RESUMEN
Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.
RESUMEN
The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing.
RESUMEN
In recent years, there has been a significant increase in the registration of drugs for nasal application with systemic effects. Previous preclinical in vitro test systems for transmucosal drug absorption studies have mostly been based on primary cells or on tumor cell lines such as RPMI 2650, but both approaches have disadvantages. Therefore, the aim of this study was to establish and characterize a novel immortalized nasal epithelial cell line as the basis for an improved 3D cell culture model of the nasal mucosa. First, porcine primary cells were isolated and transfected. The P1 cell line obtained from this process was characterized in terms of its expression of tissue-specific properties, namely, mucus expression, cilia formation, and epithelial barrier formation. Using air-liquid interface cultivation, it was possible to achieve both high mucus formation and the development of functional cilia. Epithelial integrity was expressed as both transepithelial electrical resistance and mucosal permeability, which was determined for sodium fluorescein, rhodamine B, and FITC-dextran 4000. We noted a high comparability of the novel cell culture model with native excised nasal mucosa in terms of these measures. Thus, this novel cell line seems to offer a promising approach for developing 3D nasal mucosa tissues that exhibit favorable characteristics to be used as an in vitro system for testing drug delivery systems.
RESUMEN
Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100ß, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.
RESUMEN
BACKGROUND: Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs. RESULTS: We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context. CONCLUSION: RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.
Asunto(s)
Proteínas Recombinantes/biosíntesis , Transfección/métodos , Animales , Formación de Anticuerpos , Células CHO , Células Clonales , Cricetinae , Cricetulus , Marcación de Gen/métodos , Vectores Genéticos , Humanos , Regiones Promotoras Genéticas , TransgenesRESUMEN
Eukaryotic DNA is organized into chromatin domains that regulate gene expression and chromosome behavior. Insulators and/or scaffold-matrix attachment regions (S/MARs) mark the boundaries of these chromatin domains where they delimit enhancing and silencing effects from the outside. By recombinase-mediated cassette exchange (RMCE), we were able to compare these two types of bordering elements at a number of predefined genomic loci. Flanking an expression vector with either S/MARs or two copies of the non-S/MAR chicken hypersensitive site 4 insulator demonstrates that while these borders confer related expression characteristics at most loci, their effect on chromatin organization is clearly distinct. Our results suggest that the activity of bordering elements is most pronounced for the abundant class of loci with a low but negligible expression potential in the case of highly expressed sites. By the RMCE procedure, we demonstrate that expression parameters are not due to a potential targeting action of bordering elements, in the sense that a linked transgene is directed into a special class of loci. Instead, we can relate the observed transcriptional augmentation phenomena to their function as genomic insulators.