Take a fish out of water and it will die, so the old adage goes. And if it doesn’t die immediately, then it will suffer acute metabolic and respiratory changes that completely distort its biology.
The same is true of the body’s cells, says Brian Feth, CEO of Californian, US, biotech Xcell Biosciences: displace cells from their natural microenvironment and they will be very different than before. To grow cells successfully in the lab, you need to create the right ecosystem that reflects their environment in vivo.
‘If you want to culture coral reef fish, you need to adjust the water flow and temperature and oxygen concentration in your fish tank, remove any ammonia that builds up and so on, Feth says. ‘Just like fish, different cells each have their own specific requirements. You have to build the right ecosystem,’’
Biologists routinely culture trained cell lines in laboratories around the world, for applications from drug screening and toxicology studies to fundamental biology investigations. However, as a white paper on the Xcell website (http://www.xcellbio.com/home/) points out, traditional laboratory incubators are based on decades-old technology that typically maintain cells at 37°C with 5% CO2 and high humidity – conditions that bear little if any resemblance to their native microenvironment.
It’s a ‘one size fits all’ approach that seriously compromises research results, says Xcell chief scientific officer James Lim: ‘Recent research, for example, has shown that growing cells in traditional CO2 incubators dramatically alters their gene and protein expression, ability to divide into new cells, and other important factors like metabolic processes,’ Lim points out. ‘The results generated from these studies do not accurately reflect the natural biology of cells.’
Scientists have recognised the importance of the microenvironment for cell biology for decades. While the DNA content of every cell is the same, what makes a brain cell different from a skin cell is down to differences in RNA and proteins – dictated by changes in the environment that control which genes are switched on (expressed) or off.
Even subtle variations in cell culture conditions, such as growing cells in low oxygen conditions or different pressures – or in 3D rather than on conventional 2D scaffolds, can have profound effects, Lim says – changing the expression patterns of hundreds of genes. Such differences could explain, for example, the difficulty in controlling stem cell differentiation, disparities in tumour cell signalling and the frequent inability to reproduce published biological research results.
Xcell’s new Avatar cell culture system is the biologists’ equivalent of the aquarists’ specialised fish tank. ‘It’s the first cell culture system capable of reproducing physiologically relevant conditions such as hydrostatic pressure and oxygen levels to reflect the range of in vivo environments specific to a particular sample type,’ Feth says. ‘Samples cultured under these fully customised conditions exhibit near native morphology and gene expression, making the results truly biologically relevant.’
Controls for monitoring and regulating oxygen levels, and pressure sensors – adapted from those used in deep sea diving – are a particularly novel feature. In the human body, circulating blood cells, for example, experience a pressure of roughly two pounds per square inch, roughly ten times that seen in tissues and organs, Lim elaborates, while oxygen levels drop from around 10% in the circulatory system to a mere 0.1% to 1% in the core of a cancer tumour.
In a recent study using Avatar for stem cell culture, Xcell researchers found that keeping the cells at low oxygen tension and high pressure maintains them in their undifferentiated state – before they begin to develop (differentiate) into different types of body cells. Further work is now under way, Lim says, to deploy the platform to determine the necessary conditions to ‘manipulate lineage commitment’ to decide stem cell fate.
Such carefully cultured stem cells could lead to the creation of lab grown tissues and organs, avoiding the need for organ donation, as well as yielding important information about gene expression changes in new microenvironments. In the future, Xcell researchers speculate it might even be possible to adjust culture settings to reflect the disease environment of ‘terminally differentiated’ cells – potentially revealing new targets for treatment.
‘The thing that excites me is that one day instead of taking a drug pill, we might be injecting our own, reprogrammed, engineered cells to treat disease,’ Lim enthuses.
Despite its futuristic sounding title, however, the platform name has nothing to do with the film. ‘Avatar is a Hindu word for a manifestation of a deity or released soul in bodily form on earth, which has a modern computer embodiment as a game character that represents the human player,’ Feth says. ‘We found this to be a very accurate depiction of our system in that we create a version of the person for R&D testing that is not truly the person, but rather a virtual but accurate depiction of that individual.’
Xcell itself was formed as a spinout from the University of California, Berkeley, US, in 2012, supported by funding from professors including ‘business guru’ Steve Blank and other founding employees. The company was one of the first to receive funding in 2014 from genetic analysis company Illumina’s ‘Accelerator program’, aimed at promoting technologies that use next generation sequencing. It raised a further $5m in 2015 from institutional investors based out of silicon valley.
Much of the early work centred on cancer diagnosis and therapy, which remains a key focus area as it’s ‘deeply personal’ affecting many of the company’s employees, Feth says. An ongoing project with researchers from the University of California, San Francisco (UCSF), for example, seeks to investigate what happens when cancer cells metastasise or spread and migrate to different parts of the body – a ‘Black Box’ phenomenon that he says has ‘eluded understanding and treatment for decades’.
Using the Avatar platform, the researchers isolated rare circulating tumour cells (CTCs) from patients’ blood and grew them for up for several months in the laboratory. They then studied and characterised their gene expression and metabolic profiles – something that would have been impossible with conventional protocols and CO2 incubators.
‘Many of our patients have had their prostates removed 20 years ago but only recently have seen their cancer come back,’ says Lim. ‘Being able to characterise different cancer subtypes in blood means we can get access to what’s going on without needing a surgical biopsy.’ And that, in turn, should lead to better diagnostics and treatments tailored to an individual patient’s condition.
Manipulating the microenvironments in which cells are grown, meanwhile, is also helping the team to learn more about the specific conditions most conducive to tumour growth. A bit like seeds in nature, Feth says, cancer cells will only start to proliferate once they locate an area with the right conditions for growth. So what is it, for example, about the environment in bone marrow that makes cells frequently opt to take root and grow here as opposed to somewhere else?
Lim credits much of early work to understand the balance of oxygen and pressure found in different tumour types to Gregg Semenza at Johns Hopkins University and Rakesh Jain at Harvard. The technology behind Avatar, he says, represents the culmination of years of hard work by researchers, including Xcell scientists, to understand the detailed cellular environments in which cells live, and then try to recreate those conditions ex vivo.
‘Modulating the environment using the Avatar platform is super easy,’ Feth says. ‘The instrument is designed so a person who’s never done primary cell culture could literally pick up the protocol and reagents and start culturing samples on the same day.’
The company targets its applications to academic labs and pharma companies, with the option to purchase ‘pods’ of modular Avatars to suit their requirements. Pharma companies, for example, often require larger cell volumes for drug screening or toxicology purposes.
‘Now that we’ve established proof of principle and we have a great dataset, there’s the opportunity to go to a larger scale,’ Feth says.
Commercial bioreactors are another obvious target, where optimising culture conditions could potentially improve the efficiency of these mammalian cell factories – boosting yields of lucrative biologic drugs and cell-based therapies.
Like the aquarist’s fish tank, Feth says, the Avatar platform is a continuously evolving ecosystem. Swimming around in the native microenvironments of cells and getting more data from customers promises to make it even more powerful.