Optimization of an oral mucosa in vitro model based on cell line TR146.

During the last years, the popularity of saliva has been increasing for its applicability as a diagnostic fluid. Blood biomarker molecules have to cross the blood-saliva barrier (BSB) in order to appear in saliva. The BSB consists of all oral and salivary gland epithelial barriers. Within this context, the optimization of in vitro models for mechanistic studies about the transport of molecules across the oral mucosa is an important task. Here, we describe the optimization and comprehensive characterization of a Transwell model of the oral mucosa based on the epithelial cell line TR146. Through systematic media optimization investigating 12 different set-ups, a significant increase of barrier integrity upon airlift cultivation is described here for TR146 cell layers. The distinct improvement of the paracellular barrier was described by measurements of transepithelial electrical resistance (TEER) and carboxyfluorescein permeability assays. Histological characterization supported TEER data and showed a stratified, non-keratinized multilayer of the optimized TR146 model. High-Throughput qPCR using 96 selected markers for keratinization, cornification, epithelial-mesenchymal transition, aquaporins, mucins, tight junctions, receptors, and transporter proteins was applied to comprehensively characterize the systematic optimization of the cellular model and validate against human biopsy samples. Data revealed the expression of several genes in the oral mucosa epithelium for the first time and elucidated novel regulations dependent on culture conditions. Moreover, functional activity of ABC-transporters ABCB1 and ABCC4 was shown indicating the applicability of the model for drug transport studies. In conclusion, a Transwell model of the oral mucosa epithelium was optimized suitably for transport studies.

The pivotal role of micro-environmental cells in a human blood-brain barrier in vitro model of cerebral ischemia: functional and transcriptomic analysis.

BACKGROUND: The blood-brain barrier (BBB) is altered in several diseases of the central nervous system. For example, the breakdown of the BBB during cerebral ischemia in stroke or traumatic brain injury is a hallmark of the diseases' progression. This functional damage is one key event which is attempted to be mimicked in in vitro models. Recent studies showed the pivotal role of micro-environmental cells such as astrocytes for this barrier damage in mouse stroke in vitro models. The aim of this study was to evaluate the role of micro-environmental cells for the functional, paracellular breakdown in a human BBB cerebral ischemia in vitro model accompanied by a transcriptional analysis. METHODS: Transwell models with human brain endothelial cell line hCMEC/D3 in mono-culture or co-culture with human primary astrocytes and pericytes or rat glioma cell line C6 were subjected to oxygen/glucose deprivation (OGD). Changes of transendothelial electrical resistance (TEER) and FITC-dextran 4000 permeability were recorded as measures for paracellular tightness. In addition, qPCR and high-throughput qPCR Barrier chips were applied to investigate the changes of the mRNA expression of 38 relevant, expressed barrier targets (tight junctions, ABC-transporters) by different treatments. RESULTS: In contrast to the mono-culture, the co-cultivation with human primary astrocytes/pericytes or glioma C6 cells resulted in a significantly increased paracellular permeability after 5 h OGD. This indicated the pivotal role of micro-environmental cells for BBB breakdown in the human model. Hierarchical cluster analysis of qPCR data revealed differently, but also commonly regulated clustered targets dependent on medium exchange, serum reduction, hydrocortisone addition and co-cultivations. CONCLUSIONS: The co-cultivation with micro-environmental cells is necessary to achieve a functional breakdown of the BBB in the cerebral ischemia model within an in vivo relevant time window. Comprehensive studies by qPCR revealed that distinct expression clusters of barrier markers exist and that these are regulated by different treatments (even by growth medium change) indicating that controls for single cell culture manipulation steps are crucial to understand the observed effects properly.

Autologous induced pluripotent stem cell-derived four-organ-chip.

Microphysiological systems play a pivotal role in progressing toward a global paradigm shift in drug development. Here, we designed a four-organ-chip interconnecting miniaturized human intestine, liver, brain and kidney equivalents. All four organ models were predifferentiated from induced pluripotent stem cells from the same healthy donor and integrated into the microphysiological system. The coculture of the four autologous tissue models in one common medium deprived of tissue specific growth factors was successful over 14-days. Although there were no added growth factors present in the coculture medium, the intestine, liver and neuronal model maintained defined marker expression. Only the renal model was overgrown by coexisting cells and did not further differentiate. This model platform will pave the way for autologous coculture cross-talk assays, disease induction and subsequent drug testing.