Harnessing 3D Cell Cultures for Drug Discovery and Characterization

The drug discovery and characterization field has seen vast improvements in complexity, adaptation to human models, and accuracy as cell model options have become increasingly better suited to research. While 2D cell models have been used for research since the early 1900s, they have continued to be adapted, transformed, and altered to be used in the lab as accurate models of human disease and treatment response. 

However, 2D models have a number of drawbacks because they:

• May be derived from a non-human species
• Can be transformed cancer cells, which behave differently from normal human cells
• Are a limited cell model, rather than a dynamic system, changing the resultant response upon exposure to compounds or pathogens

Thanks to the development of 3D cell cultures, the field is on the cusp of a launch into the twenty-first century, opening new horizons for drug discovery and characterization. 

The functionality of 3D cell cultures

Three-dimensional cell cultures are superior to 2D models primarily because of their complexity, making them much more applicable to a human system. In the human therapeutic research and development field, having a model that is a complete system with the most similar response to a human system to a proposed therapeutic is ideal. For example, simply treating transformed cells with a therapeutic cannot tell researchers how the compound would interact with in the complete human system it is eventually intended for. This is where 3D models can help because they:

• Are a system designed of human cells rather than an animal or transformed model
• Can be designed to contain a complete human system, such as a reproductive tract, intestinal system, or respiratory system
• Can include complete immune system responses in real time
• Are still a cell system, meaning the ethical and financial burdens of live animal experimentation do not apply 

There are a number of applications in drug discovery and development that are beginning to take advantage of 3D cell cultures, including:

• Monitoring drug adsorption and absorption
• Evaluating how a therapeutic is metabolized 
• Observing the immune response to therapeutics
• Transportation of therapeutic metabolites in a human system 

A success story in drug development 3D cell modeling

With all of these considerations and hurdles to cross when approaching drug development using 3D cell models, comfort can be found in the success stories. There are scientists that are successfully using this approach in their therapeutic development studies, such as Josip Blonder, MD, a scientist emeritus at Frederick National Laboratory. He recently published his work in Oncotarget, describing the many differences between 3D and 2D proteomic responses and validating the use of 3D models in research. Indeed, Blonder’s research suggests that 3D models are a promising way to bridge the gap between current cell culture and animal research and may even replace animal models in preclinical drug development.

In an exclusive interview with Lab Manager, Blonder commented on the positive impact of 3D models in his work. “In tumor tissue and/or normal healthy tissue cells grow in 3D microenvironment. Therefore, it is reasonable to expect that the proteome/phenotype of 3D cultured cells mimic more faithfully the proteome/phenotype of cells growing naturally in their 3D environment than the proteome/phenotype of 2D cultured cells. For this reason, 3D models represent a better preclinical testing option. 

However, there are also proteins whose expression is not affected by a particular growth condition. Hence, effective drugs discovered using 2D cultured cells certainly target proteins whose expression level is unaffected by the cell culture condition. Significantly, our proteomic approach can reveal these proteins at the high confidence level too. Consequently, I am convinced that all essential cancer cell lines used as preclinical cancer testing models should be grown in 3D culture and analyzed using the approach we described in our recent publication." 

Blonder continues: "3D culture is technically demanding and more time consuming than 2D culture ones currently. Importantly, 3D culture procedures are not yet standardized. However, these obstacles can be easily overcome by automatization of 3D cell culture and/or introduction of the 3D bioprinting in 3D cell culture systems.”

Blonder's work has largely been used to develop improved means of growing 3D matrices of cancer cells to be used in preliminary oncological therapeutic research. Blonder’s group has been using a 3D scaffold to build up NCl-H23 cells (a standard lung cancer cell line) to simulate a more realistic cellular environment because it includes cell-ECM interactions, among other features more similar to a living system, compared to 2D cell models. 

Dr. Blonder’s proteomics work has applied 3D cell cultures successfully to proteomics research. “We uncovered a subset of 1,166 (i.e., 24.4percent) of proteins to be regulated in culture dependent manner in NSCLC model cell line NCI-H23 harboring homozygous KRAS4BG12C mutant. We revealed phenotypical changes induced by 3D culture consistent with tumor stroma-like transformation and metabolic shifting towards adipogenesis/lipogenesis. These finding are coherent with hypoxic conditions taking place in 3D culture and in human tumors in vivo. Finally, we confirmed and cross-validated exclusive expression of CD99, CD146 and CD239 at the surface of 3D-grown NCI-H23 cells. Importantly, we showed for the first-time successful targeting of isoform-specific KRAS peptides using solution-based and antibody-free bottom-up MS-based proteomics, showing that 3D culture induces upregulation of both KRAS4BWT and KRAS4BG12C mutant allele in NCI-H23 cells, compared to 2D cultured ones.”

The future of 3D models for drug discovery and characterization

Because 3D scaffolds and the resultant generation of 3D models remain a relatively new technology for life sciences, there are some shortcomings in utilizing this tool. Applying 3D models to screening pipelines for novel therapeutics may speed up the pipeline of preliminary work that must be performed when finding new treatments for conditions. Because 3D models more accurately resemble the living system therapeutics will ultimately be used in, researchers hope that the use of 3D models instead of 2D cell models will reduce the false drug discovery hits. These false positive hits can be present in 2D models but then disappear when translated to more complex systems, and represent a large area of resource loss for drug discovery research groups. 

Many in the drug discovery and characterization research field hope that as bioprinting continues to improve, more long-lived and accurate models are generated. Ensuring cells can survive and thrive in an artificial environment is paramount to successful (and financially feasible) research. The effectiveness and efficiency of 3D models is improving day by day, and when these models also become more feasible and long-lived in a laboratory setting, the amount of work that can be done with them will be maximized. 

Dr. Blonder told Lab Manager: “The future is bright since big pharma is actively looking for cell culture that can truly reproduce the natural proliferation, migration, and/or drug permeation taking place in their innate 3D environment. I expect that in the future, 3D-based cell model drug discovery and characterization will be fully automatized. Our results underscore the importance and capability of direct proteomic analyses to identify/quantify deferentially expressed cell surface proteins in drug discovery and characterization, due to the limitation of genomics to provide explicit information about cell surface protein expression level, actual post-translational modification status and/or explicit subcellular location of a given target. I foresee further development of 3D culture techniques relaying on 3D bioprinting 3D coculture, and organoids couple with state-of-the-art MS-based proteomics for better understanding of biology and toxicology taking place in drug discovery and characterization research.”