Establishment and comparison of air-liquid interface culture systems for primary and immortalized swine tracheal epithelial cells

Wang, Haiyan; He, Lina; Liu, Beibei; Feng, Yanyan; Zhou, Hao; Zhang, Zhenzhen; Wu, Yuzi; Wang, Jia; Gao, Yuan; Yuan, Ting; Wu, Meng; Xie, Xiulan; Feng, Zhixin

doi:10.1186/s12860-018-0162-3

Cited by 39 publications

(36 citation statements)

References 26 publications

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“…In our previous study, primary PTECs were isolated for air-liquid interface culture by placing small tracheal tissues digested in a pronase/DNase solution at 2-8 °C overnight for 24 to 48 h [35]; however, in this study, to isolate primary PTECs, isolated tracheal tissues were digested with collagenase I and trypsin together for no more than 1 h. The shorter digestion time of minced porcine tracheal tissues with microsurgical forceps as well as microsurgical scissors allowed a large quantity of extremely pure and reliable epithelial cells. Digestion in this manner with a shorter time has obvious advantages over isolation techniques applied in other species, including animals and human [13,23,36], since an extended digestion time such as 16-48 h and weak enzymes may increase the possibility of other contaminating cells not of epithelial origin [5].…”

Section: Discussionmentioning

confidence: 99%

Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Xie

Fei

Wang

et al. 2020

Preprint

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Background: Primary porcine tracheal epithelial cells (PTECs) are an appropriate model for studying the molecular mechanism of various porcine respiratory diseases, including swine-origin mycoplasmas, which were isolated from pig respiratory tract and were mainly found on the mucosal surface surrounding swine trachea. However, the short proliferation ability of primary PTECs greatly limits their lifespan.Results: In this study, we carefully isolated and cultured primary PTECs. Besides, we constructed immortalized PTECs by transfecting primary PTECs with the recombinant constructed plasmid pEGFP-hTERT containing human telomerase reverse transcriptase (hTERT). Immortal PTECs (hTERT-PTECs) maintained both the morphological and functional characteristics of primary PTECs, as indicated by the expression of cytokeratin 18, cell-cycle analysis, proliferation assay, western blotting, telomerase activity assay, karyotype analysis and quantitative RT-PCR. hTERT-PTECs had an extended replicative lifespan, higher telomerase activity, and enhanced proliferative activity. In addition, using this cell line, the lack of transformed and grown tumors in nude mice indicated that it could be safely applied in further studies. Moreover, hTERT-PTECs were vulnerable to all swine-origin mycoplasmas and were suitable as a cell adhesion model.Conclusions: In summary, hTERT-PTECs could be widely used as an adhesion cell model for swine-origin mycoplasmas and in the infection study of various porcine respiratory pathogens.

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Section: Discussionmentioning

confidence: 99%

Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Xie

Fei

Wang

et al. 2020

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“…2A). Firstly, we investigated the integrity of mucociliary differentiated HBECs grown on Transwell inserts by the measurement of transepithelial electrical resistance (TEER) [31,32] before EMTU establishment. The TEER value was measured again in triplicates in each insert containing differentiated HBECs cultured in the presence of HBFs (in the EMTU) for 4 days.…”

Section: Emtu Cultures From Asthmatics Are More Sensitive To the Tgf-mentioning

confidence: 99%

“…presented in our study revealed more enhanced sensitivity to the TGF-β1 of the AS co-cultures than in case of the NA ones. Measurement of TEER determines the quality of differentiated pseudostratified airway epithelium with well-developed tight junctions and is often used as a marker of a barrier function of the epithelium [31,32,43,44]. However, differences in the TEER values between ALI monocultures of asthmatic and non-asthmatic HBECs remains contradictory.…”

Section: Comparison Of the Compatible Asthmatic Emtu Cultures With Thmentioning

confidence: 99%

“…A fresh medium was added to the ALI-cultured HBECs to the upper part of inserts and TEER was measured as described previously [31,32] using Millicell ERS-2 Voltohmmeter (Merck Millipore) each in triplicates. Results were presented as Ω x cm 2 values in the mucociliary differentiated HBECs after 4 days of the EMTU in relation to the control condition (before the EMTU establishment; % of control).…”

Section: Transepithelial Electrical Resistance (Teer) Measurementsmentioning

confidence: 99%

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Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and non-asthmatic donors to the transforming growth factor-β1 in epithelial-mesenchymal trophic unit model

Paw

Wnuk

Jakieła

et al. 2020

Preprint

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Background: Asthma-related airway wall remodeling is associated with i.a. damage of bronchial epithelium and subepithelial fibrosis. Functional interactions between human bronchial epithelial cells and human bronchial fibroblasts are known as the epithelial-mesenchymal trophic unit (EMTU) and are necessary for a proper functioning of lung tissue. However, a high concentration of the transforming growth factor-β 1 (TGF-β 1 ) in the asthmatic bronchi drives the structural disintegrity of epithelium with the epithelial-to-mesenchymal transition (EMT) of the bronchial epithelial cells, and of subepitheial fibrosis with the fibroblast-to-myofibroblast transition (FMT) of the bronchial fibroblasts. Since previous reports indicate different intrinsic properties of the human bronchial epithelial cells and the human bronchial fibroblasts which affect their EMT/FMT potential beetween cells derived from asthmatic and non-asthmatic patients, cultured separatelly in vitro , we were interested to see whether corresponding effects could be obtained in co-cultures of bronchial epithelial cells and bronchial fibroblasts. In this study, we investigate the effects of the TGF-β 1 on the EMT markers of the bronchial epithelial cells cultured in the air-liquid-interface and effectiveness of FMT in the bronchial fibroblast populations in the EMTU models. Results: Our results show that the asthmatic co-cultures are more sensitive to the TGF-β 1 than the non-asthmatic ones, which is associated with a higher potential of asthmatic bronchial cells for a profibrotic response, analogously to be observed in “2D” cultures. Moreover, our results indicate a noticeable impact of human bronchial epithelial cells on the TGF-β 1 -induced FMT, stronger in the asthmatic bronchial fibroblast populations in comparison to the non-asthmatic ones. Conclusions: Our data are the first to demonstrate a protective effect of human bronchial fibroblasts on the properties of human bronchial epithelial cells, which suggests that intrinsic properties of not only epithelium but also subepithelial fibroblasts affect a proper condition and function of the EMTU in both normal and asthmatic individuals.

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“…Such systems may employ perfusion or direct mechanical actuation to stimulate a 3-D construct [12,[15][16][17][18][19], where the method of delivering stimulus and system design are determined by model and application specific requirements [20,21]. Several strategies for respiratory tissue engineering have been described, including decellularized ECM scaffolds [22][23][24], air-liquid interface well inserts [25][26][27] and PDMS microfluidic chips with integrated 2-D membranes [27][28][29]. However, biologically-derived materials introduce batch-batch inconsistencies and there exists a need for a truly 3-D in vitro culture model that also provides mechanical stimuli.…”

Section: Introductionmentioning

confidence: 99%

Biomimetic system design for engineering biofidelic 3-D respiratory tissuesin vitro

Poon

Zhang

Boughton

et al. 2020

Preprint

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AbstractObjectiveThe structural and functional complexity of the respiratory system present significant challenges to capturing conditions vital for maintaining phenotypic cellular functions in vitro. Here we report a unique tissue engineering system that enables respiratory constructs to be cultured under physiological loading at an air-liquid interface (ALI).MethodsThe system consists of a porous poly-e-caprolactone scaffold mounted in a well insert, which articulates via magnetic coupling with a linear actuator device to strain attached scaffolds through a sterile barrier. For proof of concept, NCI-H460 human carcinoma cells were seeded on scaffold inserts which were subjected to 5-15% cyclic tensile strain at 0.2Hz within a six well plate. The dynamic constructs were cultured at an ALI in a standard incubator for up to 10 days along with unstimulated (static) ALI and static submerged control groups.ResultsHigh (near-100%) cell seeding efficiency was achieved within the scaffold-strain device. Both dynamic and static ALI groups yielded higher cell densities compared to the submerged control for all time points. Distinctly different patterns in cellular growth and behaviour between dynamic air-liquid interface and conventional static submerged culture groups were revealed by nuclei staining, where the actuated group displayed more uniform cellular distribution throughout the construct compared to both static controls.ConclusionAir-liquid interface culture and physiological strain are important for engineering respiratory tissue models.SignificanceThe system described allows scalable and replicable culture of 3-D tissue engineered respiratory models under biologically-relevant conditions.

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Establishment and comparison of air-liquid interface culture systems for primary and immortalized swine tracheal epithelial cells

Cited by 39 publications

References 26 publications

Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Construction of a telomerase-immortalized porcine tracheal epithelial cell model for swine-origin mycoplasma infection

Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and non-asthmatic donors to the transforming growth factor-β1 in epithelial-mesenchymal trophic unit model

Biomimetic system design for engineering biofidelic 3-D respiratory tissuesin vitro

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