Purpose We aimed to establish a rabbit model with retinal atrophy induced by an iatrogenic retinal pigment epithelium (RPE) removal, for future testing of the efficacy and safety of cell therapy strategies. Methods A localized detachment of the retina from the RPE/choroid layer was created in 18 pigmented rabbits. The RPE was removed by scraping with a custom-made extendable loop instrument. The resulting RPE wound was observed over a time course of 12 weeks with optical coherence tomography and angiography. After 4 days (group 1) and 12 weeks (group 2), histology was done and staining with hematoxylin and eosin, as well as immunofluorescence performed to further investigate the effects of debridement on the RPE and the overlying retina. Results Already after 4 days, we observed a closure of the RPE wound by proliferating RPE and microglia/macrophage cells forming a multilayered clump. This pattern continued over the observation time course of 12 weeks, whereby the inner and outer nuclear layer of the retina became atrophic. No neovascularization was observed in the angiograms or histology. The observed changes were limited to the site of the former RPE wound. Conclusions Localized surgical RPE removal induced an adjacent progressive retinal atrophy. Altering the natural course of this model may serve as a basis to test RPE cell therapeutics.

The mechanisms triggering the human immunodeficiency virus type I (HIV-1) to switch the coreceptor usage from CCR5 to CXCR4 during the course of infection are not entirely understood. While low CD4+ T cell counts are associated with CXCR4 usage, a predominance of CXCR4 usage with still high CD4+ T cell counts remains puzzling. Here, we explore the hypothesis that viral adaptation to the human leukocyte antigen (HLA) complex, especially to the HLA class II alleles, contributes to the coreceptor switch. To this end, we sequence the viral gag and env protein with corresponding HLA class I and II alleles of a new cohort of 312 treatment-naive, subtype C, chronically-infected HIV-1 patients from South Africa. To estimate HLA adaptation, we develop a novel computational approach using Bayesian generalized linear mixed models (GLMMs). Our model allows to consider the entire HLA repertoire without restricting the model to pre-learned HLA-polymorphisms. In addition, we correct for phylogenetic relatedness of the viruses within the model itself to account for founder effects. Using our model, we observe that CXCR4-using variants are more adapted than CCR5-using variants (p-value = 1.34e-2). Additionally, adapted CCR5-using variants have a significantly lower predicted false positive rate (FPR) by the geno2pheno[coreceptor] tool compared to the non-adapted CCR5-using variants (p-value = 2.21e-2), where a low FPR is associated with CXCR4 usage. Consequently, estimating HLA adaptation can be an asset in predicting not only coreceptor usage, but also an approaching coreceptor switch in CCR5-using variants. We propose the usage of Bayesian GLMMs for modeling virus-host adaptation in general.

Purpose Human induced pluripotent stem cell (hiPSC)-derived lung cell types such as alveolar epithelial cells are promising for toxicological and pharmaceutical in vitro screenings. Reproducible differentiation processes are highly demanded, but protocols which are suitable for the high-throughput generation of lung cell types from hiPSCs are lacking. Methods In this study, a new approach for the hiPSC-differentiation in alveolar epithelial-like cells type 2 under dynamic 3D-conditions in a suspension bioreactor is presented. Gene and protein expression analyses of key markers during the embryonal lung development have been performed in comparison to cells differentiated under static 2D-conditions to evaluate the differentiation efficacy of the new bioreactor-based approach. Finally, the resulting cells were infected by SARS-CoV-2 pseudotypes to demonstrate their functionality and suitability for e.g. COVID-19 drug development. Results The dynamic bioreactor is suitable to differentiate hiPSCs in spheroids, which express relevant lung markers in each developmental stage on gene and protein level. The 3D method is able to significantly increase the expression of some markers in comparison to conventional 2D differentiation. 3D-differentiated alveolar epithelial-like cells express functional SARS-CoV-2 receptors and can display the viral infection. Conclusion The presented dynamic 3D-differentiation is a promising, new approach to generate alveolar epithelial-like cells from hiPSCs as cell source for in vitro lung models.

In vitro model systems of the blood–brain barrier (BBB) play an essential role in pharmacological research, specifically during the development and preclinical evaluation of new drug candidates. Within the past decade, the trend in research and further development has moved away from models based on primary cells of animal origin towards differentiated models derived from human induced pluripotent stem cells (hiPSs). However, this logical progression towards human model systems from renewable cell sources opens up questions about the transferability of results generated in the primary cell models. In this study, we have evaluated both models with identical experimental parameters and achieved a directly comparable characterisation showing no significant differences in protein expression or permeability even though the achieved transendothelial electrical resistance (TEER) values showed significant differences. In the course of this investigation, we also determined a significant deviation of both model systems from the in vivo BBB circumstances, specifically concerning the presence or absence of serum proteins in the culture media. Thus, we have further evaluated both systems when confronted with an in vivo-like distribution of serum and found a notable improvement in the differential permeability of hydrophilic and lipophilic compounds in the hiPS-derived BBB model. We then transferred this model into a microfluidic setup while maintaining the differential serum distribution and evaluated the permeability coefficients, which showed good comparability with values in the literature. Therefore, we have developed a microfluidic hiPS-based BBB model with characteristics comparable to the established primary cell-based model.

As highlighted in the Minamata Convention, Mercury (Hg) in its various forms poses a substantial risk to human health and the environment. The health relevance of Hg is also recognized by the European Human Biomonitoring Initiative (HBM4EU), which classifies Hg as a priority substance, since considerable knowledge and data gaps on Hg exposure levels and their changes over time still exist in Europe. The German Environmental Specimen Bank (German ESB) provides valuable policy relevant data and long-term trends of substance exposure on a national level for international comparison and evaluation. In this study we analysed data of the German ESB on Hg exposure of young adults aged 20 to 29 including data on urinary Hg levels from 1995 to 2018 and whole blood Hg levels from 2001 to 2010. Results show a clear decrease in both, about 86% in urine total daily Hg excretion from 1995 (0.76 μg/L) to 2018 (0.11 μg/L) (n = 10,069) and about 57% in blood concentrations of Hg from 2001 (1.76 μg/L) to 2010 (0.77 μg/L) (n = 4085). Over the investigated timeframe only a few values exceeded the toxicologically derived health based guidance value HBM I for blood and urine, with these exceedances decreasing over time in line with the general trend. The factors mostly influencing Hg excretion identified in this study are dental amalgam as well as fish and seafood consumption. Besides other factors (e.g. age and sex), also airborne Hg exposure appears to be a low but evident influencing factor in Germany. Although a considerable decrease in internal Hg exposure is recognized in the last decades, the current low-level exposure may cause adverse health effects especially to vulnerable groups such as pregnant women and children. To further elucidate and evaluate current exposure sources and to reduce human exposure to Hg, continuous environmental and human biomonitoring is needed.

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

A better understanding of their interaction with cell-based tissue is a fundamental prerequisite towards the safe production and application of engineered nanomaterials. Quantitative experimental data on the correlation between physicochemical characteristics and the interaction and transport of engineered nanomaterials across biological barriers, in particular, is still scarce, thus hampering the development of effective predictive non-testing strategies. Against this background, the presented study investigated the translocation of gold and silver nanoparticles across the gastrointestinal barrier along with related biological effects using an in vitro 3D-triple co-culture cell model. Standardized in vitro assays and quantitative polymerase chain reaction showed no significant influence of the applied nanoparticles on both cell viability and generation of reactive oxygen species. Transmission electron microscopy indicated an intact cell barrier during the translocation study. Single particle ICP-MS revealed a time-dependent increase of translocated nanoparticles independent of their size, shape, surface charge, and stability in cell culture medium. This quantitative data provided the experimental basis for the successful mathematical description of the nanoparticle transport kinetics using a non-linear mixed effects modeling approach. The results of this study may serve as a basis for the development of predictive tools for improved risk assessment of engineered nanomaterials in the future.

Microfluidic technology is a valuable tool for realizing more in vitro models capturing cellular and organ level responses for rapid and animal-free risk assessment of new chemicals and drugs. Microfluidic cell-based devices allow high-throughput screening and flexible automation while lowering costs and reagent consumption due to their miniaturization. There is a growing need for faster and animal-free approaches for drug development and safety assessment of chemicals (Registration, Evaluation, Authorisation and Restriction of Chemical Substances, REACH). The work presented describes a microfluidic platform for in vivo-like in vitro cell cultivation. It is equipped with a wafer-based silicon chip including integrated electrodes and a microcavity. A proof-of-concept using different relevant cell models shows its suitability for label-free assessment of cytotoxic effects. A miniaturized microscope within each module monitors cell morphology and proliferation. Electrodes integrated in the microfluidic channels allow the noninvasive monitoring of barrier integrity followed by a label-free assessment of cytotoxic effects. Each microfluidic cell cultivation module can be operated individually or be interconnected in a flexible way. The interconnection of the different modules aims at simulation of the whole-body exposure and response and can contribute to the replacement of animal testing in risk assessment studies in compliance with the 3Rs to replace, reduce, and refine animal experiments.

The development of new tumor models for anticancer drug screening is a challenge for preclinical research. Conventional cell-based in vitro models such as 2D monolayer cell cultures or 3D spheroids allow an initial assessment of the efficacy of drugs but they have a limited prediction to the in vivo effectiveness. In contrast, in vivo animal models capture the complexity of systemic distribution, accumulation, and degradation of drugs, but visualization of the individual steps is challenging and extracting quantitative data is usually very difficult. Furthermore, there are a variety of ethical concerns related to animal tests. In accordance with the 3Rs principles of Replacement, Reduction and Refinement, alternative test systems should therefore be developed and applied in preclinical research. The Hen's egg test on chorioallantoic membrane (HET-CAM) model provides the generation of vascularized tumor spheroids and therefore, is an ideal test platform which can be used as an intermediate step between in vitro analysis and preclinical evaluation in vivo. We developed a HET-CAM based intestine tumor model to investigate the accumulation and efficacy of nano-formulated photosensitizers. Irradiation is necessary to activate the phototoxic effect. Due to the good accessibility of the vascularized tumor on the CAM, we have developed a laser irradiation setup to simulate an in vivo endoscopic irradiation. The study presents quantitative as well as qualitative data on the accumulation and efficacy of the nano-formulated photosensitizers in a vascularized intestine tumor model.

Biosensors become increasingly relevant for medical diagnostics, pharmaceutical industry, and environmental technology, for example, to test new drugs easily and reliably or to detect cell growth in changing environmental conditions. Novel materials like graphene are promising candidates to produce biosensors on an industrial scale by means of printing processes. To reach this aim, methods for the reliable and automated production of electrode structures and their coating are required. We present an impedance biosensor in the format of a microtiter plate, fabricated by highly efficient roll-to-roll printing of graphene-based microstructures on large-area polymer foils. Proof-of-principle experiments show the evidence of the suitability of the printed graphene biosensors for impedance-based monitoring of viral cytopathogenicity and its inhibition in the presence of antiviral drugs. The developed system is a promising approach toward cost-efficient impedimetric biosensors for high-throughput screening in vaccine research and antiviral drug development.

For the treatment of intestinal tumors by oral administration of drugs, the mucus layer of the intestine represents a barrier, but also a target possibility for drug. The use of nanoparticular drug delivery systems is an important tool to improve the transport and accumulation of the drug on the target side. Therefore, specific in vitro test systems to simulate the conditions of an intestine tumor are necessary for the screening and efficiency testing of potential new nanoparticular drug delivery systems. Here, we introduce a microfluidic 3D intestine tumor spheroid model, which allows to test functionalized nanoparticles and their ability to adsorb onto and permeate over a mucus layer under dynamic conditions. More specifically, the model consists of a 3D tumor spheroid with a mucus layer and microvilli on the surface, which is placed into a microfluidic chip system. The system incorporates a fluid flow, which is an important factor in nanoparticle residence time and uptake. To test the microfluidic 3D intestine tumor spheroid model, two different nanoparticle systems surfaces-coated with Carbopol® or Pluronic® F127 were produced to test the adsorption and permeation over the mucus layer. The data showed obvious adsorptive and permeable properties of uncoated and surface-coated nanoparticles in the static and dynamic system. The 3D intestine tumor spheroid model has the potential to significantly advance the preclinical development of new drugs like nanoparticles.

Lead is a ubiquitous pollutant with well-known effects on human health. As there is no lower toxicological threshold for lead in blood and since data gaps on lead exposure still exist in many European countries, HBM data on lead is of high importance. To address this, the European Human Biomonitoring Initiative HBM4EU classified lead as a priority substance. The German Environmental Specimen Bank (German ESB) has monitored lead exposure since more than 35 years. Using data from the early 1980s to 2019 we reveal and discuss long-term trends in blood lead levels (BLLs) and current internal exposure of young adults in Germany. BLLs in young adults decreased substantially in the investigated period. As results from the ESB sampling site Muenster demonstrate, the geometric mean of BLLs of young adults decreased from 1981 (78,7 μg/L) to 2019 (10.4 μg/L) by about 87%. Trends in human exposure closely correlate with air lead levels (ALLs) provided by the European Monitoring and Evaluation Programme (EMEP). Hence, the decrease of BLLs largely reflects the drop in air lead pollution. Known associations of sex, smoking, alcohol consumption, and housing situation with BLLs are confirmed with data of the German ESB. Although internal lead exposure in Germany decreased substantially, the situation might be different in other European countries. Since 2010, BLLs of young adults in Germany levelled out at approximately 10 μg/L. The toxicity of lead even at low levels is known to cause adverse health effects especially in children following exposure of the child or the mother during pregnancy. To identify current exposure sources and to minimize future lead exposure, continuous monitoring of lead intake and exposure levels is needed.

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric β-cyclodextrin derivates is described, which result from the attachment of a hydrophilic β-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized β-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (β-estradiol, dexamethasone) and compared to monomeric β-cyclodextrin derivates regarding solubilization and complexation efficiency. The β-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of β-estradiol, higher solubilities could be achieved by complexation with both synthesized β-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized β-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like β-estradiol.

Human biomonitoring (HBM) depends on high-quality human samples to identify status and trends in exposure and ensure comparability of results. In this context, much effort has been put into the development of standardized processes and quality assurance for sampling and chemical analysis, while effects of sample storage and shipment on sample quality have been less thoroughly addressed. To characterize the currently applied storage and shipment procedures within the consortium of the European Human Biomonitoring Initiative (HBM4EU), which aims at harmonization of HBM in Europe, a requirement analysis based on data from an online survey was conducted. In addition, the online survey was addressed to professionals in clinical biobanking represented by members of the European, Middle Eastern and African Society for Biopreservation and Biobanking (ESBB) to identify the current state-of-the-art in terms of sample storage and shipment. Results of this survey conducted in these two networks were compared to detect processes with potential for optimization and harmonization. In general, many similarities exist in sample storage and shipment procedures applied by ESBB members and HBM4EU partners and many requirements for ensuring sample quality are already met also by HBM4EU partners. Nevertheless, a need for improvement was identified for individual steps in sample storage, shipment, and related data management with potential impact on sample and data quality for HBM purposes. Based on these findings, recommendations for crucial first steps to further strengthen sample quality, and thus foster advancement in HBM on a pan-European level are given.

PURPOSE:To analyse the cytotoxic and antiproliferative effect of methotrexate (MTX) and fluorouracil (5-FU) in vitro on fibroblasts, retinal pigment epithelial (RPE) and photoreceptor cells as an adjunct for reducing the incidence of proliferative vitreoretinopathy (PVR) after rhegmatogenous retinal detachment surgery.METHODS:Methotrexate and 5-FU were dissolved separately in balanced salt solution (BSS) with concentrations ranging from 0-8000 µg/ml and 0-4000 µg/ml, respectively. All solutions were analysed in terms of pH and osmolarity and applied for 1 h to fibroblasts (BJ), RPE (ARPE-19) and photoreceptor (661W) cell lines adherently cultivated in 96-well cell culture plates (10 000 cells/well). 24 h after incubation, the proliferative (BrdU), metabolic (CellTiter-Glo) and apoptotic (Caspase 3/7) activity of the cells were examined in vitro.RESULTS:5-FU had an antiproliferative effect on BJ and ARPE-19 cells starting from low concentrations (2 µg/ml). However, the viability of 661W cells decreased and apoptosis was induced with increasing 5-FU concentration. In contrast, MTX up to a concentration of 266 µg/ml did neither result in a significant loss of viability nor in increased caspase 3/7 activity of BJ, ARPE-19 and 661W cells and inhibited the proliferation of ARPE-19 already at low concentrations starting from 8 µg/ml.CONCLUSIONS:Methotrexate dissolved in BSS is biocompatible up to a concentration of 266 µg/ml and may act as an intraoperative rinse solution to inhibit RPE proliferation in PVR-diseased eyes. Contrary, the use of 5-FU within the posterior segment of the eye is limited by its cell-damaging effect on photoreceptor cells.

A major challenge of drug discovery is to overcome the mucus barrier, which is lined the surface of the gastrointestinal tract. The ability to overcome this barrier must be tested in preclinical in vitro models, prior to in vivo studies. Therefore, a microfluidic chip was developed to examine the possibility of compounds to overcome a mucus barrier. Native porcine intestinal mucus was used as a mucus model. The mucus was loaded inside the chip comprising of a microporous membrane and a fluidic compartment. To validate the system, caffeine as a hydrophilic positive compound, fluorescein isothiocyanate-dextran (FITC-dextran) as high molecular negative compound and diclofenac sodium as poorly permeating compound were tested. Test samples were applied on the surface of the mucus and samples were collected every 30 min from the fluidic compartment underneath the membrane. The permeation was measured over 3 h and the permeated compound was detected by high-pressure liquid chromatography (HPLC) and a microplate fluorescence reader. The results show that caffeine and diclofenac sodium are permeating over the mucus layer proportionally to the time. FITC-dextran, chosen as non-permeating compound, displays equivalently no permeation. The validation provides the functionality of the developed mucus-chip system.

The anti-HIV activity of a new humic substance-derived preparation has been studied in individual pools of immune cells (CD4+ T lymphocytes, macrophages, dendritic cells). Near-complete inhibition of the HIV infection (by more than 90%) was achieved by treating each of the abovementioned cell types with non-toxic concentrations of the preparation. The inhibitory effect demonstrates the possibility of preventing the depletion of a significant portion of functionally important immune cells. A comparative study of infection inhibition in individual cell pools has allowed us to reveal the differences in the preparation's effectiveness in each of the cell populations. A R5-tropic HIV-1 infection in macrophages exhibited maximum sensitivity to the preparation: 90% and 50% inhibition of the infection were observed in the presence of concentrations as low as 1.4 and 0.35 μg/ml, respectively. A 15- and 19-fold higher concentration was required to achieve the same extent of inhibition in dendritic cells infected with the same strain. The effectiveness of the drug in CD4 + T lymphocytes is quite comparable to its effectiveness in macrophages. The drug is universally effective for both the T- and M-tropic variants of HIV-1.

Serum supplementation is crucial in in vitro cell culture to provide all the essential nutrients needed for cellular processes. Fetal bovine serum (FBS) is considered the 'gold standard', but its production raises serious ethical concerns. Human-derived alternatives to FBS exist in the form of human platelet lysates (hPLs) or human AB serum (ABS). However, these serum products are usually pooled from several donors, in order to have a standardised product without patient-specific deviations. Nevertheless, the use of patient-specific serum in cell culture might be the key to successful transplantation of the cultured cells in medical applications, particularly as it avoids the transmission of infectious components or xenogenic proteins. In addition, the production of non-pooled hPL from single donors is likely to be a cost-effective and time-saving method. The current study used hPL units isolated from single donors and tested their performance as medium supplements for cell culture in comparison with FBS or ABS. This proof-of-concept study aimed to assess the potential of non-pooled hPL for personalised serum supplementation, and thus optimise in vitro models by making them more relevant to human physiology. We showed that A549, HepG2 and Caco-2 human cell lines were generally able to adapt to the new culture conditions and maintain viability, morphology and certain cell-specific characteristics. These results indicate that non-pooled, single patient-derived hPL could be a suitable alternative for in vitro serum supplementation.