Frankenstein's followers. Maintenance of human cells outside the body. Issue 1.

Although science can endeavour to do a great many things, unachievable thus far, these activities should be, but seldom are, tempered with the question, should we really do it? This is not necessarily implying a moral code to scientific activity, but at least suggests that we probably should consider the long-term consequences of certain scientific activities to human society and the environment. Indeed, scientists have struggled with the consequences of their discoveries, not least Nobel himself, who set up the Nobel prize as a reaction to being called "The father of death", due to his discovery and financial success with dynamite. Here, we set out the basis for a series of articles entitled, Frankenstein's Followers, Maintenance and propagation of human cells outside the body.

Application of RPTEC/TERT1 cells for investigation of repeat dose nephrotoxicity: A transcriptomic study.

The kidney is a major target organ for toxicity. Incidence of chronic kidney disease (CKD) is increasing at an alarming rate due to factors such as increasing population age and increased prevalence of heart disease and diabetes. There is a major effort ongoing to develop superior predictive models of renal injury and early renal biomarkers that can predict onset of CKD.

A SAGE based approach to human glomerular endothelium: defining the transcriptome, finding a novel molecule and highlighting endothelial diversity.

Background: Large scale transcript analysis of human glomerular microvascular endothelial cells (HGMEC) has never been accomplished. We designed this study to define the transcriptome of HGMEC and facilitate a better characterization of these endothelial cells with unique features. Serial analysis of gene expression (SAGE) was used for its unbiased approach to quantitative acquisition of transcripts.

Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel.

Claudins are the major proteins of the tight junctions and the composition of claudin subtypes is decisive for the selective permeability of the paracellular route and thus tissue specific function. Their regulation is complex and subject to interference by several factors, including oxidative stress. Here we show that exposure of cultured human proximal tubule cells (RPTEC/TERT1) to the immunosuppressive drug cyclosporine A (CsA) induces an increase in transepithelial electrical resistance (TEER), a decrease in dome formation (on solid growth supports) and a decrease in water transport (on microporous growth supports).

Delineation of the key aspects in the regulation of epithelial monolayer formation.

The formation, maintenance, and repair of epithelial barriers are of critical importance for whole-body homeostasis. However, the molecular events involved in epithelial tissue maturation are not fully established. To this end, we investigated the molecular processes involved in renal epithelial proximal-tubule monolayer maturation utilizing transcriptomic, metabolomic, and functional parameters.

Application of integrated transcriptomic, proteomic and metabolomic profiling for the delineation of mechanisms of drug induced cell stress.

High content omic techniques in combination with stable human in vitro cell culture systems have the potential to improve on current pre-clinical safety regimes by providing detailed mechanistic information of altered cellular processes. Here we investigated the added benefit of integrating transcriptomics, proteomics and metabolomics together with pharmacokinetics for drug testing regimes. Cultured human renal epithelial cells (RPTEC/TERT1) were exposed to the nephrotoxin Cyclosporine A (CsA) at therapeutic and supratherapeutic concentrations for 14days.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E.

Carcinogens induce loss of the primary cilium in human renal proximal tubular epithelial cells independently of effects on the cell cycle.

The primary cilium is an immotile sensory and signaling organelle found on the majority of mammalian cell types. Of the multitude of roles that the primary cilium performs, perhaps some of the most important include maintenance of differentiation, quiescence, and cellular polarity. Given that the progression of cancer requires disruption of all of these processes, we have investigated the effects of several carcinogens on the primary cilium of the RPTEC/TERT1 human proximal tubular epithelial cell line.

Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media.

The search for alternatives to the use of fetal bovine serum (FBS) in cell and tissue culture media has become a major goal in terms of the 3R principles in order to reduce or to avoid harvesting of FBS from bovine fetuses, and, in terms of Good Manufacturing Practice (GMP), to ensure safe and animal product-free conditions for biomedical tissue engineering and (adult) stem cell therapy, respectively. In the present study, we investigated the feasibility of using platelet lysates (PL) as a substitute for FBS, based on the fact that most of the potent mitogenic factors present in serum are derived from activated thrombocytes. Platelet lysates were obtained from outdated human donor platelet concentrates.

A framework program for the teaching of alternative methods (replacement, reduction, refinement) to animal experimentation.

Development of improved communication and education strategies is important to make alternatives to the use of animals, and the broad range of applications of the 3Rs concept better known and understood by different audiences. For this purpose, the Center for Alternatives to Animal Testing in Europe (CAAT-Europe) together with the Transatlantic Think Tank for Toxicology (t(4)) hosted a three-day workshop on "Teaching Alternative Methods to Animal Experimentation". A compilation of the recommendations by a group of international specialists in the field is summarized in this report.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells).

An expert consortium review of the EC-commissioned report "alternative (Non-Animal) methods for cosmetics testing: current status and future prospects - 2010".

The European cosmetics legislation foresees a review in 2011 and possible postponement of the 2013 marketing ban to enforce the testing ban for systemic and repeated-dose animal tests. For this purpose, a 119-page report commissioned by the European Commission was published recently. Here, a group of 17 independent experts from the US, Europe, and Japan was brought together to evaluate the report.

Lactate is an ideal non-invasive marker for evaluating temporal alterations in cell stress and toxicity in repeat dose testing regimes.

Technological developments are driving in vitro methods towards integrated "omic" strategies. However, there is still an over reliance on classical viability assays for dose range finding. Such assays are not readily suited to the investigation of subtle alterations in cell function and most require termination of the experiment, which makes it difficult to monitor temporal alterations in repeat-dose long term exposure experiments.

Identification and dissection of the Nrf2 mediated oxidative stress pathway in human renal proximal tubule toxicity.

The identification and dissection of cellular stress mechanisms is fundamental to understanding the susceptibility of the kidney to chemicals and pharmaceuticals and for the development of renal biomarkers indicative of sub lethal injury. Here, we utilised whole genome DNA microarrays in an attempt to uncover molecular mechanisms of response to nephrotoxin exposure. Human renal proximal tubular cells (HK-2) were treated for 12h and 48 h with 5 μM Cadmium (Cd), 30 μM Diquat Dibromide (Diq), and 5 μM Cyclosporine A (CsA).

In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells.

Epidemiological studies show that exposure to nickel (Ni) compounds is associated with a variety of pulmonary adverse health effects, such as lung inflammation, fibrosis, emphysema and tumours. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. In the current study we used Calu-3, a well differentiated human bronchial cell line, to investigate in vitro the effect of Ni in soluble form (NiCl(2)) and in the form of micro-sized Ni particles on the airway epithelium.

Uromodulin facilitates neutrophil migration across renal epithelial monolayers.

The glycosylated protein uromodulin is exclusively found in the thick ascending limb cells (TAL) of the kidney, where it is produced on mass and apically targeted, eventually being secreted into the urine. Recently, there has been a renewed interest in this protein due to its ability to interact with the immune system, implicating this protein as a renal inflammatory molecule. Here we investigated the potential role of membrane bound uromodulin on neutrophil adhesion and trans-epithelial migration.

Serum-free cell culture: the serum-free media interactive online database.

Fetal bovine serum (FBS) is a ubiquitously used essential supplement in cell culture media. However, there are serious scientific and ethical concerns about the use of FBS regarding its harvest and production. During the last three decades, FBS could be substituted by other supplements or by the use of defined chemical components in serum-free cell culture.

Evidence for a role of uromodulin in chronic kidney disease progression.

Background: Uromodulin (also known as Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals and exhibits diverse functions including prevention of ascending urinary tract infections by binding type I-fimbriated Escherichia coli. Although uromodulin is targeted to the apical membrane of thick ascending limb (TAL) cells and secreted into the lumen, detectable levels are also found in venous blood. Uromodulin has been shown to interact with and activate specific components of the immune system, and thus, may act as a signalling molecule for renal tubular damage.

Characterisation of cadmium chloride induced molecular and functional alterations in airway epithelial cells.

Epidemiological studies show that cadmium (Cd) exposure causes pulmonary damage, such as emphysema, pneumonitis, and lung cancer. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. The aim of this study was to further investigate cadmium chloride (CdCl(2)) induced toxicity using Calu-3 cells as an in vitro model of human bronchial epithelial cells.

Differential effects of hypoxic stress in alveolar epithelial cells and microvascular endothelial cells.

Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial cells and microvascular endothelial cells are highly sensitive to hypoxia and together orchestrate a rapid and sustained adaptive response.

Inter-laboratory comparison of human renal proximal tubule (HK-2) transcriptome alterations due to Cyclosporine A exposure and medium exhaustion.

There is an acknowledged need to promote and further develop in vitro techniques in order to achieve the goal of improved risk assessment of chemicals and pharmaceuticals to humans. The EU 6th framework project "PREDICTOMICS" was established in order to contribute to the further development of in vitro toxicology, with a particular focus on emerging techniques including toxicogenomics. DNA microarray technology is being used more frequently in the in vitro field, however, only very few studies have assessed the reproducibility of this technique with respect to in vitro toxicology.

hTERT alone immortalizes epithelial cells of renal proximal tubules without changing their functional characteristics.

Telomere-dependent replicative senescence is one of the mechanisms that limit the number of population doublings of normal human cells. By overexpression of telomerase, cells of various origins have been successfully immortalized without changing the phenotype. While a limited number of telomerase-immortalized cells of epithelial origin are available, none of renal origin has been reported so far.

The surface properties of nanocrystalline diamond and nanoparticulate diamond powder and their suitability as cell growth support surfaces.

Nanocrystalline diamond (NCD) films and nanoparticulate diamond powder (DP) are the two main representatives of diamond at the nanoscale. This study was designed to investigate the suitability of these biomaterials as cell growth supports and to determine surface characteristic properties best suited to cell attachment and proliferation. Surface topography, chemical termination and wetting properties of NCD- and DP-coated borosilicate glass substrates were correlated to attachment, proliferation and differentially regulated gene expression of human renal epithelial cells (HK-2 cell line) cultured on these surfaces.

The carcinoGENOMICS project: critical selection of model compounds for the development of omics-based in vitro carcinogenicity screening assays.

Recent changes in the European legislation of chemical-related substances have forced the scientific community to speed up the search for alternative methods that could partly or fully replace animal experimentation. The Sixth Framework Program project carcinoGENOMICS was specifically raised to develop omics-based in vitro screens for testing the carcinogenic potential of chemical compounds in a pan-European context. This paper provides an in-depth analysis of the complexity of choosing suitable reference compounds used for creating and fine-tuning the in vitro carcinogenicity assays.