2018

14 projects receive grants in 2018

A total of 2,3 million SEK has been awarded in grants from the Swedish Fund for Research without Animal Experiments in 2018. It was divided between 14 projects. See recipients and project titles below.

Martin Andersson, RISE Research Institutes of Sweden
Eye-irritation, prediction, animal-free method, cornea, Hansen parameters, polymers, threads
Each fluid and polymer has three Hansen solubility parameters that can be plotted graphically in a threedimensional space called the Hansen space. Substances that are close to each other in this space are soluble in each other, and a liquid that is close to a polymer will be able to dissolve, penetrate or swell it. Our hypothesis was that substances that are highly eye-irritating but not alkaline/acidic/oxidizing/ reactive etc must irritate through penetration through the cornea and in the previous project we have been able to show that this hypothesis is correct. In the 2017-project we used a database of eye-irritation data from historical animal tests based on data recommended for validation of new methods and found a well-defined void in Hansen space where NONE of the 62 non-irritating (NoCat, CO=0) liquids in the dataset are positioned. At the same time, all four very strong (Cat1) irritants in the database that are not acidic/alkaline/reactive etc (4 substances) were found inside the void, and by expanding the radius of the void somewhat it could also encompass the two weaker irritants (Cat2A, ”Hansen irritant”-substances) that was found in the database.
We have also worked on an in-vitro method based on elongation of polymers when dipped in the liquid to be tested. The method needs to be refined and experimentally validated, but if information from three different polymers are combined the method should be able to predict the four strong irritants in the validation-dataset (will miss the less irritating Cat2A substances), but should incorrectly predict 15% of the non-irritating substances to be irritants. We now want to move on and further develop the method aiming at developing an in vitro test that can accurately predict if an unknown fluid, or complex formulation (e.g. cosmetic product), is eye-irritating or not. We need to find more suitable polymeric materials and evaluate these in both pure liquids and complex formulations.

Gunnar Cedersund, Linköping university
Knowledge-driven drug development without animal experiments
In the last few years, mathematical modelling has displayed an unprecedented impact on drug development. For instance, recent specific approvals, and even more recent general guidelines, imply that computer simulations now can be used for regulatory approvals. Since model simulations are cheaper and faster, such approvals typically end the use of test animals for that application. We have modified such an approved type 1 diabetes model to also describe type 2 diabetes, a much more widespread and rapidly growing disease. Our award winning model has been developed through numerous experimental/modelling iterations, and is already at use in several major pharmaceutical companies. However, this usage is still mostly done in traditional drug development pipelines, involving a series of cell and animal systems, which often display highly different outcomes. To allow us to move to a radically new, knowledge-driven, more efficient, and increasingly human-centered pipeline, I will use uniquely informative data to do the four most important additions still needed in the model: i) storage and release of fatty acids, ii) specific drug-targets in heart and adipose tissue, iii) multi-level and long-term translations, iv) mapping from other animal-free systems. For these groundbreaking developments, the Swedish Fund for Research without Animal Experiments awarded me the first edition of their award – ”Nytänkaren” – and I am an active participant in the steering group for the new government-initiated National Centre for 3R. Together with this new and unprecedented platform, we can put my research into the bigger context, where a major impact on both refinement, reduction, and replacement of animal experiments can be achieved at last.

Pernilla Eliasson, Linköping university
How do tendons heal? – and what happens when they don´t heal?
The ethology behind tendon pathology is multifactorial, however different drugs might influence this. Tendon injuries are common and require a long rehabilitation time. Mechanical loading improves healing, but it is unclear how. I have introduced a model for 3D cell culture studies of human primary tendon fibroblasts, in Linköping. This 3D model makes it possible to study effects on mechanical strength of the tissue after for example drug treatment or changes in loading situations. This is in contrast to conventional cell culturing. Studies on mechanical strength is an important tool in orthopedic research and this has previously only been done in animal models.
Moreover, this in vitro model also allows us to study mechanotransduction. Mechanotransduction is the way a cell converts mechanical stimulus into a biochemical response. We will use this model to study the effect of loading without micro-damage associated inflammation (which is present in animal models) to investigate the mechanotransduction component during loading. I have previously observed that tendon fibroblasts from some patients lack the ability to form tendon tissue, in vitro. We will study the differences between cells that can form tendon tissue with those that can´t. This might help to identify factors that are vital for tendon formation. I have also started a project where we study some aspects on why tendon injuries appear. We have performed an epidemiological cohort study to investigate associations between statin use and tendon injuries in different part of the body. We will also use the in vitro model to study the mechanisms why tendons are affected by statins and high cholesterol levels. The goal with these studies is to understand some factors which might contribute to the appearance of different tendon injuries, and to gain information to increase the understanding on what happens after a tendon injury, so that treatment and rehabilitation protocols can improve.

Ewa Ellis, Huddinge hospital
Large scale production of human liver spheroids
Primary human liver cells (hepatocytes) are used as screening tools in pharmaceutical industry and for academic research. As human hepatocytes are scarce many researchers employ the use of animal derived hepatocytes instead. This is unfortunate as there are significant species differences and the result may not translate into humans. Spheroid cultures of primary hepatocytes have been shown to maintain polarity and hepatocyte specific function over extended time (1 month). However, formation of spheroids has been cumbersome with the use of techniques such as low attachment plates, yielding one spheroid per well, or hanging-drop, yielding one spheroid per drop. These techniques are not feasible to employ for the vast amount (10-20 billion) of cells that are typically isolated from human donor livers.
In this project we will develop a large-scale bioreactor culture system for long-term maintenance of human hepatocytes as spheroids in a bioreactor system. With the spheroids cultures described in this project it will be feasible to give researchers/customers a time span that allows for planning, and still use unfrozen cells. Successful development of large-scale spheroid cultures and maintenance of human liver spheroids over extended time periods will allow us to make use of precious donated human liver tissue to full extent and reduce to the use of experimental animal and animal derived liver cells in lack of human primary liver cells.

 

Robert Fredriksson, Uppsala university
Establishing a proof of concept for a novel animal free method for botulinum toxin potency testing
Botulinum toxin type A (BoNT/A) is one of the most potent neurotoxins known and its mechanism of action is to cleave SNAP-25 in neurons to make them unable to signal. BoNT/A is used in the beauty industry to reduce facial wrinkles, marketed under the trade name BoTox, as well as in medicine to treat various conditions such as chronic pain and excessive sweating. Accurate potency determination is vital for its safe and effective use as a commercial substance and potency measurements must be assessed for each batch produced. Today potency measurements are done by all BoNT/A producers using LD50 assays in mice. There are a few patented protocols for animal free BoNT/A potency measurements, but these seem to be used to a limited extent in practice. The challenges has been to identify a cell-line with high enough sensitivity towards the BoNT/A and a good enough readout of the BoNT/A effect, which could be done by measuring amount of cleaved SNAP-25. We have several candidate immortal celllines as well as a stem cell derived line that has very high sensitivity towards BoNT/A as well as high expression of SNAP-25. We have also identified and tried out monoclonal antibodies that are specific enough for detection of cleaved and uncleaved forms of SNAP-25 in these cells. Within this project we will refine the methods for detecting cleaved SNAP-25 as well as the cell-culturing methods. The main outcome we are aiming for is to have a Proof of Concept protocol including cell-culture, BoNT/A treatment and detection of cleaved SNAP-25 including preliminary determination of internal assay parameters such as sensitivity, variability and reproducibility. This can be used to show that there is a possibility to establish a cell based assay for detection of BoNT/A potency determination.

Martin Hallbeck, Linköping university
Development of a modell to test future drugs to treat Alzheimer’s disease
The goal of the project is to develop a model for testing drugs against Alzheimer’s disease. It is a progressive disease and the patient suffers constant deterioration. Our knowledge of how the disease spreads in the brain is incomplete but there is a growing understanding that it is due to the spread of small protein collections. Our hope is to impede this spread.
It is difficult to study the human brain directly, good models are needed. We have developed models to study how Alzheimer’s and other neurodegenerative diseases can spread between nerve cells. Through previous funding, we have further developed the model using human induced pluripotent stem cells developed by programming skin cells from donors. If we can show that this model can be used by the pharmaceutical industry and in research on dementia and result in better results than with animal research, many animal tests can be replaced and at the same time research can take important steps forward.

Maria Karlgren, Uppsala university
Human cell based models for accurate assessments of brain drug uptake
Poor predictions of CNS drug exposure is a major problem in CNS drug development and is primarily the result of relying on animal-based models although major species differences are well-known for the blood-brain barrier (BBB) and the expression of drug transporters. Here, we will solve this problem by developing predictive models taking the drug transporters expressed in human BBB into account and thereby accurately quantify drug delivery to human brain. This will be done by combining in vitro kinetics from mechanistic transporter models and human BBB in vitro models with quantitative proteomics and physiological parameters to predict the human in vivo situation.
The project will be performed at Uppsala University in close collaboration with UDOPP (part of DDDp/SciLife Lab) giving a unique opportunity for validation in real-life drug development projects. With our model drug candidates with predictable and favorable human BBB properties will be identified and selected early in drug discovery/development. As a result an initial reduction of animal PK experiments of 50-60%, with further reduction potential, is estimated. Thereby providing the refined tools needed to substantially reduce and, in a longer perspective, replace animal BBB studies throughout the drug discovery/development process.

Hanna Karlsson, Karolinska institute
New cell models for improved assessment of genotoxicity and cancer risk of nanoparticles
The production and use of small nanoparticles is increasing in society and this leads to a requirement to test the toxicity of a substantial number of nanoparticles in the future. Despite research during recent years it is still unclear how risks associated with nanoparticles should be assessed, particularly considering chronic effects. To assess cancer risks, genotoxicity assays can be used both in cell-based assays and in animal studies. For particles, it is believed that a so-called secondary, inflammatory driven, mechanism is important for genotoxicity. This mechanism is believed to be related to release of reactive oxygen species (ROS) by immune cells (macrophages, neutrophils) that infiltrate the lung upon particle exposure (called oxidative burst) leading to DNA damage. Such a mechanism is, however, presently considered to be hard to detect in vitro and this has been used as an argument for continuing to use animal studies. The aim of this study is therefore to develop an in vitro assay that can detect an inflammatory-driven genotoxicity. The assay should also be functioning under serum-free conditions in order to avoid animal serum. We will culture both epithelial cells and macrophages and test two approaches: 1) Exposure of epithelial cells to “conditioned medium” from macrophages and 2) Co-cultures of epithelial cells and macrophages. We will test both particles considered to be carcinogenic to humans (e.g. quartz) as well as non-carcinogenic particles (iron oxide particles) as benchmark particles leading to a form of “validation” of our assay. We will then test a range of nanoparticles. The goal is to develop a standard assay that can take the interplay between different cell-types into account in order to completely avoid the use of animals.

Pekka Kohonen, Karolinska institute
Deepened mechanistic validation of toxicity pathway functionality in a patented analysis tool
This application is the follow up to the last year funded plan, submitted then as a pro posed two-year project effort. The work actions are fully on schedule relative the initial purpose and aims. A Predictive Toxicogenomics Space (PTGS) tool, being completely novel in concept to the environmental medicine field, was accordingly brought to publication [1]. Following the project plan, the tool was thereafter applied to dose-response analysis with benchmark dosing technology. Specifically, the project aims during its second and final year to apply the PTGS concept for developing much needed, novel, increasingly sensitive and mechanism-driven benchmark dosing tools. Aiming directly for regulatory and industrial applications, the effort evaluates in depth the ability of PTGS to capture tumor suppressor p53-mediated signaling within an extensive in vitro-derived transcriptomics data set. This effort has the advantage of directly addressing possibly the most central cellular defense mechanism for adequately dealing with insults of toxic and cancer-inducing agents. Overall, the project is set to generate proof-of-concept for novel, widely applicable solutions towards toxicity prediction of environmental agents. This goal covers also creating standard operating procedures for a real product-based applicable testing tool. Agreeing fully with the many years of Forska Utan Djurförsök-supported work, the ultimate goal is thereby to continue to act in a translational manner relative current societal needs of improving environmental health and safety with precise and cost-effective in vitro and in silico assays that fully replaces animal testing.

Johan Lundqvist, Swedish University of Agricultural Sciences
Aquatic toxicology, fish, in vitro toxicology
This research program aims to develop a system for animal-free testing to assess aquatic toxicity, based on the principle of toxicity pathways. Today, European regulations require that a large number of chemicals should undergo assessment of aquatic toxicity, which often include animal experiments. Assessment of the aquatic toxicity in toxicity pathway-based cell culture models instead of in animal experiments, will allow analysis of a large number of chemicals to a moderate cost and will significantly reduce the number of animals used for aquatic toxicity assessment of chemicals. The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) has recently proposed development of cell cultured based assays as a key strategy to reduce and replace the use of fish in toxicity testing. Status update This is an ongoing research project, currently funded by the Swedish Fund for Research Without Animal Experiments and the Swedish Research Council Vetenskapsrådet (3R subcall). During the first year of research (2017), we have successfully transfected multiple fish cell lines in our laboratory, showing that reporter gene assay is a feasible methodological approach also for fish cells. Further, we have establish an in vitro method to measure oxidative stress in cultured fish cells and used the method to study the toxicity of a panel of commercially used pesticides. These findings show that the established in vitro method can be used as an alternative for animal experiments when assessing fish toxicity of chemicals. Currently, these results are in final preparation for submission to a scientific journal. The results were orally presented at the 10th World Congress on Alternatives and Animals in the Life Sciences, Seattle (August, 2017).

Stina Oredsson, Lund university
Novel 3D Cell Culturing Methods in Cancer Research
Millions of animals are used in cancer research to develop new drugs for treatment. M any times initial testing of potential drugs is done in 2D cancer cell line systems and when positive results are achieved, the compounds are injected into tumour bearing animals without investigation of toxic side effects. Unfortunately, cell culture work with cancer and normal cells involves animal-derived products such as fetal bovine serum, collagen, and matrigel, the latter from tumour bearing mice. Our aim is to develop a 3D cell culturing system for co-culturing of cancer cells and stromal cells found in a tumour to replace and reduce animal experiments in cancer drug discovery. The basis is a unique collagen mimicking polycaprolactone fibrous structure made by electrospinning. Cancer cells, fibroblasts (normal or cancer-associated), and immune cells are co-cultured in medium without products derived in animalethics questionable ways. Using phase contrast and holographic time laps imaging of cultures incubated in normoxia and hypoxia we can follow cell dynamics such as cell movement. After fixation and staining with different dyes, we use confocal microscopy to investigate cell-cell and cell-matrix interactions and calibrate/compare to in vivo systems. Using a unique library of potential cancer drugs we investigate effects on cancer cell sub-populations as well as on the stromal cells. We anticipate that this system can replace many animal experiments in cancer research.
Emma Pedersen, RISE Research Institutes of Sweden
Development of an alternative method for sensitization testing of medical devices standard series ISO 10993
The sensitizing potential of medical devices is today evaluated using animal tests, primarily the GPMT (guinea pig maximization test) or alternatively the LLNA (local lymph node assay) on mice or Buhler test on guineapig. Every year, more than 14 000 animals are used for sensitization testing of medical devices in the EU, and many more worldwide.
The aim of this project is to further develop an animal free method, SENS-IS, to evaluate the sensitizing potential of medical devices. The SENS-IS assay is based on a reconstructed human skin model (Episkin) as test system and analysis of the expression of a large panel of genes relevant to the considered biological processes. The SENS-IS assay has previously been shown to accurately predict sensitization of pure chemicals and, in contrast to most other in vitro sensitization assays, can provide potency information. This makes it a promising tool for evaluation of medical device extracts, which requires a very sensitive test. This project aims to verify that a modified version of the protocol works for medical device extracts, and in particular that the assay is capable of detecting weakly sensitizing materials. This includes development of new control materials. Our goal is to include the method in the standard ISO 10993:10, replacing the current animal tests for determination of sensitizing potential.
The project team has previously been involved in the validation of a similarly modified protocol for testing of skin irritation of medical devices (financed in 2016 by Forska Utan Djurförsök). That protocol will likely replace the current animal tests for skin irritation in the next update of ISO 10993:10. Thus, the necessary contacts to translate the results into an internationally accepted standard are already established.

Peter Sartipy, University of Skövde
Human stem cell derived cardiomyocytes for toxicity testing, an alternative to animal experiments
Anthracyclines, e.g. doxorubicin, are highly efficient chemotherapeutic agents against a variety of cancers. However, anthracyclines are also among the most cardiotoxic therapeutic drugs presently on the market. Chemotherapeutic-induced cardiomyopathy is one of the leading causes of disease and mortality in cancer survivors. The exact mechanisms responsible for doxorubicin-induced cardiomyopathy are not completely known, but the fact that the cardiotoxicity is dose-dependent and that there is a variation in time-to-onset of toxicity, and gender- and age differences suggests that several mechanisms may be involved. In the present project, we investigate doxorubicin-induced cardiotoxicity in human pluripotent stem cell-derived cardiomyocytes using global expression analysis (including proteomics, mRNA- and microRNA arrays).
In addition, these different sources of omics data (protein, mRNA, and microRNA) are combined and analyzed using newly developed methods to identify differential expression in data of various origin and types. Subsequently, the results are integrated in order to generate a combined visualization of the findings and to identify panels of biomarkers that can be used assess anthracyclin induced cardiotoxicity more rapidly. The establishment of human stem cell derived cardiomyocytes as a cellular system for toxicity testing is key in order to replace currently used animal models which include several species (e.g., rat, rabbit, mouse, pig and dog). The cellular models have also most often relied on the use of cells isolated from hearts from experimental animals. One of our main objectives with this project is to demonstrate the utility of human stem cell derived cardiomyocytes, which provide an unlimited source of human cells, that can be used for in vitro toxicity testing. While additional research is needed to generate scientific evidence, human stem cell derived models are expected to be able to replace many types of experimental animal models.

Lena Svensson, Lund university
Replacing animal models by microfluidic vascular models to study cell migration
This project aims to adopt microfluidic blood vessel models from literature and adapt them suit our experimental needs. Creating model blood vessels for in vitro studies will enable the replacement of several animal experiments, in particular intravital microscopy. Further, the models will refine experimental conditions by introducing control over several parameters not possible in test animals.
The models are based around three parallel channels: a central channel containing a hydrogel to mimic the extracellular matrix and two adjoining, identical channels. The channels are separated by pillars to contain the collagen while allowing liquid exchange. By culturing endothelial cells in one side channel, it is possible to create a model blood vessel into which other cells can be introduced, e.g. leukocytes or cancer cells. The purpose is then to study how these cells adhere to, and migrate through, the endothelial layer using microscopy. Such transendothelial migration is a key process for both the immune system and cancer cells.
Currently, microchannel fabrication and endothelial cell culture have been successfully optimised and the system has been tested with leukocytes for physiological validity. We need to implement further adjustments in order for our microfluidic blood vessel model to attain full functionality, such experiments are currently underway.

Senast uppdaterad: 9 mars 2018