Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo -like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.

In early preclinical drug development, potential candidates are tested in the laboratory using isolated cells. These in vitro experiments traditionally involve cells cultured in a two-dimensional monolayer environment. However, cells cultured in three-dimensional spheroid systems have been shown to more closely resemble the functionality and morphology of cells in vivo . While the increasing usage of hepatic spheroid cultures allows for more relevant experimentation in a more realistic biological environment, the underlying physical processes of drug transport, uptake and metabolism contributing to the spatial distribution of drugs in these spheroids remain poorly understood. The development of a multiscale mathematical modelling framework describing the spatio-temporal dynamics of drugs in multicellular environments enables mechanistic insight into the behaviour of these systems. Here, our analysis of cell membrane permeation and porosity throughout the spheroid reveals the impact of these properties on drug penetration, with maximal disparity between zonal metabolism rates occurring for drugs of intermediate lipophilicity. Our research shows how mathematical models can be used to simulate the activity and transport of drugs in hepatic spheroids and in principle any organoid, with the ultimate aim of better informing experimentalists on how to regulate dosing and culture conditions to more effectively optimize drug delivery.

Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.

There are no physical or visual manifestations that define skin sensitivity or irritation; a subjective diagnosis is made on the basis of the evaluation of clinical presentations, including burning, prickling, erythema, and itching. Adverse skin reaction in response to topically applied products is common and can limit the use of dermatological or cosmetic products. The purpose of this study was to evaluate the use of human skin equivalents based on immortalized skin keratinocytes and evaluate the potential of a 22-gene panel in combination with multivariate analysis to discriminate between chemicals known to act as irritants and those that do not. Test compounds were applied topically to full-thickness human skin equivalent or human ex vivo skin and gene signatures determined for known irritants and nonirritants. Principle component analysis showed the discriminatory potential of the 22-gene panel. Linear discrimination analysis, performed to further refine the gene set for a more high-throughput analysis, identified a putative seven-gene panel (IL-6, PTGS2, ATF3, TRPV3, MAP3K8, HMGB2, and matrix metalloproteinase gene MMP-3) that could distinguish potential irritants from nonirritants. These data offer promise as an in vitro prediction tool, although analysis of a large chemical test set is required to further evaluate the system.

Chemical-induced skin irritancy or sensitivity to topically applied dermatological agents, cosmetics, skin-care products, detergents or other compounds is a common problem. Skin sensitivity relates only to those individuals who are susceptible to a particular chemical, whereupon it produces paresthetic sensations, whereas skin irritation is related to the characteristics of a chemical that, when applied, will induce irritation on all subjects. 1 Several patient or animal-based assays have been used to identify potentially irritant chemicals, but none of these tests provide a clear standardised measurable outcome and are not compatible with high-throughput testing that is required by industry, particularly for first-pass drug screening. This, along with the legislative move toward non-animal testing, has prompted the development of several in vitro assays for skin irritation.

Aim To determine the clinical and biochemical variables associated with change in HbA1c in patients with type 2 diabetes who start sodium‐glucose linked transporter (SGLT) inhibitor therapy. Methods We performed a prospective cohort study (ACTRN12616000833460) of 48 adults (30 male, 18 female) with type 2 diabetes who attended a tertiary hospital diabetes clinic. Fasting serum and urine samples, collected during clinic visits prior to and at 1, 12 and 24 weeks after commencing SGLT inhibitor treatment, were analysed for HbA1c, electrolytes, urea, creatinine and glucose. Results After 12 weeks, SGLT inhibitor therapy was associated with respective median (97% CI) decreases in weight, blood pressure, HbA1c and urine albumin/creatinine ratio of 3.0 (1.7–3.4) kg, 8 (2–16)/4 (3–9) mmHg, 6 (3–14) mmol/mol and 0.69 (0.18–1.8) mg/mmol. These effects persisted to 24 weeks. Urinary frequency and genitourinary infection were common adverse effects. Baseline HbA1c and eGFR independently predicted ΔHbA1c at 12 weeks whereas only baseline HbA1c independently predicted ΔHbA1c at 24 weeks. Urinary fractional glucose excretion and change in fasting glucose 1 week after starting SGLT inhibitor did not contribute to prediction of glycaemic response. Conclusions SGLT inhibitor therapy in a hospital clinic setting was associated with clinical improvements comparable to those observed in clinical trials but with higher incidence of genitourinary side‐effects. Baseline HbA1c and eGFR, but not urine fractional glucose excretion, predicted glycaemic response.