
As assays become more miniaturized to adapt to high-throughput screening, volume decreases, presenting measurement limitations and noise due to evaporation.
Bioinformatics and data-processing capabilities must also be considered, with adequate computing power to handle the large amounts of data generated from high-throughput experiments — but established analyses pipelines from 2D screens can be used.
Cell density
Researchers must be able to obtain a high enough density of cells to conduct high-throughput analysis of 3D models.
Speed
In order to be applicable for real-world personalized treatment selection, assays must be completed in a time frame conducive to informing patient treatment. For example, time is of the utmost importance in cancer therapy selection.
“As a consumable and equipment vendor, we closely follow the problems that scientists are facing with moving 3D models into a high-throughput environment. Assay miniaturization, reagents, standardization, automation, quantity of data—these are all key points that come up in conversation. So, we are working diligently to come up with novel materials and technological solutions that can help solve some of these issues and advance the field.”
— Elizabeth Abraham, Ph.D.,
Business Manager, Advanced Cell Culture Portfolio, Corning Life Sciences
Technological advancements in materials and resources have helped to address some of these challenges to move the field forward. Corning® Elplasia® Plates enable high-density, scaffold-free spheroid formation that has aided with scaling, facilitating the generation of spheroid quantities amenable to high-throughput analysis. Matrigel® Matrix, one of the most widely used ECMs that can be used to generate models for 2D and 3D culture in vitro, is now available pre-dispensed in high-throughput plate wells to increase ease of use and consistency. Reagents can also be improved to decrease signal-to-noise ratio, as demonstrated by Corning® 3D Clear Tissue Clearing Reagent. Advancements in microfluidics have also improved the replication of in vivo environments. But improvements in the assay must be met in equal measure with improvements in data handling and analysis.
Given that these challenges can be overcome, the potential for advancements in drug discovery and screening are vast. New therapeutics could be tested in an array of human organoids prior to clinical trials via high-throughput screening, informing patient selection, and stratification. This kind of living bio-bank could speed drug development by increasing efficiency and translation to the clinic.
Researchers are already beginning to explore this approach to advance personalized medicine in specific patient populations. In the Netherlands, one research study explored the use of organoids to inform cystic fibrosis treatment decisions.4
“In pharmaceutical drug testing, you may have a library of organoids that you are always testing, in which case you have established operating procedures for every single patient-derived organoid. In precision medicine, you may need to tailor your approach for each new patient. Regardless, high-throughput screening will be important for both areas.”
— Hervé Tiriac, Ph.D., Assistant Research Scientist, Department of Surgery,
Division of Surgical Oncology, Moores Cancer Center, University of California, San Diego
Looking to the Future
As we look back at the speed of recent scientific advancement, it becomes clear that it is a matter of when, not if 3D models, such as organoids, will be used on a high-throughput scale for drug development, drug screening, and personalized medicine.
When this is realized, more sophisticated models are sure to become the new focus. The ability to co-culture organoids with other cell types important to tumor biology, such as cancer-associated fibroblasts, macrophages, and other immune cells (and to do so in a high-throughput environment), would enable the investigation of immunotherapies within the representative microenvironment. This would involve an entirely new set of challenges, such as the ability to obtain the required variety of patient-matched samples and to do imaging on a cell-specific level.
The hope of expanding the scope of application of 3D cell models in preclinical research offers scientific possibility only constrained by the knowledge and technology of today. More representative 3D models of human disease can not only change how therapeutics are discovered and developed, but also transform the treatment paradigm by providing critical, personalized knowledge on a patient’s own disease. Bringing 3D models into a high-throughput environment will enable this technology to reach its full potential while provoking new scientific thoughts and ideas to usher in the next wave of advancements in cell culture.
References
- Narasimhan V., J.A. Wright, M. Churchill, et al. "Medium-throughput drug screening of patient-derived organoids from colorectal peritoneal metastases to direct personalized therapy." Clin Cancer Res. 26:3662–3670 (2020). doi:10.1158/1078-0432.CCR-20-0073
- Tiriac H., P. Belleau, D.D. Engle, et al. "Organoid Profiling Identifies Common Responders to Chemotherapy in Pancreatic Cancer." Cancer Discov. 8:1112–1129 (2018). doi:10.1158/2159-8290.CD-18-0349
- U.S. National Library of Medicine. "Pancreatic adenocarcinoma signature stratification for treatment." 24 Jun. 2021. Accessed 25 Jul. 2021. https://clinicaltrials.gov/ct2/show/NCT04469556
- Berkers G., P. van Mourik, A.M. Vonk, et al. "Rectal organoids enable personalized treatment of cystic fibrosis." Cell Rep. 26:1701–1708.e3 (2019). doi:10.1016/j.celrep.2019.01.068