P01 - Development of high-throughput cell migration assay using dynamic micropatterned surfaces
Almeida, F.V. (1), Costa, P.A.(2), Connelly, J.T.(1)
(1) Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry
(2) Instituto Medicina Molecular (iMM), Universidade de Lisboa (University of Lisbon), Portugal
Cell migration is a key process in several biological contexts such as in development, cancer and wound healing. Current in vitro migration assays lack in the accurate control of important variables that orchestrate cell motility. We have developed a system that allows the control of soluble and insoluble factors, as well as the mechanical cues in a high-throughout apparatus. This was achieved by using dynamic micropatterned substrates, which allow the binding of specific peptides to POEGMA polymer brushes via "click chemistry". We used collagen mimetic peptide GFOGER to induce keratinocytes migration and investigate the role of epigenetic factors in their collective movement.
P02 - Development of vascular-on-a-chip platforms for pre-clinical evaluation and screening of vascular and cancer therapies.
Bottaro, E. (1), Caine, M. (2), Lewis, A.L. (2), Carugo, D. (1).
(1) Bioengineering Sciences Research Group, Faculty of Engineering and the Environment,University of Southampton Highfield Campus, Southampton, UK, (2) Biocompatibles, UK Ltd., Farnham Business Park, Weydon Lane, Farnham, Surrey, UK.
A plethora of therapeutic agents are administered through the vascular system, either to impart a therapeutically relevant action on localized vascular segments or to be transported towards a specific target tissue. The development of artificial vascular models has recently attracted significant attention for its relevance to the investigation and screening of numerous therapeutic modalities. Given the small size of microvessels, emerging microfluidic technology could be employed to recreate a functional microvascular network. Microfluidic devices could therefore be employed as an alternative to traditional in vitro platforms and animal models, overcoming the technical, economic and ethical issues associated with these approaches.
The main scope of this work is to develop artificial, patient-specific vasculature-on-a-chip models with potential utility for pre-clinical evaluation and screening of vascular and cancer therapies.
Models will replicate the in vivo structure of vascular micro-arterioles and surrounding connective tissue constituents, to investigate the extravascular penetration and therapeutic action of biologically active compounds (i.e., anticancer drugs). Specifically, these platforms will be utilized to quantify the physical and biological performance of chemo-embolic microspherical beads employed for the treatment of hypervascularised tumours. Depending on therapeutic indication, typical pre-clinical animal models are swine, rabbit (pharmacokinetic) or ovine (physicochemical). The study builds upon previous work in which engineered models of elements of these pre-clinical models were utilised to test bead distribution and drug elution under flow conditions. A second-generation model will be developed to replicate the biological constituents of healthy and cancerous vasculature, and the results validated by comparison with in vivo histological assays.
P03 - Characterisation of extracorporeal flow in Thiel embalmed human cadavers
Burton, B. (1) (2), McLeod, H. (2), Matthew, S. (2), Houston, G. (2)
(1) University of St Andrews, (2) School of Medicine, University of Dundee
In medicine today, it is essential to test medical devices extensively before they are used on patients. Currently, many emerging devices, including stents and graphs, are tested on large living animals in pre-clinical settings. Thiel-embalmed cadavers provide an excellent alternative to animal testing, as they have life-like vascular systems and accurate human anatomy. To emulate living physiology, cadaver vessels are perfused with large quantities of commercially produced blood mimics, but these blood mimics are extremely expensive. Here, a novel blood mimic was formulated in the lab using flour that can be mixed in large quantities very cheaply. The fluid was tested for acoustic properties and imaging quality. It was found that the blood mimic was easy to image using colour Doppler ultrasound, allowing vessel structure in a human cadaver kidney to be imaged clearly. This cost effective and easy-to- produce blood mimic will allow colour Doppler ultrasound to be used for cadaveric stent-graft insertion, potentially replacing the use of some animals in pre-clinical testing.
P04 - Development of a perfused plant-based tissue engineered cell model for toxicity assessment and drug development.
Khan, F.A. (1), Garrod, M. (1), and Chau, D.Y.S. (1)
(1) Department of Pharmacy, Pharmacology and Postgraduate Medicine, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, AL10 9AB, UK
INTRODUCTION: Toxicological assessment is a key criteria used in the development of chemical compounds and frequently determined using animal-based models. In addition, the more complex 3D perfused systems that aim to mimic fluid flow are difficult to construct and often require the use of stem cells or ex vivo organs. Intriguingly, plant-derived materials may offer a potential alternative to traditional cell-supporting scaffolds due to its innate architecture, low immunogenicity, biodegradability and biocompatible characteristics.
METHODS: Brassica napus leaf sections were treated with trypsin, pectinase, cellulase, sodium bicarbonate or SDS at a range of temperatures. Histological analyses was performed using Safranin and Fast Green stains for structural components. Flowability was assessed by the injection of a synthetic dye. Human cells (BxPC3) were cultured on lignin and cellulose-derived scaffolds and characterised according to their spreading and attachment profile (0.25% (w/v) May-Grunwald and 0.4% (w/v) Giemsa stains), proliferation (CellTiter 96 AQueous One reagent) and viability (CytoTox-ONE assay).
RESULTS: Brassica napus leaf samples remained undamaged when exposed to trypsin. However, incubation for 48 hours with 20% (w/v) SDS solution removed the majority of extracellular components. Positive perfusion was demonstrated by the transition of dye along the stem sample treated with pectinase/cellulase solution. Cells cultured on lignin and cellulose-based scaffolds demonstrated characteristics comparable to traditional tissue culture plastic.
CONCLUSIONS: Results suggest that plant-derived materials may potentially be used as a framework to support cell growth, within a 3D environment, as well as offering a simple perfusion systems to mimic fluid flow in native tissues or organs.
P05 - Introducing a new model for bitter taste assessment - the social amoeba Dictyostelium
Marco Cocorocchio 1, Robert Ives 2, David Clapham 2, Paul L. R. Andrews 3, Robin S. B. Williams 1
1 Centre of Biomedical Sciences, School of Biological Sciences, Royal Holloway University of London, UK; 2 RD Platform Technology & Science, GlaxoSmithKline, Hertfordshire, UK; 3 Division of Biomedical Science, St George’s University of London, UK
A large number of therapeutically active compounds have a bitter taste. This often causes an aversive reaction - particularly in children - leading to decreased patient compliance and in some cases to severe reactions such as nausea and vomiting. Identification of bitter taste liability during drug discovery utilises the rat in vivo brief access taste aversion (BATA) test which is time-consuming and has limited throughput. Here, we investigate the suitability of using a simple non-animal model, the amoeba Dictyostelium discoideum, to examine taste-related responses and particularly to identify those molecules with a bitter taste liability. We initially analysed the acute effects of taste-related compounds on Dictyostelium cell behaviour to show no response to salty or sour conditions, or umami and sweet tasting compounds, whereas cells rapidly responded to bitter tastants. We then developed a medium-throughput assay, based in Dictyostelium, to monitor responses to a wide range of structurally diverse bitter tastants and a panel of blinded molecules. This assay employed visualising cells pre- and post-bitter tastant exposure, recording cell response with time-lapse photography and using computer-generated quantification to monitor changes in cell membrane movement. Dictyostelium showed varying responses to the bitter tastants, with IC50 values providing a rank order of potency. Comparison of Dictyostelium IC50 values to responses observed at a similar concentration range in the rat BATA test showed a significant positive correlation between the two models (p = 0.0172). In addition, the data showed a similar response of Dictyostelium to that provided by a human sensory panel assessment test. These experiments suggest that Dictyostelium might provide a suitable model for early prediction of bitterness for novel tastants and drugs, where a common response to bitter tastants appears to be conserved from a single-celled amoeba to humans.
P06 - Mucosa-mimetic materials to replace animal tissue in mucoadhesion testing
Cook, M.T. (1), Da Silva, J.B. (1,2), Bruschi, M.L. (2), Smith, S.L. (3), and Khutoryanskiy, V.V. (3).
(1) Department of Pharmacy, Pharmacology, and Postgraduate Medicine, University of Hertfordshire, U.K; (2) Department of Pharmacy, State University of Maringá, Brazil; (3) Department of Pharmacy, University of Reading, U.K.
In order to achieve site-specific mucosal delivery of drugs, dosage forms are designed so that they are “mucoadhesive”, and adhere to mucosal membranes. This adhesion process is governed by a number of chemical interactions, including hydrogen-bonding, as well as physical interactions, such as the wetting of a dosage form, or the entanglement of polymer chains. In order to assess the mucoadhesion of dosage forms, mucosa is excised from deceased laboratory animals, a large number of which are slaughtered specifically for this tissue. Given the desires of the scientific community to reduce the number of animals used in research, and the emergence of the “three Rs”, an alternative to this ex vivo mucosa is desired.[1,2]
We report a hydrogel composed of N-acryloyl glucosamine and 2-hydroxyethyl methacrylate, which is able to mimic ex vivo mucosa in the testing of solid, semi-solid, and liquid dosage forms.[2,3] It is believed that the ability of the hydrogel to mimic ex vivo mucosa is related to its chemical similarity to the heavily-glycosylated secretory mucins which line the surface of mucosal membranes.
1. Cook, M. T. & Khutoryanskiy, V. V. Mucoadhesion and mucosa-mimetic materials—A mini-review. Int. J. Pharm. 495, 991–998 (2015).
2. Hall, D. J., Khutoryanskaya, O. V. & Khutoryanskiy, V. V. Developing synthetic mucosa-mimetic hydrogels to replace animal experimentation in characterisation of mucoadhesive drug delivery systems. Soft Matter 7, 9620 (2011).
3. Cook, M. T., Smith, S. L. & Khutoryanskiy, V. Novel glycopolymer hydrogels as mucosa-mimetic materials to reduce animal testing. Chem. Commun. (2015).
P07 - Non-invasive measurement of perfusion and structure in chronic kidney disease using magnetic resonance imaging
Cox, E.F. (1), Buchanan, C.E. (1,3), Selby, N.M. (2,3), Taal, M.W. (3), Francis, S.T. (1)
(1) Sir Peter Mansfield Magnetic Resonance Centre, School of Physics & Astronomy, University of Nottingham, UK; (2) Department of Renal Medicine, Royal Derby Hospital, Derby, UK; (3) Division of Medical Sciences and Graduate Entry Medicine, School of Medicine, University of Nottingham, Nottingham, UK
Chronic Kidney Disease (CKD) is a condition in which the kidneys are damaged in function and/or structure, and cannot filter blood as well as possible. Kidney damage can occur from a physical injury or a disease like diabetes, causing wastes to build up in the body leading to other health problems. Animal models are commonly used to study CKD, and although most established animal models of chronic kidney disease bear some similarities to human chronic kidney failure, none is fully representative and ideal at replicating human disease. Numerous animal studies use invasive laser Doppler needle probes to assess renal tissue perfusion. Here, we develop non-invasive MRI (magnetic resonance imaging) methods to study both the structure and function of the kidneys without the injection of contrast agents or exposure to radiation.
We will outline the findings of this Dr Hadwen Trust funded project which include the development of MRI techniques as an alternative to the invasive measures of laser Doppler needle probes. We use arterial spin labelling to assess renal tissue perfusion, as well as blood flow measures, and diffusion-based sequences. We also develop MR longitudinal (T1) relaxation time measures to assess structural changes in the tissue linked with fibrosis and inflammation.
We will present our results of applying our MRI methods to assess microvasculature and microstructural changes in patients with CKD patients, comparing the MR measures with clinical measures obtained for the patients.
P08 - Multimodal Brain Imaging and Brain Stimulation of GABA Function in Humans
Jackson S.R. (1,2), Dyke, K. (1), Pépés, S.E. (1), Chen, C (3), & Kim, S.Y (1,2).
(1) School of Psychology, University of Nottingham; (2) Institute of Mental Health, University of Nottingham; (3) Sir Peter Mansfield Imaging Centre, University of Nottingham
Neurodevelopmental disorders such as Autism, ADHD, OCD, and Tourette syndrome (TS) are common mental health conditions that can have a devastating effect on a child’s cognitive, emotional, social and educational development, and impact negatively on the child’s immediate family and caregivers. Diagnosis of developmental psychiatric and neurological disorders has most often been based upon taxonomies of distinguishing clinical symptoms. However, there is now increasing evidence to indicate that there are mechanistic similarities with respect to the underlying genetics and neurobiology across a wide variety of diagnoses. Importantly, Autism, ADHD, OCD, and TS, which are highly co-occurring conditions, have each been associated with alterations in the balance of excitatory and inhibitory influences within key brain networks, and more specifically with the impaired operation of GABA (inhibitory) signaling mechanisms. GABA is the main inhibitory neurotransmitter in the brain. It is present in 25–50% of synapses and contributes to almost all brain functions. As a consequence, GABA is a major target for drug development and, because manipulating GABA concentrations in humans has previously proven to be difficult, it is very often the case that investigations of GABA function in health and disease make use of invasive animal experiments, including studies in non-human primates. Our research programme aims to replace the use of animals through the development of robust brain imaging and brain stimulation methods that permit GABA function in health and disease to be effectively investigated in humans.
P09 - Computational techniques informing material engineering for stem cell control in the laboratory
George Joseph (1), Rosemary Fricker (2), Paul Roach (2), Theocharis Kyriacou (3)
(1) Faculty of Natural Sciences, Keele University; (2) Institute of Science and Technology in Medicine, Keele University; (3) School of Computing and Mathematics, Keele University
Stem cells give rise to specialised (mature) cells that make up organs of our body. The use of stem cell therapies are increasing rapidly, with a need for more control over cells grown in the laboratory. This is particularly relevant for the treatment of neurological disorders, wherein clinical trials for, e.g. Parkinson's disease are limited by the number of derived and matured cells. The drive for this project is to better understand the surface chemistry of the cell’s environment and its effect on cells. The aim is to find chemical designs to enhance cell growth and function through the study of the surface-cell interface. The cell’s micro-environment is complex, being responsible for stem cell attachment, proliferation and maturation to organ cells. Relevant work to enhance cell responses have looked towards surface chemical variation although the many permutations pose a major challenge in the optimisation of the chemical design. Here we aim to use methods to capture the relationship of surface chemistry (inputs) and cell responses (outputs) computationally from laboratory cell experiments using rat tissue. This allows the simulation of cell experiments on computers and re-iterations will guide towards the optimal chemical design. Simulating cell experiments computationally to discover the optimal surface chemistry effectively reduces rat use.
P10 - Development of a 3D placental model for screening of endocrine disrupting chemicals
Sophie-Christine Jahn (1), Phoebe Maund (1), Elisabete Silva (1), Emmanouil Karteris (1)
(1) Department of Life Sciences, Biosciences, Brunel University London, Uxbridge, UB8 3PH, UK
Endocrine disrupting chemicals (EDCs) are a group of chemicals found in every day products such as food containers and consumer goods. They have the capacity to disrupt hormone-controlled systems in the body. A crucial transient endocrine organ during pregnancy is the placenta, as it maintains the pregnancy and is essential for the exchange of vital nutrients between the mother and the child. EDCs appear to exert a multitude of adverse effects including preterm labour and spontaneous abortions. To date, our understanding of the impact of EDCs on placental function is still very limited. The majority of experiments performed to study the effect EDCs on the placenta have used a plethora of animal models: from primates to mice. This possesses a number of problems in terms of interpreting the data, as there are intrinsic differences between a human and a placenta of an animal. Despite these limitations, no viable alternative has been provided. Here we propose the use of a 3D placental culture model a platform for streamlining the effects of EDCS by acquiring an “omics” approach to discover biomarkers of EDCs in a more relevant model to humans. Currently, we are using microarray analyses to as a non-biased screen to identify changes that Bisphenol-A (BPA) can exert at transcriptome level. This study will assist in replacing animal models to study the impact of EDCs in human health. We estimate that we can save approximately 300 mice and 500 rats per year in UK.
P11 - The Development of a Dynamically Perfused System for Models of the Human Airways, with Automated Feeding and Sampling Capability
Leslie, L.J. (1), Lloyd, H. (1), and Marshall, L.J. (2)
(1) Biomedical Engineering Research Group, Mechanical Engineering and Design, Aston University, Birmingham.
(2) School of Life and Health Sciences, Aston University, Birmingham.
The development of relevant in vitro models to replace outdated animal models is the focus of many life science researchers. Whilst the cellular make-up of models can be made physiologically relevant, often the delivery of nutrients to, and the collection of samples from, these models can be static and un-realistic. Combining biological expertise with engineering know-how has allowed our team to focus on the development of a dynamically perfused cell culture environment which aims to enable relevant biological functionality. There are currently no perfusion models of the upper airways, despite recent evidence recognising the role of small airways in human chronic respiratory disease. This project has three specific objectives. 1) design and build a perfused system, 2) identify the optimum flow parameters, and 3) design and build a sampling system. This is an ongoing project, enabled through the Dr Hadwen Trust Summer Studentship Scheme, and here we present the first phase of development including the prototyping of cell chambers and manifolds using 3D printing and careful materials selection processes, the advancement of programmable systems for timed sampling to meet individual user requirements and the development of prototyped automated apparatus for sample collection. Future work will combine the engineering aspects with the biological side and begin to analyse the effects perfusion will have on cell viability and the effects of introducing challenges to measure cytotoxicity. If successful, this project could provide an alternative to highly severe experiments involving dogs and rodents where airways are challenged through direct exposure or chemical damage.
P12 - Mas related gene receptors as novel molecules for pain relief
McMillan, H, Lundy, F, Curtis, T, El Karim, I
Centre for Experimental Medicine, Wellcome-Wolfson Institute for Experimental Medicine, School of medicine Dentistry and Biomedical Sciences, Queen’s University Belfast
Background: Transient Receptor Potential (TRP) channels expressed in nociceptive neurons are detectors of noxious stimuli to initiate pain sensation. TRP channel activity can be modulated by G-protein coupled receptors (GPCRs) leading to sensitisation and hyperalgesia, typically seen in neuropathic and inflammatory pain. A novel GPCR, Mas related gene receptor X1 (MRGX1), which is exclusive to humans, is a desirable target for pain relief, as it is specific to sensory nerves and is thought to modulate TRP activity. The lack of neuronal tissue and several animal species (ferret, guinea pig, rat, mouse), which do not faithfully represent the human physiology have been the burden of pain studies. We have developed and characterised a human neuronal model which uses human dental pulp stem cells (hDPSCs) to derive functional nerves. Aim: To characterise a role for MRGX1 in regulating TRP channel activity under injury and inflammatory conditions. Methods: Expression and functionality of TRP channels in neurons derived from hDPSCs will be demonstrated by immunohistochemistry (IHC) and calcium imaging, respectively. Polymerase Chain Reaction (qPCR) and IHC will determine the expression of MRGX1 in neurons, and further investigation will determine whether expression and function of MRGX1 is affected by injury and inflammation. We will also use calcium imaging to study the functionality of MRGX1 in neurons. We will finally study the effect of MRGX1 activation on TRP channel activity. Outcome: This study will provide important insights into the molecular mechanisms of inflammatory and neuropathic pain, leading to the identification of novel analgesic targets.
P13 - Trans- endothelial migration of non-small cell lung cancer cells: Role of CD15 in brain metastasis
Pilkington, G.J. 1, Jassam, S.A. 1, Maherally, Z. 1, Ashkan, K. 2, and Fillmore, H.L. 1
1 Brain Tumour Research Centre, University of Portsmouth; 2 Neurosurgery, Kings College Hospital, London
Circulating cancer cell (CCC) adhesion to brain endothelium and extravasation through the Blood Brain Barrier (BBB) are key to brain metastasis. In this study the role of CD15 (a cell adhesion molecule which correlates with metastasis) was investigated in adhesion and trans-endothelial migration in brain metastasis using human BBB modelling. The gene encoding fucosyltransferase 4 (FUT4), responsible for CD15 synthesis, was overexpressed and knocked down (KD) in 3 human cell lines: metastatic lung to brain cancer (SEBTA-001), metastatic lung to lymph node (NCI-H1299) and primary lung carcinoma (COR-L105). Their protein expression levels were then evaluated by Immunocytochemistry, flow cytometry and Western Blot as were those of human cerebral microvascular endothelial cells (hCMEC/D3). Adhesion and trans-endothelial migration were determined using a cell-cell adhesion assay and CellZscope® respectively. Overexpression and KD of FUT4 correlated with the up-regulation and down-regulation of CD15 expression respectively. CD15 overexpression significantly increased adhesion in all cells studied compared to wild type (WT) (p<0.01) while, FUT4 silencing decreased adhesion compared to WT (p<0.01). Cell lines overexpressing FUT4 added onto an endothelial monolayer caused an immediate decrease in trans-endothelial electrical resistance (TEER) compared to KD and control cells (p<0.01). KD of FUT4 in metastatic cells known to transmigrate failed to generate a decrease in TEER compared to control cells (p<0.01). Targeting CD15 may, therefore, preclude brain metastasis. Our data will help in comparison with those published on animal models eventually enabling use of our model in pre-clinical investigation of anti-metastatic agents.
P14 - Understanding Medulloblastoma Dispersal and Colonisation; direct leptomeningeal or haematogenous involvement?
Loveson,K. F., Mather, R.L.,Jones, A., Pilkington, G.J., and Fillmore, H.L.
Brain Tumour Research Centre, School of Pharmacy & Biomedical Sciences, University of Portsmouth, St Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK.
Medulloblastoma (MB) is the most common solid malignant tumour in children. Genomic research advances have led to classification based on molecular profiles into four distinct subgroups with Groups 3 and 4 having the worst prognosis due to tumour dissemination away from the cerebellum forming “drop metastasis” along the spinal cord. The current dogma suggests that tumour cells shed from the primary tumour mass into the Cerebrospinal Fluid (CSF), travel via the CSF and establish metastatic colonies on the leptomeningeal surface and spinal cord. A group in Toronto, Canada, challenges this dogma, demonstrating, by way of an animal model, that MB metastasis could occur through a systemic route or ‘true metastasis.’
To investigate the two potential routes of MB dispersal we developed two distinct all-human in vitro models: 1. ‘leptomeningeal’ cell BEN-MEN1 (CSF) and 2. Brain-endothelial cell (hCMEC) (haematogenous spread via blood stream). In addition to ‘host’ cell types, two human Group 4 MB cell lines derived from the same patient (CHLA-01-original biopsy) and one derived from a metastatic lesion (CHLA-01R) were used. Using various parameters where CHLA-01 were placed on monolayer cultures of BEN-MEN1 or hCMEC cells and treated with conditioned media from co-cultures, significant changes in motility but not cell viability were observed. Results suggest that there is a bi-directional communication between the tumour and host cells.
The introduction of an all-human in vitro model system would allow insight into the dynamic cellular and molecular pathways involved in medulloblastoma progression and provide an alternative to the animal model.
P15 - Strategies to isolate specific cell-types from adult human gastrointestinal tissues, to investigate changes during advanced age and degenerative disorders
Palmer A, Scott F, Kouassi M-A, Broad J, Knowles CH, Sanger GJ (1)
(1) Blizard Institute and the National Centre for Bowel Research, Barts & The London School of Medicine and Dentistry, Queen Mary University of London, UK
Degenerative disorders are commonly studied using rodents. For the gastrointestinal (GI) tract these include changes during diabetes, advanced age and chemotherapy. To investigate mechanisms in humans, with GI physiology markedly different from rodents, specific cell-types controlling neuromuscular functions must be isolated from mature, adult tissues. This has not previously been achieved. Manipulation of live, primary human cells could then be achieved by novel environments, pharmacological tools and techniques such as live fluorescence imaging, calcium imaging, RNA analysis and immunohistochemistry.
We are developing methods to extract and culture human enteric neurons and glia from the myenteric plexus within the smooth muscle. Muscle from human colon is digested and cultured in a neuron-specific medium, encouraging attachment and survival of neurons and glia, while discouraging growth of other cell types. We have also cultured human GI muscle for up to 48 hours for separate study and to allow neurons to interact with other cell types, such as muscle fibres and leukocytes.
Using human stomach, conditions are being optimised for dissociation of the muscle and isolation of interstitial cells of Cajal (ICC) within the muscle, using antibodies selective for ICC cell surface epitopes, cKit and Ano1, and fluorescent-activated cell sorting. Ano1 positive cells have been successfully isolated and RNA integrity is being assessed. Antibody staining is being optimised, to further enable selection of viable double positive cells for downstream analysis.
We will use these techniques to study live, mature adult, primary cells in degenerative and aging disorders, avoiding use of moderate-to-severe techniques in animals.
P16 - Surface curvature of scaffolds - implications in the development of a human, ophthalmic, cell culture model
Shafaie, S. (1), Hutter, V.(1), Cook, M.T. (1), Brown, M.(1), Chau, D.Y.S.(1)
(1) Department of Pharmacy, Pharmacology and Postgraduate Medicine, School of Life and Medical Sciences, University of Hertfordshire, College Lane, Hatfield, AL10 9AB, UK
Traditionally, rabbit, chicken and bovine eyes have been widely used to evaluate the potential ocular irritancy and toxicity associated with a test substance. Since the introduction of the principles of the 3Rs as a robust framework to minimise animal use, in vitro cell based models have been extensively developed and validated as an alternative approach for drug toxicity testing. As the geometrical configuration of a population of cells seem to have a profound influence on their growth and differentiation, there is still a need for in vitro models that are not only physiologically relevant but also take into account the native organs’ geometry and growth conditions. Herein, we aim to develop a human ocular in vitro model based on the spherical/curved surface geometry of the eyeball. The effect of the surface curvature on ocular cells’ activity, death and viability were characterised using a combination of a metabolic activity assay (CellTiter AQ/MTS), LDH release profiles (CytoTox ONE) and absolute cell counts (Guava ViaCount), respectively. In addition, cell differentiation and expression of specific marker proteins were determined examined using flow cytometry. Results confirmed that growth surface topography had a significant effect on mitochondrial activity of human retinal cells as well as effecting their phenotype and expressed specific marker proteins. Accordingly, these results highlight and enforce the requirement of mimicking the native microenvironment of human cultured cells, at a geometric surface substrate level, in order to direct their correct growth and differentiation.
P17 - The ADAPT principles can help the implementation of alternatives
Taylor, K (1)
(1) Cruelty Free International Trust
Our experience with various sectors over the last 20 years has highlighted the complexity of the ‘path to acceptance’ and the many hurdles that are placed in the path of new methodologies that replace animal tests.
Even simple, like- for –like replacements have struggled to gain acceptance and full implementation. Time scales from validation to adoption and replacement have been in excess of 10 years and for many are still not complete. This is due in part to a failing of regulatory authorities to take responsibility for identifying new methods, to assess the suitability for their sector and to then clearly notify industry of their decision.
Cruelty Free International has created the ADAPT principles help regulatory bodies identify where changes in their policies and processes are needed to ensure the more rapid implementation of alternatives. The principles are: Assessment - does the body have a proactive mandate to assess the suitability of new methods for their sector? Decision - who takes responsibility for deciding whether an alternative method is suitable? Acceptance - have all the bureaucratic steps to acceptance such as the need to revise guidance and/or legislative text been identified? Policing - are there mechanisms in place to monitor the use of alternatives and will action be taken if animal tests are done unnecessarily? Transparency - does the authority inform all stakeholders of their actions at each stage?
We provide examples of how each ADAPT stage can avoid delays with implementation, what regulators and companies can do to apply ADAPT and how animal protection groups can help.
P18 - Predicting adverse immune responses to biologics
Tulah, A.S. (1), Ahmed, S. (1) and Dickinson, A.M. (1)
(1) Alcyomics Ltd, Newcastle upon Tyne, United Kingdom
Background: No reliable human in vitro assays exist which test for sensitivity, efficacy and adverse immune reactions of biologics that are equivalent or superior to in vivo animal testing. Alcyomics has developed Skimune™, a non-artificial (non-3D) human in vitro skin test which can predict adverse immune responses to test compounds. The test gives a predictive readout of graded histopathological skin damage, correlating with inflammatory cytokine release and T-cell proliferation responses.
Methods: We tested ten antibody formulations using Skimune™Mab on healthy volunteer skin biopsies (n=10) in the presence and absence of autologous lymphocytes. Histopathological grading was compared to expected clinical response. We also show examples where Skimune™Mab was used to determine the efficacy and potency of immunomodulatory drugs and biosimilars and have modified this assay to investigate the safety of antibody drug conjugates (ADC).
Results: Antibody formulations tested in Skimune™Mab showed highly significant correlation (Pearson=0.96, p=0.0001) with clinical outcome. Additionally, data from an analogue to TGN (TGN1412) showed Skimune™Mab could have predicted the serious life threatening cytokine storm which caused the 2006 Northwick Park trial. In our analysis of antibody drug conjugates, we did not identify any immunogenicity risk at any dose tested for two different linker molecules.
Conclusions: Skimune™ aids the development of therapeutics by the early detection of adverse immune reactions prior to phase I clinical trials. Overall, our results show that Skimune™Mab can provide vital information regarding drug efficacy, potency, dose responses and safety of therapeutics including monoclonal antibodies, biosimilars and ADC.
P19 - Rapid detection of adverse reactions to novel drugs using in vitro assays
Tulah, A.S. (1), Ahmed, S. (1) and Dickinson, A.M. (1)
(1) Alcyomics Ltd, Newcastle upon Tyne, United Kingdom
Background: We present two non-animal assays: Skimune™ a non-artificial (non-3D) human skin test to predict adverse immune reactions and Skimune™3D, a full-thickness 3D autologous human skin equivalent model developed as an in vitro testing platform.
Methods: Fifteen small molecule drugs were tested with Skimune™ to assess their sensitising capacity. Briefly, monocyte derived dendritic cells were incubated with or without the test compound. These primed cells were then incubated with autologous skin to induce tissue damage which was histopathologically graded. T-cell proliferation and interferon-gamma analysis was also completed. Autologous primary fibroblasts and keratinocytes were used to generate the full-thickness skin equivalent model. Histology and immunofluorescence for key protein markers was completed for comparison to normal skin. We co-cultured the model with activated periperhal blood mononuclear cells (PBMCs) and observed similar immune damage to that in our skin explant assay, Skimune™. Immunofluorescence staining for heat shock protein 70 (HSP70) as a marker of apoptosis was used to confirm these findings.
Results: Skimune™ was shown to correctly predict the sensitising capacity of small molecule drugs via T-cell proliferation and interferon-gamma release as well as Skimune™ assays. Histology demonstrated that Skimune™3D was representative of normal skin, showing positive staining for protein markers of epidermal differentiation and could be used to detect immune damage as shown by positive HSP70 staining.
Conclusions: Skimune™ provides vital information regarding the efficacy, potency, dose response and adverse reactions of novel drugs. We have also generated an autologous 3D skin equivalent model which is representative of normal human skin, a useful additional platform for testing.
P20 - Towards an in-vitro multi-cellular human airways model for evaluating the effects of electronic cigarettes
Vasanthi Bathrinarayanan, P. (1), Marshall, L.J. (2), Brown, J. E.P. (2), Leslie, L.J. (1)
(1) - School of Engineering and Applied Science, Aston University, Birmingham, B4 7ET, UK; (2) - School of Life and Health Sciences, Aston University, Birmingham, B4 7ET, UK
Despite being poor representations of human airways architecture, innumerable animals, especially rodents, have been used in cigarette smoking studies. The advent of Electronic-cigarettes (ECs) could herald a further escalation, with data from in-vivo experiments already increasingly published. The current study aims to demonstrate the application of a human in-vitro model for evaluation of EC, providing alternatives to the outdated in-vivo models.
The human airways model consists of relevant cell types that would be directly encountered during vaping. Human bronchial epithelial cells and pulmonary fibroblasts were co-cultured at air-liquid interface (ALI) under conditions that promote mucociliary differentiation, tight junction formation and mucin production. An in-house built smoking machine was used to deliver vapour from a commercially available EC (ECV) or whole cigarette smoke (WCS) to the co-culture model according to ISO standard. This methodology closely mimics human smoking behaviour, as opposed to enforced nasal inhalation in rodent smoking/vaping model.
24h post exposure, XTT cell viability analysis showed that WCS caused a significant decrease (p<0.0001) in cell viability (<70%) compared to control cells exposed to air only. ECV on the other hand did not have a significant impact on cell viability, thus suggesting low cytotoxicity. This difference in effect correlates with a number of existing in-vitro and in-vivo ECV/WCS studies, illustrating that the current model is a relevant, more realistic platform for EC studies compared to animal models.
Further, such an airways model resembling in-vivo physiology can be used to study COPD progression and development, a condition difficult to replicate in rodents.