The capacity for social media to influence the utilization of re-purposed medicines to manage COVID-19, despite limited availability of safety and efficacy data, is a cause for concern within health care systems. This study sought to ascertain links between social media reports and utilization for three re-purposed medicines: hydroxychloroquine (HCQ), ivermectin and colchicine. A combined retrospective analysis of social media posts for these three re-purposed medicines was undertaken, along with utilization and clinical trials data, in South Africa, between January 2020 and June 2021. In total, 77,257 posts were collected across key social media platforms, of which 6884 were relevant. Ivermectin had the highest number of posts (55%) followed by HCQ (44%). The spike in ivermectin use was closely correlated to social media posts. Similarly, regarding chloroquine (as HCQ is not available in South Africa), social media interest was enhanced by local politicians. Sentiment analysis revealed that posts regarding the effectiveness of these repurposed medicines were positive. This was different for colchicine, which contributed only a small number of mentions (1%). Of concern is that the majority of reporters in social media (85%) were unidentifiable. This study provides evidence of social media as a driver of re-purposed medicines. Healthcare professionals have a key role in providing evidence-based advice especially with unidentifiable posts.

Hepatotoxicity remains a major challenge in drug development despite preclinical toxicity screening using hepatocytes of human origin. To overcome some limitations of reproducing the hepatic phenotype, more structurally and functionally authentic cultures in vitro can be introduced by growing cells in 3D spheroid cultures. Characterisation and reproducibility of HepG2 spheroid cultures using a high-throughput hanging drop technique was performed and features contributing to potential phenotypic variation highlighted. Cultured HepG2 cells were seeded into Perfecta 3D® 96-well hanging drop plates and assessed over time for morphology, viability, cell cycle distribution, protein content and protein-mass profiles. Divergent aspects which were assessed included cell stocks, seeding density, volume of culture medium and use of extracellular matrix additives. Hanging drops are advantageous due to no complex culture matrix being present, enabling background free extractions for downstream experimentation. Varying characteristics were observed across cell stocks and batches, seeding density, culture medium volume and extracellular matrix when using immortalized HepG2 cells. These factors contribute to wide-ranging cellular responses and highlights concerns with respect to generating a reproducible phenotype in HepG2 hanging drop spheroids.

Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may influence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to define the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the influence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profiling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatography-tandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confidently identified, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.

Background: Standardizing in vitro pre-clinical hepatotoxicity is confounded by diverse cellular origins which often lack representative hepatocellular function. Dedifferentiation and loss of cellular polarity are inherent limitations introduced by long term cell culture. HepG2 cells, with a stable phenotype in early passage, have been extensively used in hepatotoxicity screening despite an apparent poor metabolic competence and "foetal-like" hepatic phenotype. Three dimensional (3D) culture techniques may serve to better preserve the cellular phenotype. One strategy to assess the differences in hepatic phenotypes is assessing the proteome. The aim of this research was to determine abundance changes in the hepatic proteome of HepG2 cells when cultured in "hanging drop" 3D cultures. Methods: Replicates of HepG2 cells (as monolayers and spheroids) were collected as whole cell lysates. Fifty micrograms of sample protein was reduced, alkylated, precipitated, digested with trypsin and labelled using isobaric tagging. Samples were analysed using a Dionex Ultimate 3000 RSLCnano LC system coupled to a Thermo Scientific Fusion Orbitrap Mass Spectrometer. Peak lists were searched against a UniProtKB/Swiss-Prot human database using SearchGUI version 2.3.1 with X!Tandem, MS-GF+ and Comet search engines. Targeted acquisitions were carried out using Skyline software. Results: Once digitally filtered, 4701, 5030 and 4932 proteins were identified and quantified across replicates 1 to 3 with a protein overlap of 3760. While distinct hepatic marker profiles varied between samples, several notable proteins were upregulated across culture models. Among these were hallmark hepatic proteins albumin and alpha-fetoprotein which showed 3.6 and 6.1-fold increases respectively. Canalicular specific marker proteins such as dipeptidyl peptidase 4 were identified with a 2-fold up-regulated in spheroid cultures. Hepatic structural markers such as hepatocyte cell adhesion molecule, collagen, laminin and fibronectin were also up-regulated by more than 2-fold. Several phase I metabolizing enzymes and membrane transporter proteins showed variable up-regulation. Conclusion: Hallmark hepatic proteins were largely up-regulated, demonstrating alterations in the hepatic phenotype, when cultured in 3D spheroid free from synthetic surfaces. Quantitation of large protein cohorts demonstrated the ability of 3D spheroids to induce changes in the HepG2 cell proteome, these changes could be advantageous in promoting maintenance of a stable hepatic phenotype.