Roadblocks to Realistic Organoids

In 2009, researchers realized that adult stem cells could self-assemble into organized three-dimensional biological structures known as organoids.¹ The discovery led to an explosion of research and the creation of numerous organoids from different tissue types. Today, organoid technology offers unprecedented opportunities for modeling human biology in vitro, accelerating drug discovery, and advancing regenerative medicine. Yet, despite their promise, organoids remain far from perfect and face several key challenges in recapitulating in vivo human organ physiology.

Vascularization and innervation

Vascularization and innervation are the most well-known unsolved limitations of organoid models. While pluripotent stem cells and adult stem cells naturally self-assemble into the various cell types found in an organ, they do not naturally form blood vessels or nerve cells. Blood vessels supply oxygen and nutrients to cells, while nerves regulate cell signaling and nutrient secretion. Without these components, organoid cells cannot fully mature and develop the complex functionality of their in vivo adult counterparts.^2,3

To solve the lack of vascularization, a research team at Stanford University simultaneously co-differentiated different cell types to form blood vessels, but these vessels remained on the outer edges of the organoids.⁴ To make blood vessels and nerves that penetrate through organoids, some research teams use 3D printers to deposit cells layer by layer. Others use microfluidics to embed organoids in chips with tiny channels that mimic blood flow.

Reproducibility

Reproducibility is another key challenge in organoid formation. The natural self-assembling ability of organoids appears stochastic, sparked by spontaneous cell sorting that relies on internal cellular developmental cues. Small variations in growth factor timing, matrix stiffness, or other environmental changes can yield different organoid compositions. Additionally, genetic and epigenetic differences between starting cell lines can affect cell composition, leading to a high degree of organoid variability from the same tissue. This can make it difficult to draw meaningful conclusions that remain consistent across different research groups.

To tackle this problem, some research teams use controlled fluid dynamics to get more consistent organoid architectures and transcriptional profiles.⁵

Standardization

Standardization may be one way to help researchers tackle batch-to-batch variation in organoid research. However, organoid researchers have yet to agree about what qualifies as a good organoid, in terms of its size, shape, and characteristic biomarkers, or the functional benchmarks to define, test, and validate organoids. Experts in the field have also yet to agree on metadata requirements, including how to report cell source, media, and growth factor timing, so that others can reproduce results. To address this issue, some researchers have suggested standardizing starting cell populations, media formulations, and extracellular matrix substitutes used for organoid research.⁶

At the institutional level, the Korean Organoid Standards Initiative has developed standardized guidelines for human intestinal organoids, detailing culture conditions, quality control assays, and functional testing protocols to enable cross-laboratory consistency. The NIH also recently launched the Standardized Organoid Modeling Center to develop standardized, reproducible organoid protocols. The FDA is also working with the NIH’s Interagency Coordinating Committee on the Validation of Alternative Methods to create a roadmap for integrating validated organoid systems into safety and efficacy evaluations. These initiatives signal a growing commitment to making organoid research more reliable and scalable.

Since their initial discovery, organoids have made significant progress in becoming translational research models, but they still have a long way to go in modeling true human biology. Researchers continue to innovate, using the field’s challenges as opportunities to find solutions, drive collaboration, and make discoveries that can help organoid research meet its full potential of transforming medicine.

References

1. Sato T., Vries, R.G., Snippert, H.J., et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265 (2009).

2. Zhao, X., Xu, Z., Xiao, L., et al. Review on the vascularization of organoids and organoids-on-a-chip. Frontiers in Bioengineering and Biotechnology (2021).

3. Das, S., Gordian-Velez, W.J., Ledebur, H.C., et al. Innervation: the missing link for biofabricated tissues and organs. Regenerative Medicine 5 (2020).

4. Abilez, O.J., Yang, H., Guan, Y., et al. Gastruloids enable modeling of the earliest stages of human cardiac and hepatic vascularization. Science 388 (6751) 2025.

5. Ge, J-Y., Wang, Y., Li Q-L., et al. Trends and challenges in organoid modeling and expansion with pluripotent stem cells and somatic tissues. Biochemistry, Biophysics and Molecular Biology (2024).

6. Sandoval, S.O., Cappuccio, G., Kruth, K., et al. Rigor and reproducibility in human brain organoid research: Where we are and where we need to go. Stem Cell Reports 19, 796-816 (2024).