Two-dimensional (2D) cell cultures have played a role in the drug discovery and development process for decades, however in recent years three-dimensional (3D) cell culture has taken center stage, due to its ability to mimic the in vivo state much more closely. Here we take a look at the evolution of cell culture, the rise of 3D, and how the industry’s shift from “static” to “fluidic” is inspiring the next generation of in vitro models – "plug-and-play" microphysiological systems.
“Applications of primary cell culture started in the 1980s. For those working in the field, you could say cell culture was more of a “curiosity” than a robust and reproducible technique for testing drug compounds,” explains Prof. Armin Wolf, PhD, Chief Scientific Officer at InSphero, “Most of the original applications came from basic research. At that time there was no scientific consensus nor confidence within the pharma companies as to whether the generated data would add value to the drug companies’ objectives.”
As Wolf explains, initially there was a clear lack of standard operating procedures available to drug discovery researchers, however the fact that it was possible to grow and test cells in culture was a huge milestone. With the advent of cell culture techniques, it was now possible to monitor cell growth, differentiation and response to various stimuli – including drug compounds. “One important milestone in the application of cell culture for drug development was achieved when we figured out how to grow cells from a living organ in a dish, so that the cells not only survived, but you could also treat them,” says Wolf.
Whilst it was possible to obtain some meaningful and translatable information from these early monolayer (2D) cultures, expectations of what these systems could deliver were much higher than the reality of what they were actually capable of delivering, cautions Wolf: “Over time people began to understand that a cell in a dish – even if it is surviving – doesn’t help much when it comes to making predictions about what would happen in an in vivo environment."
“We quickly learned 2D cell culture had limitations.”
Advances in cell culture
The origin of cell culture is generally linked to Ross Harrison, who developed a technique for studying neural tissue growth in vitro in 1907 – more than 100 years ago. By the mid-1950s both the “L” cell line (Earle, 1948) and human HeLa cell line (Gey, 1951) had been established – propelling cell culture capabilities forward.
It is indisputable that monolayer cultures were fundamental for advancing cellular research, Wolf explains: “When you grow cells in a dish, however, they may survive, but lose their original phenotype, morphology, and specific function.”
“A 2D environment is not able to support the complex cell-cell interactions and specific needs of the cells, meaning they are unable to maintain their correct function.”
“Hepatocytes, for instance, constitute ~80% of the liver cell population and play a critical role in the cellular metabolism of protein, carbohydrate, lipids and bile, and in the detoxification of xenobiotics. In native liver, hepatocytes have a distinct structural polarity that is required for active bile acid uptake and secretion into bile canaliculi. The cytochrome P50 activity must be high to maintain drug metabolism, However, most of the structural and functional properties of hepatocytes disappear after 24 hours in 2D cell culture.”
“They lose their specific hepatocellular functions, such as the ability to metabolize xenobiotics. After ~24 hours, they rearrange in such a way that they look more like fibroblasts –due to the cells attaching to the plastic.” The 2D cultured cells that adhere to the plastic adopt unreliable behaviors due to them being unable to maintain their differentiated state.
Wolf touches on the development of the collagen sandwich configuration and its impact on hepatocyte differentiation and function: “We then learned how to put hepatocytes in a collagen hydrogel “sandwich” configuration, in which hepatocytes retained most of their cellular functions over a longer period of time. This sandwich model was a step forward for drug development, but it still had limitations. We could not create stable 3D co-culture structures of hepatocytes with other cell types involved in core liver functions, namely Kupffer cells, endothelial cells, and stellate cells (collectively known as non-parenchymal cells or NPCs). It was difficult to handle in the extracellular matrix environment due to unspecific binding of drugs—and it was expensive due the sheer number of NPCs required to achieve liver-life organotypic cell distributions in vitro.”
“The next big milestone in the field was the application of hanging drop technology for hepatocytes,” explains Wolf. This method allows for the placement of cells in a drop, meaning they have no interaction with the plastic, preventing attachment to it. The cells can reassemble and aggregate into tightly packed “tissue-like” spheroids via the gravitational force in the fluidic environment of the drop, which allows the cells to maintain a more in vivo-like state. “With hanging drop, we could integrate NPCs into the liver spheroids in a co-culture. Overall, this approach was also more cost effective than sandwich culture.”
Wolf highlights that phenotypic and morphological changes previously observed in the 2D cultured cells that adhered to plastic did not occur using this special fluidic gravitation condition: “In the hanging drop environment the cells only established contact with themselves.”
3D spheroid formation of cells in hanging drops. Credit: InSphero
“In general, this type of [hanging drop] cell culture turned out to be superior to any type of 2D culture, primarily because it allowed us to successfully co-culture hepatocytes with NPCs.” says Wolf. Kupffer cells are important for immune processes that occur in the liver and stellate cells for the fibrogenic processes which can take place in the livers too under certain pathophysiological conditions.
In vitro cell models are frequently used to determine the “stop/go” decisions made by drug discovery researchers early in the development process. More specifically, they are used to ascertain basic safety by evaluating toxicity and pharmacodynamics of the drug compound.
The development of 3D cell culture has highlighted a number of shortcomings associated with 2D culture, for example, differences in cell morphology, receptor expression, and architecture, and a lack of cell–cell and cell–extracellular matrix signaling, compared to that which is observed “naturally” in vivo.
“3D cultures are better at predicting not only toxicity, but also efficacy of treatment,” explains Wolf – due to them more accurately displaying the phenotype of cells within target tissues.
“It is clear to see that now we have entered a new era of cell culture – 3D – which is more functional and more morphologically comparable to in vivo systems.”
Microfluidics – Turning 3D culture into physiological systems
Perhaps the most exciting development in 3D cell culture today is the application of microfluidic technology, which enables interaction between multiple tissue types and organ systems, and allows you to image and analyze chemical, biological, and physical processes in extremely small samples of fluid. Its ability to miniaturize and optimize culturing conditions makes this technology an ideal tool for drug discovery testing.
- Small diffusion distances
- Fast reaction times
- High-throughput through miniaturization
- Only small amounts of drug compound required for testing (beneficial for preserving precious/costly samples)
Body-on-a-chip – Linking multiple microtissues
Advances in microfluidic technology soon led to the development of “organ-on-a-chip” systems that recreate the mechanics and physiological responses of human organs. This development subsequently fueled interest in “body-on-a-chip” in vitro solutions – designed to simulate in vivo multi-tissue interactions – helping experts form a broader and more comprehensive understanding of how a drug compound works throughout the body.
“The systems we were talking about before were static systems, the culture plate, the sandwich culture, spheroids – the next generation of in vitro model is the microphysiological system (MPS) – this type of system allows you to culture even longer and enhances the capabilities of the microtissues,” explains Wolf.
Wolf touches on the basic principles of “body-on-a-chip” technology: “An MPS system consists of “connected” wells, and within each well you can put a microtissue from a particular organ of interest.”
“You can design MPS networks to answer specific scientific questions. For example, you could put liver microtissue in one well in a row of pancreatic islet microtissues to create a miniature metabolic system. Then use this system to investigate how insulin secreted from islets influences the longevity of liver cells, when stimulated by glucose.” The key benefit of MPS says Wolf is that the system can be designed and adjusted to test the types of hypotheses that typically arise during drug development.
“This is an ideal platform where we have media flow from one well to another. It allows you to mimic the crosstalk between specific organs in the body – it mimics the body in a way that enables us to observe the direct effects of a drug compound in a particular organ as well as its indirect effects on neighboring organs,” concludes Wolf.
Prof. Armin Wolf, PhD is the Chief Scientific Officer of InSphero, overseeing the company’s 3D-cell-based organ-on-a-chip, metabolic disease, oncology, and toxicology programs. Wolf was speaking to Laura Elizabeth Lansdowne, Science Writer for Technology Networks.