Primary Nasal Cell Research Articles

In situ-gelling hydrophobized starch nanoparticle-based nanoparticle network hydrogels for the effective delivery of intranasal olanzapine to treat brain disorders

Intranasal (IN) delivery offers potential to deliver antipsychotic drugs with improved efficacy to the brain. However, the solubilization of such drugs and the frequency of required re-application both represent challenges to its practical implementation in treating various mental illnesses including schizophrenia. Herein, we report a sprayable nanoparticle network hydrogel (NNH) consisting of hydrophobically-modified starch nanoparticles (SNPs) and mucoadhesive chitosan oligosaccharide lactate (COL) that can gel in situ within the nasal cavity and release ultra-small penetrative SNPs over time. Hydrophobization of the SNPs enables enhanced uptake and prolonged release of poorly water soluble drugs such as olanzapine from the NNH depot through mucous and ultimately into the brain via the nose-to-brain (N2B) pathway. The hydrogel shows high in vitro cytocompatibility in mouse striatal neuron and human primary nasal cell lines and in vivo efficacy in an amphetamine-induced pre-clinical rat schizophrenia model, with IN-delivered NNH hydrogels maintaining successful attenuation of locomotor activity for up to 4 h while all other tested treatments (drug-only IN or conventional intraperitoneal delivery) failed to attenuate at any time point past 0.5 h. As such, in situ-gelling NNHs represent a safe excipient for the IN delivery of hydrophobic drugs directly to the brain using customized SNPs that exhibit high penetration and drug complexing properties to maximize effective drug delivery.

Activin-A Is a Pro-Inflammatory Regulator in Type-2-Driven Upper Airway Disease

Background: Allergic upper airway disease involves pro-inflammatory type-2 cytokines such as IL-5 and regulatory tissue repair mediators, in particular transforming growth factor (TGF)-β₁. The TGF-β-superfamily member activin-A displays multiple biological functions and shares certain signalling pathways with TGF-β₁. We aimed to examine the coregulation of mucosal activin-A and TGF-β₁ in acute allergic and chronic Th2-driven upper airway disease. Methods: We investigated mucosal cytokine expression profiles and kinetics using RT-PCR after nasal allergen challenges in patients with seasonal allergic rhinitis. Furthermore, we analysed mucosal specimens from patients with chronic upper airway disease with nasal polyps using ELISPOTs and confocal microscopy. In addition, we stimulated nasal mucosa ex vivo from patients with nasal polyps as well as primary nasal cell cultures from healthy donors. Results: Mucosal activin-A expression revealed increasing correlation with IL-5 and TGF-β₁ at 0.25, 6, and 24 h, respectively, and was significantly upregulated at 6 h after allergen challenge. The correlated expression was found to be more pronounced in chronic disease with nasal polyps, showing substantially (48-fold) increased activin-A-producing cells in nasal polyps by ELISPOT, while submucosal downstream signalling as determined by confocal microscopy was decreased. Ex vivo stimulations of nasal tissue suggested that activin-A and TGF-β₁ mutually regulate each other’s expression at the mRNA level and, when combined, enhance IL-5 expression. Conclusion: Activin-A in allergic upper airway disease acts as a pro-inflammatory mediator and TGF-β₁ modifier. Our data in the upper airways oppose the view of potentially anti-inflammatory properties in contrast to lymphatic compartments.

Application of RPMI 2650 nasal cell model to a 3D printed apparatus for the testing of drug deposition and permeation of nasal products

The aim of this study was to incorporate an optimized RPMI2650 nasal cell model into a 3D printed model of the nose to test deposition and permeation of drugs intended for use in the nose. The nasal cell model was optimized for barrier properties in terms of permeation marker and mucus production. RT-qPCR was used to determine the xenobiotic transporter gene expression of RPMI 2650 cells in comparison with primary nasal cells. After 14days in culture, the cells were shown to produce mucus, and to express TEER (define) values and sodium fluorescein permeability consistent with values reported for excised human nasal mucosa. In addition, good correlation was found between RPMI 2650 and primary nasal cell transporter expression values.The purpose-built 3D printed model of the nose takes the form of an expansion chamber with inserts for cells and an orifice for insertion of a spray drug delivery device. This model was validated against the FDA glass chamber with cascade impactors that is currently approved for studies of nasal products. No differences were found between the two apparatus.The apparatus including the nasal cell model was used to test a commercial nasal product containing budesonide (Rhinocort, AstraZeneca, Australia). Drug deposition and transport studies on RPMI 2650 were successfully performed.The new 3D printed apparatus that incorporates cells can be used as valid in vitro model to test nasal products in conditions that mimic the delivery from nasal devices in real life conditions.

Marked repression of CFTR mRNA in the transgenic Cftrtm1kth mouse model

Primary nasal septal epithelia cultures allow mechanistic comparisons that correspond well with the in vivo nasal potential difference assay, a mainstay of cystic fibrosis transmembrane conductance regulator detection in vivo, and have been viewed as suitable models for testing the activity of CFTR correctors and potentiators in “personalized medicine” type approaches to cystic fibrosis (CF) therapeutics [1–3]. Primary nasal septal epithelia from mice (MNSE) have substantially greater CFTR activity in Ussing chamber analysis than comparably processed tracheal monolayers, and allow direct analysis of otherwise genetically identical (congenic) animals without the confounding effects attributable to mixed genetic backgrounds [4]. There are three widely utilized F508del transgenic mouse models—Cftrtm1Kth (Utah), Cftrtm2Cam (Rotterdam), and Cftrtm1Eur (Cambridge). The Cftrtm1Kth mutated allele contains a 3-bp (CTT) deletion between bases 1656 and 1660, which results in a loss of a phenylalanine residue in exon 10 at a position which corresponds to human position 508 [5]. This exon 10 replacement strategy left a selective marker gene inserted in one of the introns. This was a similar strategy to the creation of the Cftrtm2Cam mouse model [6]. This marker gene is likely to have an effect on transcription activity or mRNA levels of the mutant allele, but does not affect the protein product. The other widely utilized F508del mouse model is Cftrtm1Eur, which was made with a “hit and run” procedure that resulted in a mutant exon from which transcriptional activity of the mutant allele is identical to the normal allele [7]. While the Cftrtm2Cam mouse was noted to have 1/3 of the transcriptional activity in all tissues tested, prior studies concerning Cftrtm1Kth transgenic mice have indicated tissue specific differences in CFTR expression—specifically lower levels of F508del CFTR mRNA in the upper intestine compared to wild type animals. Normal levels of CFTR mRNA were reported previously in F508del murine lung, testes, and submaxillary glands in the same strain [5]. Given the high interest in evaluating misprocessing correctors of F508del CFTR in a relevant in vivo and in vitro model of disease, proper F508del CFTR expression is critical to promote maturation of a similar pool of misprocessed protein. After the University of Alabama at Birmingham Institutional and Animal Care and Use Committee approval, MNSE tissues from wild type (C57BL/6), heterozygous F508del, and homozygous F508del/F508del transgenic (Cftrtm1Kth) were cultured on permeable supports for 14 days to confluence and full differentiation. This F508del mouse strain was obtained from Jackson Labs (stock # 002515) and backcrossed to the C57BL/6 line 6 times. The mice are greater than 98% on the BL/6 background. Quantitative RT-PCR analysis (reported as relative mRNA ± S.D.) showed dramatic differences in CFTR mRNA among homozygous, heterozygous, and wild type both in MNSE cultures and tissues (Fig. 1A). Whether these differences are mediated by higher expressivity in a pure epithelial cell population (compared to mixed cells from native tissues), decreased CFTR transcription, or message instability will require further investigation. In addition, there was minimal F508del CFTR-mediated anion transport following 24 hour low-temperature correction (27 °C) in Ussing chambers (Fig. 1B). Fig. 1 Relative CFTR mRNA expression in wild type, F508del heterozygous, and homozygous murine nasal tissues and cell cultures (A). CFTR mRNA expression in F508del homozygous murine nasal tissues (n = 6 per condition) and cultures (n = 9 per condition) were ... Our findings suggest significant limitations of nasal airway cultures from Cftrtm1Kth mice, and previous studies of F508del CFTR from the tm1 Kth background should be re-evaluated in this light [8]. For example, potentiator activation profiles observed in F508del CF mice may underestimate potency as a result of diminished levels of mRNA in this murine genotype. While murine CFTR is insensitive to many agents that enhance the activity of human CFTR (e.g. ivacaftor) [9], agents (e.g. flavonoids) known to increase channel opening in both murine and human CFTR should be appraised in this light. Furthermore, this mouse model should not be utilized for evaluating F508del misprocessing correctors with upper airway experiments in vivo (e.g. NPD measurements) or in vitro (primary nasal cell culture electrophysiology) endpoints. The findings also provide a cautionary note indicating the potential for highly discordant levels of CFTR mRNA from certain CFTR genotypes should nasal cells be developed as a universal screen meant to “personalize” CFTR modulation therapy in patients with rare genetic defects.

Primary cell culture from the nose of a marine organism, the banded houndshark, Triakis scyllium

Animal cells have been widely and continuously studied due to their usefulness in biological researches and production of pharmacological agents as well as food additives. Nevertheless, there are several problems such as the existence of viruses which introduce the possibility of mammalian-infection. In this reason, recently, animal cells derived from marine organisms have emerged to overcome these problems by many researches. However, marine animal-derived cells have not yet been well developed. The banded hound shark occupies an important position in an evolutionary perspective and currently a few biological substances have been used for medicines or food additives such as shark squalene and cartilage extract. In this study, primary cells were cultivated from a banded hound shark nose containing many extracellular matrix (ECM) materials. After successful isolation of one type of primary nasal cell, we optimized culture conditions including coating materials, media composition, serum concentrations and pH. Additionally, these cells demonstrated proliferation ability in vitro, generating secretions of collagen and sulfated polysaccharides. The cultivated primary cells are useful in the study of cellular biology and may be used to create a variety of ECM-originated bioactive substances.