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European Biopharmaceutical Review

Super Models

One of the biggest drivers for innovation is necessity, and this is nowhere truer than in the pharmaceutical industry, which has come under increasing pressure to overhaul the drug discovery and development process. The challenge is to fix the high rate of attrition, whereby too many promising drug candidates fail at late stages in the process. In 2010, bringing each new molecular entity to market cost approximately $1.8 billion. If failure in clinical trials could be avoided, this figure would drop to $0.4 billion (1). Recent analysis has shown that 70% of Phase 2 and 87% of Phase 3 failures are due to problems with safety and efficacy, which is believed to correlate to the low concordance of animal data with human: the positive human toxicity rate for rodents is just 43% (2,3). Advances in in vitro testing offer one of the most promising solutions to tackle this huge attrition problem. There is much speculation about the potential of organ- or human-on-a-chip technology. This article examines the more fundamental requirements for better – more physiologically relevant and predictive – in vitro models. It points to some of the requirements that must be met before these future visions can become a practical reality.

Animal Models

Until new techniques reach the point where they are demonstrably better than animals, we are unlikely to see any uptake in their use. Animal tests are a regulatory requirement in preclinical testing, and will continue to be so until researchers can demonstrate that in vitro testing is a more accurate and cheaper alternative. Despite ethical arguments, animals do have some advantages as an experimental method: they represent a homeostatic environment prior to the start of dosing and, in theory, provide a systemic model that includes the potential for organ-to-organ interactions. Long-term studies and repeat dosing are also possible. However, there are challenges in deconstructing the mechanisms of toxicity when cell-to-cell signalling is important, as well as in translating from animal to human.

Major Obstacles


If in vitro testing is to replace animal testing, there are a number of problems – scientific and legislative – that must be tackled. The investigations should be physiologically relevant, easy to run and, importantly, deliver reductions in cost and attrition rates by not generating misleading data.

A major obstacle for in vitro methods is to create a physiological and homeostatic environment. In the past, emphasis has been on high-throughput screening in 96- or 384-well plates, which rarely provide long-term viability or a representative cell environment. Most assays were designed to be fast end-point assays as a simple indication of go/no go toxicity and do not take into account that cells should function in a similar fashion to how they behave in vivo.

There are at least three important variables that contribute to this. The first is the choice of cells: many in use today are tumour-derived cell lines (HepG2, Caco-2) or are derived from animals (rat PC12, mouse 3T3) with questionable relevance. Researchers are moving towards primary human cells and great promise is being shown by human-induced pluripotent stem cell-derived mature cell lines, but these can be difficult to culture in a way that maintains or restores function, particularly after cryopreservation (4).

The second variable is the time period over which the cells are cultured, which could be periods of up to 28 or even 90 days, in order to create an appropriate homeostatic environment for drug challenge and follow outcomes for a reasonable amount of time.

The third is to control the hidden variables in the experiment. Temperature and CO2 levels are usually assumed as being stable within a standard incubator, but even in this environment, cells can be subject to mechanical and optical stimulation when removed for visual inspection under a microscope.

Cell Culture Environment

The importance of the cell culture environment should also be emphasised, be it the cell culture dish, gel coating, scaffold material or coverslip. Different surfaces influence cell behaviour and, more importantly, can have a dramatic influence on the availability of the drug with which the cells are being dosed. Cells adapt to culture in a two-dimensional environment by modifying gene expression and remodelling the cytoskeleton, which can lead to irrecoverable changes in cell shape, function and interactions. However, cells cultured in a three-dimensional (3D) environment have improved morphology, adhesion and cell-to-cell signalling (5).

In the human body, cells are surrounded by extra cellular matrix (ECM), and are provided with nutrients and oxygen by perfusion-driven pressure and concentration gradients. Static cell culture in well plates misses this important mechanism, so it is unsurprising that cells can quite rapidly de-differentiate and lose function (6). In addition, there is concern that the cell culture chambers themselves may interfere with experimental outcomes due to non-specific binding of proteins or drugs to plastics. Such effects must be understood, minimised and modelled effectively.

Improving Strategies

The introduction of medium perfusion into a static system represents an improved strategy for better in vitro testing. In the vasculature, a high level of flow stress is essential to maintain healthy endothelial cells and tight junctions between cells, but it is accepted that elsewhere in the body, interstitial flow – established by pressure and concentration gradients introduced via blood flow – is an absolute requirement for correct cell functioning (7). Many cell types – such as hepatocytes – are highly sensitive to flow stress, but the introduction of a correct flow level to cell culture systems has been shown to be beneficial, not only for hepatocytes, but also for endothelial cells and adipose tissue, and osteoblasts with chondrocytes (8-12).

Many simple mechanisms have been designed to move media through systems, from simple rocking plates and syringe pumps, to the more complicated microfluidic and perfusion systems (13-15). In some of these approaches, the variation of media flow velocity and resultant flow stress is enormous: some cells will be in turbulent conditions, others in a stagnant backwater. A perfusion system that allows the researcher to model, predict and control flow reproducibly is an essential ingredient of future advanced in vitro culture systems. Equally important is ease of use, such as making the fl ow system leak-proof and free of air bubbles.

Chamber Size

In theory, the above requirements for better in vitro testing strategies could be met by a variety of flow systems ranging in size from micro to macro scale. Ease of use is one factor that will determine which systems are practical and easily translatable from current techniques. End-point assay sensitivity is also an important consideration in the selection of scale, as the experiment needs to produce enough viable cells for assays, such as Western blots or microscopy. Strangely, the physics of flow does not help the move to smaller dimensions in the way that it did with electronic microchips; small (μm scale) capillaries require high pressures to load in medium or reagents, and are incompatible with gels. Major hazards for future organ-on-achip systems include bubbles and surface tension, but these are simple to overcome when the scale is 10 times larger (16).

Overall size is important in microfl uidic systems – like experimental organ-ona- chip – where the tubing between the chambers dominates the volume and surface area. A group at Harvard Medical School is currently modelling different scale systems, investigating the ways in which drug concentrations vary with the volume and surface area of the cell culture chamber and associated tubes. In response to these challenges, a system has been designed to build complex physiologically relevant models at a scale that can be reduced later, once the key protocols and methods have been established.

3D Models

Introducing flow into a cell culture system is just one way of improving physiological relevance. Another involves the creation of 3D or more tissue-like models. The last decade has seen rapid growth in 3D cell culture techniques, with limited standardisation. The first step was the introduction of non-biological scaffolds, such as Reinnervate’s Alvetex polystyrene scaffold or SpheriTech’s more complex super-macroporous polymer scaffolds. These provide mechanical support and variable porosity, but have no biological function; however, these scaffolds do allow cell growth in a 3D environment and result in better cellto- cell interactions (17).

The development of gels that more appropriately mimic the ECM has encouraged the move from simple gels – like collagen – to more complex gels – such as Matrigel, Matrigen Softwell, Biogelx’s peptide gels and Cellendes’ hydrogels. Importantly, many of these new hydrogels are derived from animal-free materials. It is also possible to generate 3D structures without the use of extracellular scaffolds by using hanging drop systems and spheroids (18). Spheroids have emerged as one of the leading 3D methods; nonetheless, they have limitations such as variable size, complex protocols and sensitivity to vibration.

Another approach is to use tissue slices that can be taken directly from animal or human tissues and maintained in the laboratory. These can be preferable to other methods because they allow complicated cell interactions that exist in vivo, although slices can be diffi cult to culture. In these kinds of systems, flow becomes not just desirable, but essential, because any tissue thicker than 300μm will suffer from necrosis. In contrast, culture in a microperfusion chamber with flow has been shown to improve the viability of thick brain slices (19).

Co-Culture

Once flow has been introduced into the in vitro system, then the move to co-culture and more systemic models becomes much simpler. For example, flow allows for the connection of hepatocytes, adipocytes and endothelial cells in the construction of a diabetes model (20). Although there has been concern that the choice of media to suit multiple cell types would be problematic, collaborators have established protocols that enable the co-culture of a number of different cells, for instance, in the creation of blood brain barrier and cardiovascular models.

Long-Term Analysis


Rather than simple end-point assays that often require termination of the culture, it is essential to monitor properties such as cell health, cell-to-cell interactions and excreted signals during an experiment. Flow systems can simplify the taking of aliquots of extractable conditioned media without terminating the investigation, meaning that long-term trials with multiple time-points are feasible. At the moment, sampling is done manually, but the next stage of development is to integrate automation into the flow systems, ultimately with attachment to an analytical machine for fully automatic analysis.

Microscopy can also provide valuable information about cells, and it is possible to integrate flow systems into live cell imaging platforms to produce informative time lapse images. Taken together, these data will provide pharma companies with information about cell health, signalling and interactions.

Commercial Reality

In vitro cell culture is a rapidly developing field, with lots of innovation and numerous solutions to create more representative and complex systems. Those under development are often restricted to niche uses in academia, but if the methods are to be transferred into pharma companies for use in drug development, they must become robust, repeatable, and able to be run by technicians or robots. The major challenge facing the adoption of in vitro testing as a gold standard in drug development is the current dominance of animal testing in toxicity studies. The animal testing market is thought to be worth over $9 billion per year. If even a small percentage of this spending was fed into developing advanced in vitro methods, then ‘human-on-a-chip’ technology might become a commercial reality, much faster than the 10 or so years currently projected.

References

1. Paul SM et al, How to improve R&D productivity: The pharmaceutical industry’s grand challenge, Nat Rev Drug Discov (9): pp203-214, 2010
2. Khanna I, Drug discovery in pharmaceutical industry: Productivity challenges and trends, Drug Discov Today (17): pp1,088-1,102, 2012
3. Olson H et al, Concordance of the toxicity of pharmaceuticals in humans and in animals, Regul Toxicol Pharmaco (32): pp56-67, 2000
4. Medine CN et al, Developing high-fidelity hepatotoxicity models from pluripotent stem cells, Stem Cells Transl Med (2): pp505-509, 2013
5. Zhang S, Beyond the petri dish, Nat Biotechnol (22): pp151-152, 2004
6. Baker BM and Chen CS, Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues, J Cell Sci (125): pp3,015-3,024, 2012
7. Swartz MA and Fleury ME, Interstitial flow and its effects in soft tissues, Annu Rev Biomed Eng (9): pp229-256, 2007
8. Tilles AW, Baskaran H, Roy P, Yarmush ML amd Toner M, Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor, Biotechnol Bioeng (73): pp379-389, 2001
9. Vinci B et al, Modular bioreactor for primary human hepatocyte culture: Medium flow stimulates expression and activity of detoxification genes, Biotechnol J (6): pp554-564, 2011
10. Chao P, Maguire T, Novik E, Cheng KC and Yarmush ML, Evaluation of a microfluidic based cell culture platform with primary human hepatocytes for the prediction of hepatic clearance in human, Biochem Pharmacol (78): pp625-632, 2009
11. Vinci B et al, An in vitro model of glucose and lipid metabolism in a multicompartmental bioreactor, Biotechnol J (7): pp117-126, 2012
12. Kuiper NJ, Wang QG and Cartmell SH, A perfusion co-culture bioreactor for osteochondral tissue engineered plugs, J Biomater Tissue Eng (4): pp162- 171, 2014
13. Weltin A et al, Cell culture monitoring for drug screening and cancer research: A transparent, microfluidic, multi-sensor microsystem, Lab Chip (14): pp138-146, 2014
14. Nichols JE, Niles JA, Vega SP and Cortiella J, Novel in vitro respiratory models to study lung development, physiology, pathology and toxicology, Stem Cell Res Ther (4) Suppl 1, S7, 2013
15. Kim D, Wu X, Young AT and Haynes CL, Micro fluidics-based in vivo mimetic systems for the study of cellular biology, Acc Chem Res, pp1,165- 1,173, 2014 16. Mattei G, Giusti S and Ahluwalia A, Design criteria for generating physiologically relevant in vitro models in bioreactors, Processes (2): pp548-569, 2014
17. Vallés G et al, Topographical cues regulate the crosstalk between MSCs and macrophages, Biomaterials (37): pp124-133, 2015
18. Kratschmar DV, Messner S, Moritz W and Odermatt A, Characterization of a rat multi-cell type 3D-liver microtissue system, J Tissue Sci Eng (4), 2013
19. Rambani K, Vukasinovic J, Glezer A and Potter SM, Culturing thick brain slices: An interstitial 3D microperfusion system for enhanced viability, J Neurosci Methods (180): pp243-254, 2009
20. Iori E et al, Glucose and fatty acid metabolism in a 3 tissue in-vitro model challenged with normo- and hyperglycaemia, PLoS One (7): pp1-9, 2012

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Dr J Malcolm Wilkinson is the Chief Executive Offi cer of Kirkstall Ltd. He has a BA from Oxford University, an MSc from Southampton and did his PhD research at Middlesex University, all in the UK. Malcolm has been a visiting Lecturer for the Swiss Foundation for Research in Microtechnology on micro and nanotechnology in biomedical engineering for over 10 years, and has co-authored several papers and a book on in vitro models of toxicity. He is a champion for the use of cutting-edge technology to replace animal testing for the development of safe drugs, nutraceuticals, chemicals and cosmetics.

Dr Kelly Davidge is Research and Development Manager at Kirkstall Ltd. She completed her BSc in Microbiology at Cardiff University and her PhD studies at the University of Sheffi eld, both in the UK. Following on from post-doctoral research posts at the universities of Sheffi eld and Nottingham, Kelly joined Kirkstall in January 2015, where she is currently working with academia and industry to develop alternatives to animal testing through the innovative application of in vitro technology.
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