Valid in vitro cell culture models are pivotal to biomedical research and drug development. While most cells exist in a three-dimensional (3D) microenvironment, standard two-dimensional (2D) culture uses a flat, unnaturally stiff surface. This rigid, synthetic structure fails to support normal cell and protein interactions that occur in vivo.

To address the limitations of current 2D cell culture models, there is a growing focus on improving traditional methods of in vitro cell culture. Capturing cell behavior within a 3D structure that includes physiologically relevant microenvironmental signals and components could help better inform mechanism of disease to identify drug targets, predict cell response to stimulus, and characterize tissue architecture for engineering.

3D cell cultures bring in vitro closer to in vivo

Techniques to develop 3D culture have advanced during recent years. The rapid production of reproducible 3D models that are compatible with laboratory-standard microplates is making research with 3D culture more accessible. Different 3D forms are available depending on application and the cells being studied, with the two most broad categories being scaffold-based or scaffold-free.

In the growth of scaffold-based cultures, a structure holds, interacts with, and regulates cells in a way that models a physiological extracellular matrix and enables tissue formation. The scaffold configuration and material are customized based on research needs. Scaffolds can be made of natural or synthetic compounds and vary in porosity, permeability, and mechanical characteristics. The matrix serves as a physical support system and can also provide a source for growth factors, hormones, and other biochemical molecules found in native tissues. Scaffold-based cultures have shown promise in tissue regeneration and have also been used to model tumors.

Scaffold-free cultures include cell aggregates that freely form in media, on plates, or in a bioreactor. Spheroid 3D cultures are an increasingly popular scaffold-free culture model used in research. These clusters include a defined mass of cells with oxygen, nutrient, and metabolite gradients representative of in vivo tissue. Spheroids were initially developed from tumor cells to measure response to radiation and chemotherapy, as their shape can help model the hypoxic core of cancer masses. 3D spheroid models have since been used for a variety of 3D cell culture applications, including toxicity studies, disease modelling, and tissue engineering.

Organoids are similar to spheroids but rely on stem cell self-organization, resulting in a tissue culture with organ-specific cell types and microanatomy. Organoids can be developed with either scaffold-based or scaffold-free techniques. These cultures are currently being used to study organ development, organ-specific disease characteristics, drug toxicities, and tissue formation.

Regardless of the technique, 3D cell culture methods and technologies are designed to model the heterogeneity, morphology, and function of human tissues for the physiologically relevant study of natural cell signaling and response.

A 3D matrix enlightens drug discovery, clinical trials, and patient care

The superiority of 3D culture in allowing cells to better express their original phenotype has been demonstrated in studies relating to both drug safety and efficacy across a variety of cell types. 3D models of primary hepatocytes, for example, have been found to maintain a more functional cell population than 2D cultures. In 2D models, primary hepatocytes quickly lose their differentiated phenotype, cytochrome P450 (CYP450) enzyme expression falls dramatically, and cells no longer produce albumin or urea.1 Studies of hepatocytes in 3D cell culture show that cells cultured in more complex spheroid systems maintain high and stable metabolic activity for up to two weeks.2

As CYP450 and other liver enzymes serve as primary pathways for xenobiotic metabolism, accurately modeling hepatocyte activity in culture is critical to understanding drug dosing and toxicity. Additionally, many drug compounds either inhibit or induce CYP450 enzyme activity, contributing to significant drug interactions. Better in vitro mapping of enzyme activity, drug metabolic pathways, and metabolite effects on organs and tissues leads to a greater understanding of drug tolerance and interactions in vivo.

The enhanced physiological similarity of 3D versus 2D cell culture to in vivo conditions has shown promise in studying and predicting drug efficacy against solid tumors. Patient-derived spheroids have been used to analyze effective HER2-negative breast cancer treatments and generated results matching current clinical guidelines.3 3D scaffold culture of breast cancer cells has also shown distinct growth profiles and drug resistance patterns compared with those seen in monolayer culture.4 These scaffold cultures can serve as a platform for studying mechanisms of drug resistance and, in turn, possible drug targets to combat it.

In medicine, knowing what won’t work can be as important as finding what will. Treatment failure can result in lost time, unnecessary expenses, disease progression, and unwanted side effects with no clinical gain. When exposed to chemotherapies, 3D cell culture models of colon cancer have revealed drug resistance while 2D culture showed the same line of cells to be treatment sensitive.5 This crucial information supports targeted treatments in the era of precision health and stratified medicine.

Combining the benefits of a 3D matrix with cultured induced pluripotent stem cells (iPSCs) is a promising application for disease modelling and broad or patient-specific drug testing. The unlimited source of cells provided from iPSCs as well as the ability to produce patient-specific spheroids of any cell type make 3D iPSC culture a novel platform for research. iPSCs in combination with 3D cell culture provide an especially exciting option for studying differentiated adult human cells that are notoriously difficult to culture, such as cardiomyocytes.

With a more realistic environment supporting natural cell behavior and interactions, 3D cell culture methods have the potential to make clinical and preclinical studies more robust, helping investigators fail fast by identifying therapeutic and toxicity limitations early. Animal testing, while informative, poses ethical issues and fails to offer a fully relevant model for drug performance in humans. An enhanced 3D view of cell behavior can help define disease pathology and identify target populations for which treatments could be effective. More relevant toxicity studies promote a safer introduction of drugs to study cohorts.

Unique challenges in imaging 3D cell culture models

Creating a more relevant environment for cellular interactions is only useful if the more detailed information generated from such models can be captured and analyzed. Biochemical and genomic analyses of 3D cell culture models are relatively straightforward and can use similar protocols as 2D models, but imaging these new structures is more complex.

3D cell culture models are thicker than 2D systems and contain a higher proportion of matrix proteins. These characteristics lead to increased light scattering and blurred images. Historically, there have been challenges generating clear images within 3D cultures using a high-content imager. The gross structure of spheroids was used as an indicator of general treatment effect, but details at the cellular level were left undetected. Attempts to image spheroids using confocal imagers improve data quality, but high-volume image analysis systems are not typically optimized for 3D data analysis.

Another issue with imaging spheroids is that they are discrete, compact structures that occupy only a small part of the well in 96-well plates. For many imaging systems, time and data storage space are wasted imaging parts of the well that do not contain cells. The excess data that is collected adds to the data processing overhead and slows down the throughput of high-content image analysis. In addition, only a portion of the spheroid may be detected within the field of view, making image analysis still more difficult and leading to reduced statistical power.

Realizing 3D cell culture’s potential to accelerate biomedical advances

By creating an in vitro microenvironment representative of a natural extracellular matrix, 3D cell culture can bolster research, preclinical investigations, and clinical studies. With more informative ways to study treatments in the lab, drugs can be made safer, more targeted, and more effective along their journey to market. Technical and logistical challenges remain in developing and imaging 3D cell culture, but novel solutions are making this an increasingly viable option in drug research and development.

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  1. Beckwitt CH, Clark AM, Wheeler S, et al. Liver 'organ on a chip'. Exp Cell Res. 2018;363(1):15–25. doi:10.1016/j.yexcr.2017.12.023
  2. Bell CC, Dankers ACA, Lauschke VM, et al. Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study. Toxicol Sci. 2018;162(2):655–666. doi:10.1093/toxsci/kfx289
  3. Halfter K, Hoffmann O, Ditsch N, et al. Testing chemotherapy efficacy in HER2 negative breast cancer using patient-derived spheroids. J Transl Med. 2016;14(1):112. Published 2016 May 3. doi:10.1186/s12967-016-0855-3
  4. Ding, Y, Liu, W, Yu, W, et al. Three‚Äźdimensional tissue culture model of human breast cancer for the evaluation of multidrug resistance. J Tissue Eng Regen Med. 2018; 12: 1959– 1971.
  5. Karlsson, H., et al. Loss of Cancer Drug Activity in Colon Cancer HCT-116 Cells during Spheroid Formation in a New 3-D Spheroid Cell Culture System. Exp. Cell Res. 2012, 318, 1577–1585. doi:10.1016/j.yexcr.2012.03.026.