2D vs 3D cell culture techniques

Cell culture techniques are ubiquitous in areas of developmental biology, drug discovery, regenerative medicine and protein production. Since the introduction of cell culture techniques, cells have been cultured in two-dimensions, attached to tissue culture plasticware or ECM attachment proteins. Cells in the physiological environment have constant interaction with the extracellular matrix, regulating complex biological functions like cellular migration, apoptosis, transcriptional regulation, and receptor expression1. In-vitro experimental data cannot be translated into clinical trials completely2 when cells are grown in 2D conditions since complicated cellular signals between cells and its matrix cannot be reproduced3. Three-dimensional cell cultures address this challenge and serve as a better model representing in vivo physiological conditions closely. Table 1 indicates the difference between 2D and 3D cell culture systems.

Table 1Cell behavior: 2D vs 3D cell culture conditions

Moreover, several studies have reported a difference in gene and protein expression profiles of the cells grown in 3D environment when compared to its 2D counterpart. Also, expression profiles in 3D culture conditions are thought to be more physiologically relevant than 2D cell culture conditions.

Advantages of 3D cell culture

Cellular events in 3D culture resemble physiological conditions closely and have the following distinct advantages over the 2D culture conditions

  • Stem cells grown in 3D exhibit significantly higher differentiation potential15.
  • Drug safety and efficacy studies are efficient and relatively easier to perform in 3D cultures reducing the time spent in drug discovery by pharmaceutical companies16. Drug-induced hepatotoxicity can be efficiently studied in 3D cell models16.
  • 3D cultures provide better data in the prediction of drug resistance. Alkylating agents demonstrated resistance in a 3D culture that was comparative with in-vivo tumors17.
  • Viral pathogenesis including viral growth, infection and pathogen-host interactions can be studied with reduced hazard levels using 3D models18.

Overview of 3D cell culture techniques

The choice of 3D cell culture technique should depend on several parameters, including the nature of the cells themselves (cell line, primary cell, tissue origin), or the final aim of the study. It’s crucial to evaluate these parameters before choosing the most relevant 3D cell culture technique.

Broadly, 3D cell culture techniques are classified as Scaffold-based or non-scaffold-based techniques.

Scaffold based techniques

In scaffold based techniques cells are grown in presence of a support. 2 major types of support can be used:

  1. Hydrogel-based support: hydrogels are by definition polymer networks extensively swollen with water. Cells can be embedded in these hydrogels or simply coated at the surface. Depending on the nature of the polymer, hydrogels can be classified in to different categories (ECM protein-based hydrogels, natural hydrogels and synthetic hydrogels) with distinct properties.
  2. Polymeric hard material based support: cells are cultivated in presence of fibers or sponge-like structures: cell recover a more physiological shape because they are not plated on a flat surface. Materials used for these supports can be polystyrene (adapted for imaging studies because of its transparency) but also biodegradable tools like polycaprolactone.

    Attributes related to these scaffolds are summarized in the following table:
Table 2Scaffold-based 3D techniques overview with attributes

Suitability: +++ = High; ++ = Medium; + = Low; - = Unsuitable; +/- = varies with scaffold components

Scaffold free techniques

Scaffold-free techniques allow the cells to self-assemble to form non-adherent cell aggregates called spheroids. Spheroids mimic the solid tissues by secreting their own extracellular matrix and displaying differential nutrient availability. Spheroids grown via non-scaffold based techniques are consistent in size and shape and are better in-vitro cellular models for high-throughput screening. Different platforms, from specialized plate to more integrated systems, can be used to generate spheroids: attributes of these techniques are described in the following table.

Application Note: Cancer Stem Cell Proliferation in Human Prostate and Breast Cancer Cell Lines Utilizing a New Defined Serum-Free 3D Spheroid Media.

Protocol Guide: Cancer Stem Cell Tumorsphere Formation Protocol.

Table 3Scaffold-free 3D technique overview with attributes

Suitability : +++ = High; ++ = Medium; + = Low; - = Unsuitable

Conclusion

The evolution of 3D cell culture has the potential to bridge the gap between in vitro and in vivo experiments. The convenience of handling cells in vitro while obtaining results that reflect in-vivo condition and avoiding ethical concerns of animal usage is making 3D cell culture techniques increasingly popular among researchers, but choosing the right system to develop a 3D cell culture model is not a trivial question.

The future will see the emerging of some more complex and advanced technologies like 3D bioprinting, an offshoot of 3D printing, helpful to print both biomaterials and living cells. 3D bioprinting has a wide medical application like skin grafting, which avoids a second wound site, characteristic of the traditional grafting methods. The major components for 3D bioprinting, like bio-inks, scaffold material, and biomaterials, are relatively well known to the scientific world. By configuring the order and position of these components various tissue products can be developed while simulating the physiological environment19. At the moment, the technique is in the early stage but has the potential to evolve as an indispensable tool for drug discovery and toxicity studies.

Materials
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