Reuse of Cell Culture Inserts for In Vitro Human Primary Airway Epithelial Cell Studies.
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ABSTRACT:
SUBMITTER: Kato T
PROVIDER: S-EPMC8456889 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mucociliary clearance (MCC) is an important innate defense mechanism that continuously removes inhaled pathogens and particulates from the airways. Normal MCC is essential for maintaining a healthy respiratory system, and impaired MCC is a feature of many airway diseases, including both genetic (cystic fibrosis, primary ciliary dyskinesia) and acquired (chronic obstructive pulmonary disease, bronchiectasis) disorders. Research into the fundamental processes controlling MCC, therefore, has direct clinical application, but has been limited in part due to the difficulty of studying this complex multicomponent system in vitro. In this study, we have characterized a novel method that allows human airway epithelial cells to differentiate into a mucociliary epithelium that transports mucus in a continuous circular track. The mucociliary transport device allows the measurement and manipulation of all features of mucociliary transport in a controlled in vitro system. In this initial study, the effect of ciliary beat frequency and mucus concentration on the speed of mucociliary transport was investigated.
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Project description:In this study, we compared 12 mm cell culture inserts with permeable polyester membranes (0.4 μm pores) from two different manufacturers: CELLTREAT® and Corning®. Physical dimensions and masses of the inserts were found to be very similar between the two brands, with CELLTREAT® inserts having a slightly smaller diameter and growth area (11.91 mm; 1.11 cm2 ) compared to Corning® Transwells® (12 mm; 1.13 cm2 ). We compared cell differentiation outcomes of human nasal epithelial cells (HNECs) at air-liquid interface grown on inserts from the two different manufacturers, including trans-epithelial electrical resistance, ciliary beat frequency, ciliated area, and gene expression. HNECs from three male donors were used for all endpoints. No statistically significant differences were observed between paired cultures grown on different brands of insert. In conclusion, these inserts are comparable for use with airway epithelial cell model systems and likely do not impact cellular differentiation or cell culture quality.
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Project description:BackgroundCell models are key instruments for in vitro studies of the thyroid. Permanent thyroid cell lines that are widely used in laboratory research typically originate from tumors. For many purposes, it is desirable to compare tumor cells with cells originating from normal tissue. However, such cultures grow slowly, have a highly limited life-span, and are known to lose their thyroid characteristics. The aim of the present study was to type coding and noncoding thyroid markers in different culture systems in an attempt to determine the optimal conditions for in vitro experimentation.MethodsHuman primary thyroid cells were isolated from histologically non-tumorous tissues. Two alternative media (6H and h7H) were used. The morphology and behavior of the ensuing monolayer (two-dimensional) cultures was monitored by microscopy. The expression of key thyroid-related genes (n = 9) was monitored by reverse transcription polymerase chain reaction on days 8, 21, and 43 after initiation. As a pilot study, the same markers were studied in a three-dimensional hanging-drop culture system.ResultsIn the cultures with 6H or h7H medium, the primary thyroid cells displayed growth in numbers and size. Most cells retained the main morphological characteristics of thyroid cells throughout the first two weeks of culture, and fibroblast-like cells appeared around day 19. By day 21, most thyroid gene markers were retained, but by day 43, several markers were no longer present. The lncRNA transcripts PTCSC2 (spliced) and PTCSC3 were the first to disappear. There were no fundamental differences between the two media in the early period of culture. In the three-dimensional system, most thyroid markers were retained by day 21.ConclusionCultures of thyroid cells retain many thyroid characteristics up to day 21. Thereafter, fibroblast-like dedifferentiated cells begin to dominate.
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Project description:Objectives:Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological model for the study of OM. Methods:We adapted a cell reprogramming protocol using irradiated fibroblast feeder medium in addition to Rho kinase inhibitor to proliferate pMEEC collected during cochlear implant surgery. Cells were plated on transwell membranes, proliferated with conditionally reprogrammed culture medium, and transferred to air-liquid interface (ALI). Cultures were maintained for 4?weeks at ALI, photos were taken and cell lysates and secretions were collected over time for characterization analysis using quantitative polymerase chain reaction, Western bolt, and proteomics. Keratins, MUC5B and MUC5AC mucins, and beta tubulin (TUBB) were analyzed at the mRNA and protein level. Results:Cultures took a mean of 2?weeks to proliferate before transwell plating and forming a tight epithelium at ALI from 2 to 4?weeks. Although mRNA expression of MUC5B, MUC5AC, TUBB, and keratin 5 (KRT5) were variable depending on the differentiation stage and the patient, both TUBB and KRT5 proteins were detected until week 2. Conclusion:We demonstrate a novel method to proliferate and differentiate pMEECs that express epithelial markers and that are able to secrete mucins for the study of OM. Level of Evidence:NA.
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Project description:BackgroundIn vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators.MethodsPrimary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor).ResultsCF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661.ConclusionsCF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.
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Project description:In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of '3Rs', new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air-liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.
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Project description:Mast cells (MCs) are found in increased numbers at airway mucosal surfaces in asthmatic patients. Because human airway epithelial cells (HAECs) actively participate in airway inflammatory responses and are in direct contact with MCs in the mucosa, we hypothesized that HAEC-MC interactions may contribute to the differentiation and survival of MCs in the airway mucosa. Here, we show that HAECs express mRNA and protein for soluble and membrane-bound stem cell factor, releasing soluble stem cell factor into the cell culture supernatant at a concentration of 5.9 +/- 0.1 ng per 10(6) HAEC. HAECs were able to support MC survival in coculture in the absence of any exogenous cytokines for at least 4 d. Before the initiation of coculture, MCs were uniformly tryptase and chymase (MC(TC)) double positive, but by 2 d of coculture the majority of MCs expressed tryptase (MC(T)) alone. MCs supported in coculture generated low amounts of cysteinyl-leukotrienes (cys-LT) after FcepsilonRI-dependent activation (0.2 +/- 0.1 ng of cys-LT per 10(6) cells) and required priming with IL-4 and IL-3 during coculture to achieve a quantity of cys-LT generation within the range expected for human lung mucosal MC (26.5 +/- 16 ng of cys-LT per 10(6) cells). In these culture conditions, HAECs were able to direct mucosal MC protease phenotype, but T cell-derived Th2 cytokines were required for the expression of a functional airway MC eicosanoid phenotype. Thus, distinct cell types may direct unique aspects of reactive mucosal MC phenotype in the airways.
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Project description:Cross-talk between lung epithelial cells and their microenvironment has an important physiological role in development. Using an in vitro model of differentiation of human induced pluripotent stem cells (iPSCs) to air-liquid interface (ALI)-cultured lung epithelial cells, we investigated the contribution of the microenvironment to maintenance of the lung progenitor cell state. Our protocol modeled in vivo cell-to-matrix and cell-to-cell interactions. These included growth of iPSCs on inserts coated with different basement membrane proteins (collagen I, IV, fibronectin, heparan sulfate or Matrigel plus collagen IV) and co-culture with human pulmonary microvascular endothelial cells (HPMECs). Marker gene expression was measured by RT-qPCR and protein expression and localization was confirmed by immunocytochemistry. The results showed that iPSCs grown on collagen IV had the highest success rate in terms of differentiation to robust ALI-cultured lung epithelial cells, followed by fibronectin, collagen I and heparan sulfate. Coating with Matrigel mixed with collagen IV further increased the success rate to > 97 %. Co-culture of iPSCs with HPMECs enhanced the expression of key airway lineage markers (NKX2.1, KRT5, TP63, MUC5AC, MUC16, FOXJ1, CFTR and SCGB1A1) during ALI culture. Cross-talk between iPSCs and their microenvironment during cell differentiation had a significant effect on lung epithelial cell differentiation in these 3D in vitro models. Both matrix proteins and endothelial cells play critical roles in the differentiation of lung progenitor cells.
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Project description:Amplification of measles virus (MeV) in human airway epithelia may contribute to its extremely high contagious nature. We use well-differentiated primary cultures of human airway epithelial cells (HAE) to model ex vivo how MeV spreads in human airways. In HAE, MeV spreads cell-to-cell for 3-5 days, but then, infectious center growth is arrested. What stops MeV spread in HAE is not understood, but interferon (IFN) is known to slow MeV spread in other in vitro and in vivo models. Here, we assessed the role of type I and type III IFN in arresting MeV spread in HAE. The addition of IFN-β or IFN-λ1 to the medium of infected HAE slowed MeV infectious center growth, but when IFN receptor signaling was blocked, infectious center size was not affected. In contrast, blocking type-I IFN receptor signaling enhanced respiratory syncytial virus spread. HAE were also infected with MeV mutants defective for the V protein. The V protein has been demonstrated to interact with both MDA5 and STAT2 to inhibit activation of innate immunity; however, innate immune reactions were unexpectedly muted against the V-defective MeV in HAE. Minimal innate immunity activation was confirmed by deep sequencing, quantitative RT-PCR, and single-cell RNA-seq analyses of the transcription of IFN and IFN-stimulated genes. We conclude that in HAE, IFN-signaling can contribute to slowing infectious center growth; however, IFN-independent processes are most important for limiting cell-to-cell spread. IMPORTANCE Fundamental biological questions remain about the highly contagious measles virus (MeV). MeV amplifies within airway epithelial cells before spreading to the next host. This final step likely contributes to the ability of MeV to spread host-to-host. Over the course of 3-5 days post-infection of airway epithelial cells, MeV spreads directly cell-to-cell and forms infectious centers. Infectious center formation is unique to MeV. In this study, we show that interferon (IFN) signaling does not explain why MeV cell-to-cell spread is ultimately impeded within the cell layer. The ability of MeV to spread cell-to-cell in airway cells without appreciable IFN induction may contribute to its highly contagious nature. This study contributes to the understanding of a significant global health concern by demonstrating that infectious center formation occurs independent of the simplest explanation for limiting viral transmission within a host.
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Project description:The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population.Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the "human airway basal cell signature" as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels.The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem/progenitor cells of the human airway epithelium.
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