Industrial-scale organoid production: challenges, advantages and solutions
The realization that 2D cell lines lack the complexity to represent human biological systems encouraged scientists to seek more advanced in vitro culture tools for biomedical research. Recent advances have led to the creation of 3D models such as spheroids and organoids.
Spheroids are generally free-floating aggregates of multiple cell types and are arguably of low complexity in mirroring tumor organization. By contrast, organoids are defined by their ability to self-assemble from differentiated stem cells into a spatial arrangement that mirrors the bodily organ they represent, recapitulating the structure and biological function of the organ, but on a miniature scale. Organoids are thus a physiologically relevant model for studying human diseases and evaluating drug efficacy and safety – as an example, organoids mimic the patient response when both are treated with the same drugs. Organoids can be produced that represent diseased or healthy tissue from most of the organs in the body.
As a result, organoid cell models offer the possibility of accelerating the drug development pipeline, reducing the high attrition rate in clinical trials and the associated costs.
However, for this to become widespread and compatible with high-throughput screening applications, organoid production at scale is required.
Scaling the production of organoids to ensure consistency, reproducibility and statistical relevance when testing is challenging. In this podcast, organoid experts Victoria Marsh Durban, Director of Custom Organoid Services at Molecular Devices and Magdalena Kasendra, Director of Research and Development at the Center for Stem Cell and Organoid Medicine at Cincinnati Children’s Hospital discuss the advantages – and challenges – of industrial-scale organoid production.
Basics of organoid production
Organoids typically arise from pluripotent or adult stem cells.
Pluripotent stem cells (PSCs) can be obtained from patients’ skin fibroblasts or peripheral blood mononuclear cells. They are self-renewing and can be reprogrammed to differentiate into multiple cell types. When using PSCs, one needs to recapitulate the embryonic development in vitro in a complex multistep process, involving the application of different growth factors to generate features of multiple cell lineages. This induces differentiation into multiple organoid types (e.g. brain, lung, cardiac, kidney and liver) from the same original cells, hence the term Induced Pluripotent stem cells (iPSCs).
Examples of 3D cell models from the top left - brain organoid, lung organoids, cardioid (heart organoid), and 3D liver model.
Adult stem cells, from patient biopsy samples or resected tissue, have an innate cellular mechanism which ‘forces’ them to differentiate down the required pathway to recreate the tissue from which they have originated. In other words, they can form into organoids spontaneously in the laboratory when cultured in extracellular matrices and given appropriate tissue-specific growth factors.
In general, manufacturing of organoids from adult stem cells isolated from primary patient tissue biopsies is more straightforward than using iPSCs.
Challenges involved in organoid production
Organoid research is still new and growing, so scientists are faced with challenges in optimization and scaled-up organoid production regardless of the stem cell type used as a starting point.
One of the main challenges in using organoids derived from adult stem cells is related to the cell culturing process that needs to be carried out entirely in 3D using hydrogel. This process is much more demanding and time-consuming than 2D culture.
PSC-based workflows bring additional complications because of the difficulty of manipulating them into the desired differentiation patterns. Magdalena states: “When using iPSCs, we need to recapitulate embryonic development in vitro through the introduction of different growth factors to mimic various cell lineages.” Fortunately, these limitations can be overcome today with the development of highly automated state-of-the-art bioprocesses.
Bioreactors have been widely used in culturing organoids (both iPSC and adult stem cell-based) in carefully monitored environmental conditions; however, additional challenges are associated with their use. Specifically, the transition from initial growth in hydrogel-based matrices to suspension in bioreactors requires great care.
While suspended in a hydrogel, iPSCs are not exposed to any mechanical stimulations. However, placing them in a 3D suspension subjects them to rapidly changing shear stress, which can impact growth rate and differentiation. While low levels of shear stress promote differentiation, excessive levels can induce cell damage and death. Thus the bioreactor shear stress needs to be carefully optimized to achieve the desired differentiation states without losing the organoid yield. Current strategies include regulating bioreactor rotation speed or using a shear-stress free bioreactor. The optimal strategy is dependent on the exact growth and differentiation requirements for the organoid of interest and ensuring that the hydrogel carrying the initial stem cells is compatible with the bioreactor. Soft hydrogels, for example, can break down in bioreactors due to shear stress, jeopardizing the integrity of the stem cell aggregates.
Another challenge is the difficulty of obtaining the source material for the adult stem cells, i.e., finding a suitable original organoid line or primary patient biopsy material. Currently, most organoid research is conducted in research institutions affiliated with academia, where organoids are produced solely for non-profit research purposes. However, the translation from academia to the commercial environment, such as pharmaceutical companies, will inevitably raise ethical concerns. Ethical consent will have to be taken into consideration when commercializing organoid cultures from patient samples.
Colorectal organoids can be used to study diseases such as inflammatory bowel disease (IBD).
Recreating organoid cultures from original organoids can also be cumbersome because of the variations between different cell line optimization protocols. For example, cerebral (brain) organoid culturing involves the transfer of IPCSs to a neuronal induction medium before growing them in hydrogel droplets. According to Victoria, “The protocols can vary significantly depending on the source tissue type, e.g., whether it’s a cancerous tissue and the cancer type-subtype it belongs to. That’s why you may need to optimize the production workflow on a line-by-line basis, which calls for an expert eye.”
A single misstep in protocol implementation can push the cell line to differentiate into an unwanted phenotype.
Advantages of scaling up organoids
Although scaling up organoid production can prove challenging, the rewards can be considerable.
According to Magdalena, one of the main advantages is the ease of translation into the biopharmaceutical landscape: “Scaling up makes organoids amenable to industry standards and regulatory practices so they can be employed more readily in drug discovery, stem cell therapy, and personalized medicine applications.”
Large-scale batch production of organoids can benefit both academic and industrial research. Making large batches of organoids enables scientists and manufacturers to conduct larger experiments with high-throughput assays.
Victoria adds that large organoid batches can mitigate batch-to-batch variability: “In particular, animal-derived reagents used in organoid production often come from a variety of sources. These growing reagents are derived for small scale experiments specifically. This means that every time one wants to grow organoids, they are going to use a different reagent, which points to a lack of organoid standardization.”
Lastly, deploying compatible bioreactors can help researchers monitor environmental conditions, such as shear stress, more easily, ultimately improving consistency and thus reproducibility.
Organoids: The predictive power of 3D
The increased emphasis on organoid research is mainly because organoids unlock deeper insight into tissue architecture and cell-cell interactions, which increases predictive power in the drug discovery process. For example, ‘mini-guts’ have been used to identify the most effective drugs for patients with specific forms of cystic fibrosis (1). Similar models, grown from patients suffering from gastrointestinal cancers, likewise proved predictive of the impact of therapies in the patients themselves (2). More recently, a vast array of organoid types were used to model the impact of SARS-CoV-2 infection in various tissues and identify various therapies (3).
Intestinal organoids are 3D microtissue models that recapitulate structures in the intestinal lumen and on the surrounding intestinal epithelium. They can simulate essential intestinal functions, such as nutrient absorption and mucus secretion.
This improved predictivity means organoids can identify drug candidate failures earlier in the drug discovery pipeline and eliminate false positives (i.e., candidates that failed in clinical settings despite their promise in preclinical research) much earlier in the process.
Organoids also hold promise within personalized medicine. Patient-specific stem cells give rise to organoids congruent with the genetic makeup and physiological characteristics of the patient. Thus, organoids will become invaluable when developing tailored therapies for individuals with rare genetic disorders and treatment-resistant cancers.
More widespread use of organoids can also reduce the use of animal models. Much like 2D cell models, animal models are also inadequate in predicting patient response, so their wide use poses a risk of clinical failure. There is also the added pressure of ethical concerns around animal use in drug discovery. With the establishment of the FDA modernization act, researchers have begun to seek alternative strategies to animal models.
The use of organoids as an in vitro platform for drug development and the study of disease mechanisms, together with changes in the law, will enable us to reduce the number of animals used in research. The adoption of organoids will gain traction as scientists realise their value and gain the necessarily skills and experience to use them in the lab. Access to organoid lines and the development of assays to make full use of this complex, human-centric in vitro model will widen the organoid field substantially and take us into a new era of life science research.
- Saini, Angela. "Cystic fibrosis patients benefit from mini guts." Cell Stem Cell 19.4 (2016): 425-427.
- Vlachogiannis, Georgios, et al. "Patient-derived organoids model treatment response of metastatic gastrointestinal cancers." Science 359.6378 (2018): 920-926.
- Han, Yuling, et al. "Human organoid models to study SARS-CoV-2 infection." Nature Methods 19.4 (2022): 418-428.