Brain organoids, illustration. Organoids are miniature, simplified versions of organs grown in the laboratory to allow testing of targeted therapies.

There is an ever-increasing myriad of disease-modeling technologies that allow us to visualize disease processes in clearer detail than we ever thought possible. In this article, we will look at 3D culture systems, which are designed to help us understand signaling pathways in certain disease processes. These are especially relevant for chronic liver diseases such as Alagille syndrome (ALGS). 

3D culture systems have a number of uses, including modeling disease microenvironments, and potentially play a role in helping scientists develop regenerative medicine. However, let’s start with the obvious: What is 3D cell culture? To answer this question, we will look at a study written by Jensen and Teng and published in Frontiers in Molecular Bioscience. 

Jensen and Teng gave us an excellent summary of the role of 3D cell culture in medical research today: “Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study,” they explained. “When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell in vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases.” 

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Read more about ALGS etiology

The key phrase here is that 3D culture experiments mimic a cell environment in vivo. That gives us an excellent tool to understand how disease processes affect cell environments in vivo: a new form of medical imaging, if you will. 

Comparing 3D and 2D Cell Cultures 

The primary thrust of Jensen and Teng’s article is illustrating the superiority of 3D cell culture to 2D cell culture. In doing so, they provide a comprehensive list of what 3D cell culture can achieve: 

  • The natural cell shape, as well as cell growth, is preserved.
  • 3D culture cells grow into 3D aggregates/spheroids, allowing for more detailed analysis of the effect of disease processes on human cells. 
  • 3D cell culture allows the mimicking of the behavior and structure of a tumor cell in vivo. 
  • 3D cells are well-differentiated. 
  • Drug metabolism using 3D cells is superior to that of 2D cells. 
  • The gene and protein expression levels resemble that from cells in vivo. 
  • 3D cell culture minimizes the differences between in vivo and in vitro drug screening, which reduces the need of using animal models for conducting studies. 
  • 3D cell cultures provide an accurate representation of response to the mechanical stimuli of cells. 

In summary: in vitro 3D cells allow us to observe how cells most likely respond in vivo. And that is good news for understanding disease processes and for the testing of new experimental drugs. 

Exploring the Notch Signaling Pathway 

Marconi et al are equally optimistic about the role of 3D cell culture. They wrote, “3D culture systems opened up new horizons in studying the biology of tissues and organs, modeling various diseases, and screening drugs. Producing accurate in vitro models increases the possibilities for studying molecular control of cell-cell and cell-microenvironment interactions in detail.” 

One such use is the illumination of the workings of the Notch signaling pathway, a pathway heavily implicated in ALGS. On ALGS, Marconi and colleagues wrote, “Mutations in the JAGGED1 (JAG1) ligand and more rarely in the NOTCH2 receptor cause the Alagille syndrome that affects multiple organs, including heart, liver, kidney, and craniofacial organs.” 

Read more about ALGS treatment 

And how do 3D cultures illustrate this in vivo? “Different 3D culture platforms such as spheroids, organoids, and “organ-on-a-chip” devices can be used to dissect the roles and the regulation of this pathway in various healthy and pathological cell populations,” Marconi and colleagues wrote. 

These spheroids from hepatic cells allow scientists to study the chronic and acute conditions of liver diseases (these differ from classical hepatocyte monolayer models, which results in cell dedifferentiation and subsequent changes in their metabolic activities). 

Genetic defects in the Notch pathway can result in severe liver malformations, so carrying out testing in vivo is out of the question. For example, mutations in the JAG1 gene can result in bile duct reduction, eventually leading to biliary tree dysfunction. “Hence, the generation of organoids from healthy and pathological liver tissues provides an additional tool for further exploring the potential therapeutic roles of Notch signaling,” the research team wrote.

“Exploring the potential therapeutic roles of Notch signaling”—that is perhaps one of the most exciting uses of 3D cell cultures. 3D cultures allow scientists to test out the efficiency of different approaches to blocking Notch activity without causing bodily harm. 

“Thus, developing 3D in vitro models that preserve the tissue complexity might constitute a safe way for molecular analysis and understanding the big potential of Notch signaling modulation in disease treatment,” Macaroni and colleagues wrote. 

Unlocking The Future 

3D culture systems have given humanity a valuable key to develop therapeutics against mutations of the Notch signaling in a way that might yet constitute a medical breakthrough.

“These technologically advanced tools are extremely useful for studying specific molecular cues and analyzing the role of specific signaling pathways, including the Notch pathway, in the context of specific disease models, drug responses and tissue regeneration,” Marconi et al concluded. 


Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. Published online March 6, 2020. doi: 10.3389/fmolb.2020.00033

Marconi GD, Porcheri C, Trubiani O, Mitsiadis TA. Three-dimensional culture systems for dissecting Notch signalling in health and disease. Int J Mol Sci. Published online November 19, 2021. doi:10.3390/ijms222212473