Introduction: Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment.
Methods: 3D cell Culture
In previous years, three dimensional (3D) cell culture technology has become a focus of research in tumor cell biology, using a variety of methods and materials to mimic the in vivo microenvironment of cultured tumor cells ex vivo. These 3D tumor cells have demonstrated numerous different characteristics compared with traditional 2D culture. 3D cell culture provides a useful platform for further identifying the biological characteristics of tumor cells, particularly in the drug sensitivity area of the key points of translational medicine. A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. It promises to be a bridge between traditional 2D culture and animal experiments, and is of great importance for further research in the field of tumor biology. Agitation-based culture systems are commonly proposed as an alternative method for 3D cell culture.
Results: Hanging Drop Culture System
There are several 3D cell culture methods available for assessment of drug metabolism and adverse/ toxic effects for industrial drug discovery, e.g agitation-based approaches, matrices and scaffolds, hanging drop and microfluidic cell culture platforms. Most of them are expensive and require specialized equipment, whereas, hanging drop method is relatively simple, inexpensive and has been reported to have 100% reproducibility for producing 3D cell per drop for different cell lines. The hanging drop method also offers advantages over other 3D culture approaches, as cells are not in contact with an artificial matrix, supporting the maintenance of in vivo–like cell morphology and behavior. The hanging drop culture forms tissue-like cellular aggregates, which provide optimum conditions for the measurement of biomechanical properties, as well as allowing for molecular and biochemical analysis in a physiologically relevant model. Hanging drop is a drop of liquid suspended from a cover glass usually placed over the cavity of a depression slide and containing cells.
Conclusion: Studies have shown that culturing cells in a 3D context produces distinct cellular morphology and signaling when compared to a rigid 2D culture system. Hanging drop method here requires no specialized equipment or reagents and is therefore highly cost-effective. The simplicity of the method minimizes potential pitfalls. Consequently, the learning curve is relatively shallow and the method can be mastered easily within a relatively short period of time. However, due to the short duration of tubular viability with the conventional protocol, the application of this method to study tubular function is limited. Future studies are required to improve the culture medium which is capable of maintaining long term viability for many types of cells, such as by adding extracellular components and growth factors in the culture medium.