Spotlight on Three Rs Progress

3-D Printing Used to Create a Diabetic Skin Model

A human cell-based in vitro model of diabetic skin has been generated by using 3-D printing. An article published in Biomaterials¹ describes how the team hypothesised that a dermal layer loaded with diabetic human dermal fibroblasts (dHDFs) would guide normal human epidermal keratinocytes toward a diabetic phenotype, while forming mature epidermal layers. The aim was that this process would lead to the development of a mature diabetic full-thickness (epidermis/dermis) skin model. By exploiting the versatility of the 3-D printing process, the authors were able to include a hypodermal layer containing diabetic human preadipocytes and a perfusable vascular channel underneath the dermal layer. Histological and gene expression analysis of the resulting stratified epidermal layers revealed delayed re-epithelialisation, which is a key feature of diabetic skin. They also reported that the diabetic skin model exhibited increased insulin resistance, vascular dysfunction, adipose hypertrophy and pro-inflammatory responses. The functionality of the in vitro model was assessed by administering test drugs through the vascular channel, which reduced the pro-inflammatory response and restored normal epidermal function.

Reference

1. Kim BS, Ahn M, Cho WW, et al. Engineering of diseased human skin equivalent using 3D cell printing for representing pathophysiological hallmarks of type 2 diabetes in vitro. Biomaterials 2021; 272: 120776. DOI: 10.1016/j.biomaterials.2021.120776

Parkinson’s Disease In Vitro Model Generated with Human Cells

Parkinson’s disease (PD) is a yet incurable disease, mainly caused by the degeneration and eventual loss of dopaminergic neurons of the substantia nigra in the ventral mid-brain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD (with G2019S being the most common LRRK2 mutation), which makes this kinase an interesting target for drug development. To fully understand the role of LRRK2 in PD, and to help identify and test potential therapies, appropriate disease models are crucial. Neuroprotective compounds are usually tested on cell models, such as induced pluripotent stem cell-based models. However, these current systems either lack physiological relevance to dopaminergic neurons or are too complex and/or costly to be used for screening purposes. To address this unmet need, Calamini et al.¹ developed a PD model with Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons that overexpressed wild-type and G2019S LRRK2 proteins. The team showed that LRRK2 overexpression did not interfere with normal LUHMES neuronal cell differentiation. However, in the G2019S cell line, overexpression of mutant LRRK2 caused PD-relevant phenotypes, such as nuclear, mitochondrial and lysosomal alterations.

The use of LUHMES cells offers several advantages, such as physiological relevance, the possibility of genetic manipulation to study genes of interest, and ease of maintenance and expansion (i.e. they are amenable to high-throughput screening of compounds). This model combines biological relevance and higher throughput — it effectively permits the study of mechanisms linked to G2019S LRRK2-mediated dopaminergic neural dysfunction and phenotypic screening. However, as it is a relatively simple model, findings will need to be further validated in more-complex systems.

Reference

1. Calamini B, Geyer N, Huss-Braun N, et al. Development of a physiologically relevant and easily scalable LUHMES cell-based model of G2019S LRRK2-driven Parkinson’s disease. Dis Model Mech 2021; 14(6): dmm048017. DOI: 10.1242/dmm.048017

Brain Organoids Used To Study Gene–Environment Interactions in Autism

Autism spectrum disorder (ASD) is caused by complex (epi)genetic and environmental interactions, but the actual mechanisms involved remain unclear. Furthermore, research into environment–gene associations and possible synergies is complicated by the susceptibility of the developing brain to numerous insults and the heterogenous genetics of ASD. In an attempt to uncover links between a single toxicant and a specific gene, a recent study employed an in vitro human brain cell model with a mutation in the gene encoding chromodomain helicase DNA binding protein 8 (CHD8), which is a high-risk gene for ASD, to determine the effects of exposure to an organophosphate pesticide (chlorpyrifos; CPF). The model was generated with human induced pluripotent stem cells (iPSC)-derived 3-D brain organoids (BrainSpheres) with CRISPR/Cas9-engineered CHD8 heterozygous knockout. Their findings — that cells with the CHD8 mutation exhibited metabolic perturbations upon exposure to the toxicant — suggest a synergy between a high-risk gene and environmental exposure. This model could be particularly useful for the study of the molecular mechanisms of such interactions, as the authors showed that CHD^+/− cells exposed to CPF had lower levels of the CHD8 protein but not mRNA levels, suggesting post-translational interaction. Their metabolomics analysis, which showed that alterations of some metabolites associated with ASD were also affected by the CHD8 mutation and CPF treatment, demonstrated the value of comparing in vitro findings with human clinical data, and further confirmed potential synergies between the CHD8 mutation and CPF exposure. The authors pointed out that: “This study demonstrates the usefulness of an approach comparing human data with data obtained in vitro to identify possible biomarkers of exposure, as well as to identify ASD-relevant molecular networks perturbed by chemical exposure.”¹

Reference

1. Calamini B, Geyer N, Modafferi S, Zhong X, Kleensang A, et al. Gene–environment interactions in developmental neurotoxicity: A case study of synergy between chlorpyrifos and CHD8 knockout in human BrainSpheres. Environ Health Perspect 2021; 129: 77001. DOI: 10.1289/EHP8580.

Winner of NC3Rs 2020 Prize Announced

The 2020 NC3Rs 3Rs Prize has been awarded to Dr Laura Pellegrini and colleagues, at the MRC Laboratory of Molecular Biology, for work on the development of cerebral organoids representative of the choroid plexus, which is the protective barrier between the blood and the cerebrospinal fluid (CSF).¹ The winning paper, which was published in Science,² describes several novel elements in in vitro neurological research, from accurately modelling the choroid plexus barrier function to the ability of the organoids to produce and store cerebrospinal-like fluid. The authors pointed out that: “Human choroid plexus organoids provide an easily tractable system to study the key functions of this organ: CSF secretion and selective transport into the CNS. As such, they can predict CNS permeability of new compounds to aid in the development of neurologically relevant therapeutics. They also provide a source of more authentic CSF and can be used to understand development of this key organ in brain development and homeostasis.”²

This prize awards a piece of primary research, published in the last three years, that describes outstanding and original work on any of the Three Rs. It is open to researchers in academia or industry, including those based outside of the UK, and consists of a £28,000 prize grant and a £2000 personal award.

Reference

1. Cerebral organoid model wins 3Rs Prize, https://nc3rs.org.uk/news/cerebral-organoid-model-wins-3rs-prize (2021, accessed 15 July 2021).

2. Pellegrini L, Bonfio C, Chadwick J, et al. Human CNS barrier-forming organoids with cerebrospinal fluid production. Science 2020; 369: eaaz5626.

Review on New Approach Methodologies

EU legislation advocates for a change in the way toxicity testing is conducted, and aims to progressively shift in vivo studies toward non-animal methods. However, a thorough understanding of the current legislative requirements regarding human health effects is essential to identify knowledge gaps and determine how non-animal methods can be incorporated into testing practices. While several endpoints (e.g. skin corrosion and irritation, mutagenicity) can be assessed with in vitro and computational approaches, systemic effects (e.g. acute toxicity) still tend to use animal studies. A review by Pistollato and colleagues¹ summarises the EU regulatory framework under REACH and the Cosmetic Products Regulation, and the requirements for the assessment of risk to human health. More specifically, they highlight the critical human health-related endpoints (skin corrosion and irritation, serious eye damage/eye irritation, photo-induced toxicity, mutagenicity/genotoxicity, acute toxicity, skin sensitisation, repeated dose toxicity, carcinogenicity, reproductive and developmental toxicity, absorption, distribution, metabolism and excretion (ADME) and toxicokinetics), and identify the main challenges in introducing alternative methods into regulatory testing practice. In addition, the authors also mention recent initiatives at the EU level that aim to promote the development and the dissemination of alternative methods and approaches.

Reference

1. Pistollato F, Madia F, Corvi R, et al. Current EU regulatory requirements for the assessment of chemicals and cosmetic products: challenges and opportunities for introducing new approach methodologies. Arch Toxicol 2021; 95: 1867–1897.

New OECD In Vitro Test Guidelines Accepted

The Organisation for Economic Co-operation and Development (OECD) has accepted two new in vitro test guidelines (TGs) under the OECD Guidelines for the Testing of Chemicals, Health Effects. These new guidelines, TG 497 and TG 498, can be used to identify and characterise the potential hazard of chemicals in terms of skin sensitisation and phototoxicity, respectively.

TG 497¹ covers defined approaches on skin sensitisation, which consist of the use of optimised combinations of information sources (e.g. in silico, in chemico or in vitro data) to come to a rules-based conclusion on the potential dermal sensitisation hazard of a chemical. The defined approaches in this guideline have been shown to provide at least the same level of information (i.e. sensitiser versus non-sensitiser) as the murine Local Lymph Node Assay (OECD TG 429).

TG 498² addresses phototoxicity with the Reconstructed Human Epidermis Phototoxicity test method. This guideline advises on the assessment of the phototoxic potential of a chemical after topical application on reconstructed human epidermis (RhE) tissue in the presence and absence of simulated sunlight. The chemical’s photo irritation potential is determined by the relative reduction of cell viability in the presence of simulated sunlight compared to the cell viability in the absence of simulated sunlight.

Reference

1. OECD. Guideline No. 497: Defined approaches on skin sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4. Paris, France: OECD Publishing, 2021, 54 pp.

2. OECD. Test No. 498: In vitro phototoxicity — reconstructed human epidermis phototoxicity test method, OECD Guidelines for the Testing of Chemicals, Section 4. Paris, France: OECD Publishing, 2021, 28 pp.

EPAA Refinement Prize 2021: Call for Submissions

The European Partnership for Alternative Approaches to Animal Testing (EPAA) has announced its Refinement Prize 2021, which will be granted to laboratory technicians, animal caretakers or technologists, for their work on novel approaches to advancing the implementation of refinements in animal testing and/or raising awareness of refinement issues. The purpose of this prize is to target those individuals who carry out much of the work for regulatory safety and efficacy testing, and are ideally placed to apply refinement strategies.

The award has a monetary value of €6000, and is open to applications from individuals or teams (teams must consist of at least one laboratory technician, technologist, or animal caretaker). Applications close on 9 September 2021.

Reference

1. EPAA. Refinement Prize 2021: Call for submissions, https://ec.europa.eu/growth/content/refinement-prize-2021-call-submissions_en (2021, accessed 20 July 2021).