A Europe-wide collaboration is developing in vitro cell cultures as a more ethical, cost-effective way of testing the safety of manufactured nanoparticles.
Manufactured nanoparticles are a new class of substance, and can be made from almost any solid material. Typical examples include silver particles used as an antibacterial and antifungal agent in water bottles, zinc oxide used to block UVA and UVB sun rays in sunscreen, and the various materials used as pigments in paints and cosmetics. The variety in their chemical compositions, shapes and surface properties means that they can interact with the body in very different ways, and their small size allows them to cross barriers in the body that hold back larger materials.
Researchers and industry are faced with the almost impossible task of analysing the safety of thousands of new materials in a very short time frame. It has become clear that new animal-free testing methods are necessary from both ethical and financial viewpoints. Many of the animal-free cell culture or "in vitro" models currently in use do not represent the human body accurately. The interactions between different organs, or the transport of a material through barriers, for example from the intestine into the bloodstream, cannot yet be modeled in a realistic, or "physiological", way.
The EU-sponsored project InLiveTox (www.inlivetox.eu) has brought together engineers and biologists from universities and research institutes across Europe in order to develop an improved cell culture system. This model will help to find out how nanoparticles taken up in food or accidentally swallowed can make their way out of the intestine and into the bloodstream, how they can affect the different cell types they encounter along the way, and how these cells communicate with each other.
There are some essential requirements for transport of particles and communication between cells to take place. The system needs to involve different types of cells which must be spatially separated, but connected in a way that allows both the particles and the messenger molecules produced by the cells to be transported between them.
Arti Ahluwalia from the University of Pisa is an expert on "fluidic systems", cell culture models which connect different cell types by circulating medium. She describes the first experiments, which dealt with nutrient metabolism in diabetes.
"It became apparent that diabetes could not be studied in single cell types because it is a systemic disease and needs to be represented by more physiological models. The presence of flow firstly increases nutrient turnover and removes metabolic waste products. It provides a mechanical stimulus as the moving fluid imparts shear forces to cells. Finally, the flow connects different cell types together in the system, so promoting cross-talk between cells."
Ahluwalia's group has achieved some major breakthroughs with their fluidics systems. "We have shown that the fluidics system behaves as a mini metabolic system, keeping glucose and lipid levels stable, by linking up liver cells, fatty tissue and cells from blood vessels," she says. "When the system is destabilised by giving it too much glucose and not enough insulin, it shows signs of inflammation and vascular stress, just as we observe in diabetes."
The InLiveTox researchers have modified the Pisa fluidics setup to model the uptake of nanoparticles from the intestine, and their transport to and effects on other tissues. Project manager Martha Liley, from the Centre Suisse d'Electronique et de Microtechnique (CSEM) in Neuchâtel, explains the need for cell culture systems examining different types of cells and their interactions, and how InLiveTox approaches the problem.
"Unfortunately, the in vitro tests that are currently available are too simple to replace most of the testing on animals. Most in vitro tests work with one type of cell in culture, but this is, of course, not the way cells work in our body, where the surrounding environment, including other cell types, has a strong influence on how cells behave.
"In InLiveTox, the tissue models in the system have also been chosen to be relevant to human exposure," Liley continues. "When we eat something, anything we absorb will first pass through the intestinal wall, then into the bloodstream, and it will then be transported to the liver. This is what we are modelling."
In the present configuration three "bioreactors" are used for cell culture. They are small closed chambers with tubes allowing a flow of cell culture medium in and out of them.
The first bioreactor contains the cells that model the intestine. These cells have to be grown outside the chamber on a membrane first, as it takes three weeks for them to reach maturity. When they are ready to be used in experiments, the membrane is transferred to the chamber. It is inserted so that it divides the chamber in a top compartment, which represents the intestine, and a bottom compartment, which represents the bloodstream.
The second and third chambers hold cells from the blood vessel wall and cells derived from the liver. The three bioreactors are connected to each other by tubes, and a peristaltic pump circulates cell culture medium. The complete setup fits on a tray which can be used in a standard cell culture incubator, so the experiment can run at 37°C.
The microporous membrane on which the intestinal cells are grown is an integral part of the setup. It offers support for cells, and contains regular little holes through which nanoparticles and messenger molecules can be transported to reach the bottom compartment of the chamber and from there the other cell types.
The reusable membranes produced by Liley's group in CSEM are especially suited to experiments with nanoparticles. "The problem with commercially available microporous membranes is that they are relatively thick," says Liley. "They work fine with small molecules that diffuse quickly, but there are problems with nanoparticles.
"Many researchers find that even in the absence of cells, they cannot detect any particles crossing the membrane but instead find them sticking to it, so it is impossible to study transport across a model biological barrier. CSEM's membranes are thinner, only half a micron thick, so it is much easier for nanoparticles to diffuse across them."
The project also involves Claus-Michael Lehr's group at the University of Saarbrücken, experts in intestinal cell culture models, and Wolfgang Kreyling's group from the Helmholtz Centre in Munich, experts in biokinetics – the analysis of the distribution of substances throughout a model organism.
With such diverse influences, the consortium had to overcome a number of challenges on the engineering and biology fronts. Ahluwalia, whose group is mainly concerned with the design and adaptations required to make fluidics systems work for a specific problem, explains: "In microfluidics, the biggest problem is the presence of air bubbles due to the high surface area of the fluidic circuit. The other problem is designing a biologist-friendly system. We have discovered that biologists do not like to change from their routine protocols."
Liley describes some other difficulties in making the culture of the three cell types in one system possible and life-like"One major achievement of the project is that conditions have been found that allow all three cell types to be maintained in culture together. As an engineer, I did not realise how difficult this would be when we put together the project. Finding a common culture medium and parameters such as medium flow rate and acceptable levels of shear stress for the different cell types really is an achievement.
"A second success is that we have found a way to carry out measurements of the tightness, and intactness, of the intestinal cell layer in the running system by so-called transepithelial electrical resistance or TEER. This is 'only' engineering, but there were a lot of challenges in terms of electrical contacts, watertight seals, fragile membranes and questions of ease-of-use."
The project has now progressed to the stage where a patent is being filed, and an industrial interest group has been established to examine how end-users could benefit from the InLiveTox system, and how the system could be modified to accommodate a variety of applications.
Malcolm Wilkinson of Kirkstall Ltd in Sheffield, the industry partner in the InLiveTox project, started working with Ahluwalia's group in 2006, when the researchers from Pisa were exploring ways to commercialise their multi-chamber cell culture.
"Kirkstall design, manufacture and sell advanced in vitro cell culture equipment under the Quasi-Vivo® brand name," says Wilkinson. "Our goal is to support cell culture models that more accurately reflect the clinical environment and have full metabolic capacity.
"We have filed three additional patents and created a manufacturing partnership with Parker Hannifin, a world-wide company with a turnover of £10bn. Our technology is now used in more than 12 university labs in Europe and is being evaluated by two leading pharmaceutical companies.
"At the moment there is tremendous interest in improving in vitro cell culture techniques to provide more realistic predictions of toxicity and long-term cell culture for modelling disease," Wilkinson continues. "These models require 3D tissue and barrier models - flow of nutrients is very important. I would see fluidics, or more particularly, Quasi-Vivo® and the developments from the InLiveTox project as essential."
Ahluwalia further explains why advanced in vitro models are essential in areas not limited to toxicity testing, and how InLiveTox can contribute to reducing the need for animal experimentation in the future"Complex diseases, the study of toxic side-effects, multiple pathways that a drug may take as it is distributed in the body, are currently studied using animal models. In many cases animal studies do not reflect the response in humans, as has been demonstrated for several drugs, for example Viox or Rosiglitaxone.
"Animal models are usually healthy and young. Better models, based on fluidics and multiple cell types and simulating diseased states, are going to be crucial for reducing animal tests, saving costs and human lives."
Liley points out the versatility of the InLiveTox system"When substances are added to the system, it can respond – changes in cell function, inflammation or even cell death can be observed as responses to toxicants. While some aspects of the system, such as the membranes, have been chosen especially to make the system suitable for nanoparticles, there is nothing to limit it to nanoparticles.
"So the InLiveTox system could also be used to evaluate the toxicity of chemicals by ingestion, or the pharmacological effects of candidate drugs."