The high failure rate in drug development is well known, and many drug candidates could be destined to fail from the earliest stages of the process. “Classic tissue culture is failing repeatedly to predict the efficacy and toxicity of new drug compounds because in this setting cells don't function the way they should,” says Henning Mann, Vice President, Science at Nortis, a biotechnology company based in Seattle, Washington. This is especially problematic because human cell-based screens can more accurately represent certain aspects of human physiology than animal testing. “Animals often don't experience the same disease processes that humans do,” says John O’Neill, co-founder and Chief Scientific Officer of Xylyx Bio, a New York City-based biomaterials company.
But don’t blame the cells — it’s how they are being cultured. Cells have evolved to thrive in their specific physiologic niche within the human body. For in vitro culture, cells are procured from the human body and transferred to an artificial, minimalistic environment ill-suited to their needs. However, approaches to cell culture are starting to change. For example, rather than providing all cells with the same generic mix of nutrients, many researchers now tailor their media preparations for specific cell types. Nevertheless, the physical substrate environment in which the cells grow remains relatively neglected. “Traditional tissue culture in 2D dishes and wells is pretty much obsolete at this point,” says Mann. “Those cells just don't get all the cues they need to act as they do in vivo.” O’Neill compares it to how differently a person might behave if plucked out of the comforts of their home and dropped into a bare plastic box. Accordingly, a growing number of researchers are striving to elicit more naturalistic behaviour from cells by providing microenvironments that better recapitulate native tissues.
Structure and function
In the human body, cells dwell within three-dimensional networks of proteins and other macromolecules collectively known as the extracellular matrix (ECM). “This is the biological material that literally holds multi-cellular organisms together,” says O’Neill. With compositions and structures unique to each tissue, the ECM provides essential biochemical and physical support and communicates key signals to cells. “If your cell culture substrate doesn’t have the right mechanical properties, for example, your cells could differentiate into fibroblastic phenotypes, or you could even induce cell death,” says Tommaso Sbrana, CEO of Massarosa, Italy-based biotechnology company IVTech.
Many cell-based systems give only the barest nod to the ECM by culturing cells on a thin coating of one or a few constituent proteins, like collagen type I. Extending the house analogy, O’Neill compares collagen-only substrates to a room where everything is made of bricks — an excellent material for home construction, but not sufficient for a comfortable living room environment. Such conditions may be acceptable for immortalized cell lines, which are genetically modified for continuous proliferation and typically less sensitive to their surroundings than ‘primary’ cells procured directly from tissues. But immortalized cell lines in culture are often a poor reflection of human biology. “It’s important to understand the basics of cell culture when we're talking about producing and screening cells at scale,” says Dmitry Shvartsman, Director of Research & Development at Cellaria Biosciences, a biotechnology company in Wakefield, Massachusetts. “When those basics are overlooked, it results in huge costs and a loss of time and efficiency.”
Those looking beyond the basics often utilize substrates containing multiple ECM components, such as basement membrane extracts like Matrigel and Geltrex, which offer a richer 3D environment and are therefore a popular choice for the cultivation of cells. These products are composed of factors secreted by the cells of a mouse tumour known as the Engelbreth-Holm-Swarm sarcoma. “This tumour produces huge amounts of ECM,” says Shvartsman. “But that doesn't mean the chemical composition of the extracellular matrix is appropriate for a specific tissue, or even relevant for the models you're trying to develop.” In fact, since these products are derived from a rapidly growing tumour, basement membrane extracts risk promoting aberrant growth or behaviour. “If you’re aiming to grow and differentiate stem cells, you don't want exotic, tumour-associated growth factors in the culture environment,” says O’Neill.
“Conventional disease models are lacking in their ability to incorporate multiple complex components within the system,” says Mike Mattie, Associate Director of Translational Sciences at Kite Pharma, a California-based company developing cell therapies for cancer immunotherapy. “Ideally disease models would be as realistic as possible. For instance, in cell therapy development, the ultimate goal is to recreate the tumour environment in the most comprehensive and realistic way. Unfortunately, current solutions are either too limited or too expensive.”
Setting up home
It was a frustrating experience with cell culture that led O’Neill to pursue a more physiologically appropriate cellular environment. As a doctoral student in Gordana Vunjak-Novakovic’s lab at Columbia University Medical Center, he sought to cultivate a subpopulation of kidney stem cells, which remain dormant unless roused by injury or disease. “If cells are removed from their natural home and put into a petri dish, they don't maintain their normal phenotype. This limits the ability to effectively develop and test life-saving drugs,” he says. As a solution to this pressing need, O’Neill and colleagues developed strategies to extract ECM from native tissue, yielding a cell-free, physiologic extracellular matrix that can then be repopulated with fresh cells. The resulting tissue-specific substrates provide scientists studying cells for early stage drug discovery and development with confidence that their systems are more predictive of human physiology.
“Tissue-derived matrices increase the correlation between in vitro models and reality,” says Sbrana. “The structure of this ECM is native, and the mechanical properties are more or less the same as the physiological scenario.” For example, Xylyx and Cellaria recently demonstrated that patient-derived lung cancer cells exhibit markedly different growth rates, drug responses, and gene expression profiles in lung ECM than in Matrigel or on tissue culture plastic. The differential responses of lung cancer cells in lung ECM were found to be significantly more consistent with the characteristics of the primary lung tumour cells than cells cultured in Matrigel or on plastic. In addition, multiple ECM product formats and disease-specific substrates provide scientists with additional options for their cell culture systems.
Performance can also be bolstered in culture systems that reproduce other aspects of human physiology. For example, ‘organ-on-chip’ devices developed by Nortis link together multiple organ models in a dynamic perfusion system that replicates fluid flow. Mann notes that such cultures regain biological activities that are typically lost in static systems and can even recreate the interplay between organs. “We can show the toxic effects of a drug on kidney cells involved with transport, and we can also show drug metabolism in a liver chip connected to that kidney chip,” he says.
Although many cell biologists still favour the culture methods they learned as students, Mann sees change on the horizon. “In a few years, we will be thinking ‘how could we ever have done 2D culture?’” he says. It may be a hard sell at first convincing researchers to shift from entrenched products and existing models and invest in more physiologically relevant cell culture systems, but compelling proof of greater reproducibility and biological fidelity — and thus the potential for greater success rates in clinical testing — will likely shift that equation. Mattie concludes, “The development of physiologically relevant 3D culture systems holds great potential to allow for testing different therapies in a more predictive microenvironment at a lower cost.”
For more information on Xylyx Bio’s cell culture technologies, visit https://xylyxbio.com/.