Article reviewed by Julía Crispim da Fontoura, a PhD candidate at the Federal University of Health Sciences of Porto Alegre, Brazil who uses organoids to study drug resistance.

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A scientist cultures organoids in a multi-well plate filled with red cell culture media
By allowing more cell-cell interactions, 3D cell cultures more closely reflect the biological complexities of tissues and organs than traditional 2D cultures. 
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What Is 3D Cell Culture?

3D cell culture is an in vitro technique where scientists grow cells in conditions that allow them to interact with each other and the surrounding environment.1 These conditions are able to closely mimic complex tissues, organs, or tumors.2 

There are several different 3D cell culture types, including scaffold-free spheroids and scaffold-based organoids.3 The combination of 3D cell culture with microfluidics has yielded the organ-on-a-chip model.4 

2D Versus 3D Cell Culture

2D cell culture primarily involves growing cells in a monolayer on the flat surface of a plastic flask or plate.5 These culture conditions lack complexity, affect cell phenotypes, and severely limit the cell-cell interactions that underpin biological processes.6

In contrast, 3D cell culture models are much closer to in vivo conditions because they allow cells to interact with each other as well as the extracellular matrix.7 Additionally, cells in 3D culture are able to functionally differentiate and form layers of various cell types.3 

“3D cell culture is taking cells out of that 2D environment, and [allowing them to have] more interactions with each other. Those interactions can happen in a number of different ways and a number of different environments,” said Julia Crispim da Fontoura, a PhD candidate at the Federal University of Health Sciences of Porto Alegre who has used organoids to study drug resistance. 

In terms of culture conditions, 3D cell culture typically requires specific growth factors and little to no serum in the cell culture medium compared with 2D cell culture, in order to grow primary and differentiating cells.8 Specific culture conditions, such as which growth factors to use and their concentration depend on the cell type.9  

3D Cell Culture Model Types

     Schematic illustrating three examples of 3D cell cultures and their applications: Brain spheroid can model tumor microenvironments; lung organoids can model disease such as COPD; and liver tissue grown on microfluidic chips can be used to test drug toxicity.
3D cell culture types include scaffold-free spheroids, scaffold-based organoids, and microfluidic-based organ on a chip devices. These are useful in disease research applications, such as studying the brain tumor microenvironment, modeling inflammatory conditions like chronic obstructive pulmonary disease (COPD), and investigating drug toxicity in vitro.
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3D spheroid cell culture

Researchers grow spheroids in scaffold-free 3D cell culture conditions that do not provide any surface or structure for cell attachment.5 Cells that cannot attach to a 2D surface will instead attach to each other, aggregating and forming a floating spherical clump known as a spheroid.5 Spheroids are more complex than 2D cell cultures, but less complex than other 3D cell culture models, such as organoids.3

3D cell culture organoids

Cells in scaffold-based 3D cell cultures self-assemble into structural units known as organoids, which resemble miniature organs.3 Scaffold-based 3D cell culture conditions include a substrate or matrix for cells to bind to.5

“Cells growing in [organoid culture] have a structure to hold them on to, but they also tend to have different types of cells within those organoids,” said Crispim da Fontoura. “That gives them a more similar structure to real tissue than a spheroid.”

Microfluidic 3D cell culture: organ on a chip

Microfluidic chips are microchips engraved with a series of channels.10 3D tissues grown on these devices form organs on chips,4 which mimic the structure and function of human tissues.11 Organs on chips can easily be integrated with other engineering techniques, including automation or biosensor technologies.11 Like other 3D cell culture approaches, organs on chips can also be personalized by using patient-specific iPSCs.11

Advantages of 3D Cell Culture

One of the key advantages of 3D cell culture is that it better mimics real biological conditions for studying complex tissues, organs, and diseases in vitro.12 “In 3D cell culture, we are generally seeing cells in an environment that is more similar to what we see in vivo, so we have cells that are organized in a way that is more similar to an organ,” explained Crispim da Fontoura. For example, organoids can be generated for a variety of organs, including brain, liver, lung, kidney, pancreas, retina, thyroid, and stomach, and can accurately recapitulate their gene and protein expression, tissue structures, and cellular interactions of those organs.13

Because 3D cell culture techniques more closely resemble in vivo conditions, they can also provide more accurate indications of how human cells will respond to novel therapies than 2D cell culture or preclinical animal models,2 meaning they can maximize efficiency in drug discovery and reduce the attrition rate of new drugs in clinical development.1 “By using these 3D-cultured cells in these types of assays, you can even help with decreasing mice usage in research,” Crispim da Fontoura added.

Additionally, 3D cell culture models such as organoids and organs on chips can be integrated with high-throughput and high-content screening, as well as computational biology and machine learning.13

3D Cell Culture Applications

One key 3D cell culture application is disease modeling, which recreates complex disease phenotypes that cannot be accurately modeled using 2D cell culture techniques. For example, researchers can generate organs on chips that recapitulate the alveolar-capillary interface in lungs, allowing them to study complex infection and inflammation responses.14

Scientists often use 3D cell culture for cancer research, both to understand the complex tumor microenvironment and to study treatment responses in vitro.15 For example, Crispim da Fontoura uses 3D cell culture techniques in her cancer drug research. She has demonstrated that 3D models have more similar gene expression levels to tumors and are more resistant to chemotherapy drugs than 2D models.5

Researchers are also working with complex tumor organoids to understand the tumor immune microenvironment, as well as to develop and test precision immunotherapies.16 “You can create organoids and co-culture them with immune cells, and then use a therapy to try to get a general idea of how a patient might respond to that therapy,” said Crispim da Fontoura. 

Because responses to immunotherapy are limited by intertumoral heterogeneity and treatment resistance, predicting patient-specific responses is a key goal of precision medicine in cancer.17   Scientists use 3D tumor-derived organoids to screen immunotherapy efficacies, as well as to understand the role of specific cells in tumor progression or remission.17 With further research, 3D cell culture techniques will continue to bridge current gaps between 2D cultures and in vivo models, enabling developments in precision medicine and maximizing efficiency in drug development.3

References

1. Padmalayam I, Suto MJ. Chapter twenty-four - 3D cell cultures: Mimicking in vivo tissues for improved predictability in drug discovery. In: Desai MCBT-AR in MC, ed. Annual Reports in Medicinal Chemistry. Vol 47. Academic Press; 2012:367-378. 

2. Edmondson R, et al. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol. 2014;12(4):207-218. 

3. Gunti S, et al. Organoid and spheroid tumor models: Techniques and applications. Cancers (Basel). 2021;13(4).

4. Fontoura JC, et al. Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance. Mater Sci Eng C. 2020;107:110264. 

5. Leung CM, et al. A guide to the organ-on-a-chip. Nat Rev Methods Prim. 2022;2(1):33. 

6. Park Y, et al. Applications of biomaterials in 3D cell culture and contributions of 3D cell culture to drug development and basic biomedical research. Int J Mol Sci. 2021;22(5). 

7. Ravi M, et al. 3D cell culture systems: Advantages and applications. J Cell Physiol. 2015;230(1):16-26. 

8. Urbischek M, et al. Organoid culture media formulated with growth factors of defined cellular activity. Sci Rep. 2019;9(1):6193.

9. Zhou C, et al. Standardization of organoid culture in cancer research. Cancer Med. 2023;12(13):14375-14386. 

10. Pattanayak P, et al. Microfluidic chips: recent advances, critical strategies in design, applications  and future perspectives. Microfluid Nanofluidics. 2021;25(12):99. 

11. Ma C, et al. Organ-on-a-chip: A new paradigm for drug development. Trends Pharmacol Sci. 2021;42(2):119-133. 

12. Cacciamali A, et al. 3D cell cultures: Evolution of an ancient tool for new applications. Front Physiol. 2022;13:836480. 

13. Lampart FL, et al. Organoids in high-throughput and high-content screenings. Front Chem Eng. 2023;5. 

14. Huh D, et al. Reconstituting organ-level lung functions on a chip. Science. 2010;328(5986):1662-1668. 

15. Stock K, et al. Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery. Sci Rep. 2016;6(1):28951.

16. Sun C-P, et al. Organoid models for precision cancer immunotherapy. Front Immunol. 2022;13:770465. 

17. Magré L, et al. Emerging organoid-immune co-culture models for cancer research: from oncoimmunology to personalized immunotherapies. J Immunother Cancer. 2023;11(5):e006290.