The use of three-dimensional (3D) cell culture in the drug discovery field has accelerated over the past ten years. Running in parallel with advances in 3D cell culture is the growing use of human cells derived from induced pluripotent stem cells (iPSCs). Researchers are interested in testing drugs in the most physiologically relevant models possible, so, it was only a matter of time before these two approaches converged – providing optimized systems for disease modeling and drug toxicity testing.
“It was natural for researchers to say, ‘well, if 3D culture grows cells in the best physiological condition and as a result of that you get the best physiological data, how about using iPSC-derived cells in 3D culture,’” explains Richard Eglen, Vice President and General Manager, Corning Life Sciences.
“The science that really spurred this convergence was the greater adoption of human iPSCs; as a result of that, we now have patient-specific, disease-specific HiPSCs.”
In other words, researchers can now take advantage of the benefits of each technique, providing them with the best models possible, “for everything from disease research, to drug discovery, through, in some cases, to diagnostics,” explains Eglen.
3D cell culture and iPSCs in drug discovery and disease modeling
3D culture is now emerging as a standard practice for both disease modeling and drug discovery. When it comes to determining the effect of a drug, or mimicking human disease, animal models are often inadequate, and there are also ethical implications of using them.
3D culture allows human cells to grow and interact with their surroundings in a three-dimensional artificial environment that more closely resembles the in vivo state.
Human cells are obviously far more physiologically relevant compared to animal derived tissue, and drug toxicity testing is a concrete example that demonstrates the clear benefits of using human cells. “If you can use human iPSC derived liver cells grown in 3D, you can get a much better prediction of what's going to happen in preclinical testing,” says Eglen.
“Animal models don't predict well what's going to happen in the human. You can measure drug metabolism, but human liver cells express different enzymes at different levels to what one sees with animal cells. And of course, as a result, drugs behave differently in animal cells compared to human cells.”
The fact that iPSCs can be made available in virtually unlimited quantities is an additional benefit for drug discovery researchers. Once you have obtained the cells, you can then derive them into unlimited amounts of cells that are highly reproducible.
But, undoubtedly one of the greatest advantages of using 3D cultured human iPSCs is that “you can work with patient-specific cells.” Using patient-specific cells provides you with data on the efficacy and safety of a drug compound in a very specific patient population.
“You can now grow heart cells from patients who have got a particular mutation in some of the proteins in their heart cells, and you can then test compounds in those heart cells,” explains Eglen.
Modeling progressive diseases with 3D cell systems
By working with patient-derived cells in 3D it is now possible to generate models that provide valuable insight into the mechanisms underlying many diseases. According to Eglen, neuroscience is an area that is making major inroads: “Parkinson's disease, Huntington's disease, and Alzheimer’s disease, are all benefiting greatly from brain 3D models based upon cells that are derived from iPSCs.”
Cell cultures in 3D can be long-lived, meaning it is now possible to create models that mimic the progressive changes observed in diseases that develop over time – for example neurodegeneration.
This temporal aspect of cell culture has been described as the “fourth dimension”. This added dimension in cell culture is critical when modeling progressive diseases – helping determine both the acute and long-term effects of exposure to a drug.
“If you think about developing compounds to treat Alzheimer's disease for example, you really want to have drugs that can influence the progression of the disease as opposed to ones that will have an acute effect,” says Eglen.
Using iPSCs – advantages over cell lines derived from traditional sources
Both immortalized and primary cell lines are examples of traditional sources. The main challenge with primary cells is that there is limited abundance, due to being taken directly from a specific patient or from a cadaver-type source.
Immortalized cells, on the other hand, proliferate indefinitely and can, therefore, be cultured over several generations. However, Eglen is quick to point out a separate drawback that should be considered when using immortalized cell lines: “If they're immortalized cells they're just that – they're immortal. And as a result, they are very grossly changed relative to what they were when they were first isolated.”
“Consider the HeLa cell line, originally isolated from Henrietta Lacks – after generations and generations of those cells being immortalized they have changed greatly from when they were first isolated and as a result of that you have to ask the question: ‘how relevant are they now relative to when they were first discovered?’.”
To avoid the issues associated with immortalization, as well as simply the abundance of the cells, researchers are turning to iPSCs.
“You can take iPSCs from multiple sources, such as skin cells, for example, or blood cells. So, they're easily isolated – it really is a breakthrough technology in terms of disease modeling and drug discovery,” explains Eglen.
Fields of research benefiting from these techniques
There are numerous areas of research that are benefiting from the use of iPSCs and 3D cell culture. Here we highlight three examples:
Diabetic retinopathy affects the blood vessel within the retina that lines the back of the eye. In people with diabetes, it is the most common cause of vision loss.
Advances in developmental and stem cell biology have made it possible to successfully differentiate pluripotent stem cells into retinal cells.
The first description of 3D retinal structures derived from PSCs appeared back in 2009, when Meyer et al. showed human embryonic and induced pluripotent stem cells could be used to model retinal development and mimic early retina formation in 3D.
“The ability to grow iPSC-derived retinal cells in 3D is allowing for people to understand the fundamentals of retinal degeneration in diabetes, as well as to think about growing cells to replace the dead cells. And as a result of that, try to replace some of the vision that occurs in diabetic patients.”
Achberger et al. recently discussed the use of stem cell-based retina models in a 2018 paper and highlight the available retina stem cell models and their current applications.
Cell-based therapies are offering new hope for patients who have experienced myocardial infarctions (MI) – also known as heart attacks. MI is caused when the blood flow to the heart becomes blocked. This interruption to the hearts blood supply can cause damage to the heart muscle that can be life-threatening.
Lalit et al. published in 2014 on the use of iPSCs for post-myocardial infarction repair. They highlight numerous preclinical studies of post-MI cell therapy using iPSCs. The iPSCs can be differentiated into the desired cardiac lineage cells, which can then be transplanted into the damaged heart (either injection or tissue engineered cardiac patches).
“After a heart attack, you get a number of cells that have died as a result of the angina attack. The ability to look at what's happening in those cells and replace them, potentially with iPSC-derived myocytes grown in 3D is a rapidly advancing field,” explains Eglen.
In May 2018 Japan’s health ministry granted scientists permission to treat patients with heart disease with cells derived from iPSCs.
Glioblastoma multiforme (GBM) – also called glioblastoma, is a fast-growing type of brain tumor that develops from a glial brain cell. GBM is an aggressive cancer that typically results in death within fifteen months of diagnosis; subsequently there is an unrelenting need to develop novel therapeutics to improve survival.
Whilst many drugs have entered clinical trials, only four are currently approved by the FDA for GBM. This lack of success could be attributed to the fact that the preclinical disease models used do not recapitulate the features of human GBM – meaning they poorly predict the efficacy of the drug once it enters the human stage of clinical testing.
Wilson et al. published a paper last year detailing their generation of “oncospheres” (spheroids cultured in serum-free media with growth factors) generated from a human glioblastoma. These oncosphere (spheroid) models more accurately mimic the genetic and phenotypic characteristics of the original tumor, compared to traditional models of the disease. The researchers were able to demonstrate that the oncosphere cell lines were amenable to high-throughput cell viability drug screening – indicating that they have the potential to help identify lead compounds.
Linkous et al. reported in March 2019, the development of a cerebral organoid glioma (GLICO) model to retroengineer patient-specific GBMs, with an aim to overcome the limitations of current preclinical glioblastoma models. By using patient-derived glioma stem cells and human cerebral organoids the team were able to produce a GLICO model that helps scientists more accurately study GBM biology in a human brain environment.
Identifying and overcoming challenges associated with using iPSCs in 3D
Whilst 3D culture has been around for almost a decade and iPSCs have certainly been known of since John B. Gurdon and Shinya Yamanaka received the Nobel Prize back in 2012, the combination of the two approaches is driving scientists to develop more sophisticated and novel protocols for their cooperative use. In some cases, explains Eglen these protocols “vary from lab to lab, and they're not reduced to routine practice. People are still working out the optimal technical conditions to do this.”
With that in mind, Eglen advises that the field is very much contained in the research laboratories at the moment, and is yet to make it into “mainstream drug discovery”.
“The main challenge is the complexity of developing standardized protocols, which you can then take forward into more routine research. I think it will get there, I just think it reflects the novelty and the fast-growing pace of the field of the moment.”
Richard Eglen was speaking to Laura Elizabeth Lansdowne, Science Writer for Technology Networks.