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1.
Microsyst Nanoeng ; 10: 18, 2024.
Article in English | MEDLINE | ID: mdl-38268774

ABSTRACT

In healthy individuals, the intestinal epithelium forms a tight barrier to prevent gut bacteria from reaching blood circulation. To study the effect of probiotics, dietary compounds and drugs on gut barrier formation and disruption, human gut epithelial and bacterial cells can be cocultured in an in vitro model called the human microbial crosstalk (HuMiX) gut-on-a-chip system. Here, we present the design, fabrication and integration of thin-film electrodes into the HuMiX platform to measure transepithelial electrical resistance (TEER) as a direct readout on barrier tightness in real-time. As various aspects of the HuMiX platform have already been set in their design, such as multiple compressible layers, uneven surfaces and nontransparent materials, a novel fabrication method was developed whereby thin-film metal electrodes were first deposited on flexible substrates and sequentially integrated with the HuMiX system via a transfer-tape approach. Moreover, to measure localized TEER along the cell culture chamber, we integrated multiple electrodes that were connected to an impedance analyzer via a multiplexer. We further developed a dynamic normalization method because the active measurement area depends on the measured TEER levels. The fabrication process and system setup can be applicable to other barrier-on-chip systems. As a proof-of-concept, we measured the barrier formation of a cancerous Caco-2 cell line in real-time, which was mapped at four spatially separated positions along the HuMiX culture area.

2.
Sci Data ; 9(1): 26, 2022 01 27.
Article in English | MEDLINE | ID: mdl-35087120

ABSTRACT

Confocal microscopy offers a mean to extract quantitative data on spatially confined subcellular structures. Here, we provide an imaging dataset of confocal z-stacks on endothelial cells spatially confined on lines with different widths, visualizing the nucleus, F-actin, and zonula occludens-1 (ZO-1), as well as the lines. This dataset also includes confocal images of spatially confined endothelial cells challenged with different glucose conditions. We have validated the image quality by established analytical means using the MeasureImageQuality module of the CellProfilerTM software. We envision that this dataset could be used to extract data on both a population and a single cell level, as well as a learning set for the development of new image analysis tools.


Subject(s)
Endothelial Cells , Glucose , Animals , Humans , Image Processing, Computer-Assisted , Microscopy, Confocal , Software
3.
Sci Rep ; 11(1): 19608, 2021 10 04.
Article in English | MEDLINE | ID: mdl-34608232

ABSTRACT

This work reports on an effort to decipher the alignment of brain microvasculature endothelial cells to physical constrains generated via adhesion control on hydrogel surfaces and explore the corresponding responses upon glucose level variations emulating the hypo- and hyperglycaemic effects in diabetes. We prepared hydrogels of hyaluronic acid a natural biomaterial that does not naturally support endothelial cell adhesion, and specifically functionalised RGD peptides into lines using UV-mediated linkage. The width of the lines was varied from 10 to 100 µm. We evaluated cell alignment by measuring the nuclei, cell, and F-actin orientations, and the nuclei and cell eccentricity via immunofluorescent staining and image analysis. We found that the brain microvascular endothelial cells aligned and elongated to these physical constraints for all line widths. In addition, we also observed that varying the cell medium glucose levels affected the cell alignment along the patterns. We believe our results may provide a platform for further studies on the impact of altered glucose levels in cardiovascular disease.


Subject(s)
Brain/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Glucose/metabolism , Hydrogels , Actins/metabolism , Biocompatible Materials , Cell Adhesion , Cell Culture Techniques , Cell Shape , Cells, Cultured , Endothelial Cells/drug effects , Glucose/pharmacology , Hyaluronic Acid/chemistry , Hydrogels/chemistry , Tissue Engineering
4.
Cell Mol Bioeng ; 14(1): 121-130, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33633814

ABSTRACT

INTRODUCTION: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments. METHODS: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 µM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARγ (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated. RESULTS: Daily delivering 5-AzaC caused a higher upregulation of PPARγ, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPARγ (tenfold-change) could indicate priming of hMSCs towards adipocytes. CONCLUSIONS: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.

5.
HardwareX ; 10: e00245, 2021 Oct.
Article in English | MEDLINE | ID: mdl-35607686

ABSTRACT

Microfluidic devices are widely used for biomedical applications but there is still a lack of affordable, reliable and user-friendly systems for transferring microfluidic chips from an incubator to a microscope while maintaining physiological conditions when performing microscopy. The presented carrier represents a cost-effective option for sustaining environmental conditions of microfluidic chips in combination with minimizing the device manipulation required for reagent injection, media exchange or sample collection. The carrier, which has the outer dimension of a standard well plate size, contains an integrated perfusion system that can recirculate the media using piezo pumps, operated in either continuous or intermittent modes (50-1000 µl/min). Furthermore, a film resistive heater made from 37 µm-thick copper wires, including temperature feedback control, was used to maintain the microfluidic chip temperature at 37 °C when outside the incubator. The heater characterisation showed a uniform temperature distribution along the chip channel for perfusion flow rates up to 10 µl/min. To demonstrate the feasibility of our platform for long term cell culture monitoring, mouse brain endothelial cells (bEnd.3) were repeatedly monitored for a period of 10 days, demonstrating a system with both the versatility and the potential for long imaging in microphysiological system cell cultures.

6.
RSC Adv ; 11(47): 29859-29869, 2021 Sep 01.
Article in English | MEDLINE | ID: mdl-35479529

ABSTRACT

Some of the most fundamental chemical building blocks of life on Earth are the metal elements. X-ray absorption spectroscopy (XAS) is an element-specific technique that can analyse the local atomic and electronic structure of, for example, the active sites in catalysts and energy materials and allow the metal sites in biological samples to be identified and understood. A microfluidic device capable of withstanding the intense hard X-ray beams of a 4th generation synchrotron and harsh chemical sample conditions is presented in this work. The device is evaluated at the K-edges of iron and bromine and the L 3-edge of lead, in both transmission and fluorescence mode detection and in a wide range of sample concentrations, as low as 0.001 M. The device is fabricated in silicon and glass with plasma etched microchannels defined in the silicon wafer before anodic bonding of the glass wafer into a complete device. The device is supported with a well-designed printed chip holder that made the microfluidic device portable and easy to handle. The chip holder plays a pivotal role in mounting the delicate microfluidic device on the beamline stage. Testing validated that the device was sufficiently robust to contain and flow through harsh acids and toxic samples. There was also no significant radiation damage to the device observed, despite focusing with intense X-ray beams for multiple hours. The quality of X-ray spectra collected is comparable to that from standard methods; hence we present a robust microfluidic device to analyse liquid samples using synchrotron XAS.

7.
J Phys Chem B ; 124(51): 11573-11583, 2020 12 24.
Article in English | MEDLINE | ID: mdl-33315405

ABSTRACT

Interfacing solid-state nanopores with biological systems has been exploited as a versatile analytical platform for analysis of individual biomolecules. Although clogging of solid-state nanopores due to nonspecific interactions between analytes and pore walls poses a persistent challenge in attaining the anticipated sensing efficacy, insufficient studies focus on elucidating the clogging dynamics. Herein, we investigate the DNA clogging behavior by passing double-stranded (ds) DNA molecules of different lengths through hafnium oxide(HfO2)-coated silicon (Si) nanopore arrays, at different bias voltages and electrolyte pH values. Employing stable and photoluminescent-free HfO2/Si nanopore arrays permits a parallelized visualization of DNA clogging with confocal fluorescence microscopy. We find that the probability of pore clogging increases with both DNA length and bias voltage. Two types of clogging are discerned: persistent and temporary. In the time-resolved analysis, temporary clogging events exhibit a shorter lifetime at higher bias voltage. Furthermore, we show that the surface charge density has a prominent effect on the clogging probability because of electrostatic attraction between the dsDNA and the HfO2 pore walls. An analytical model based on examining the energy landscape along the DNA translocation trajectory is developed to qualitatively evaluate the DNA-pore interaction. Both experimental and theoretical results indicate that the occurrence of clogging is strongly dependent on the configuration of translocating DNA molecules and the electrostatic interaction between DNA and charged pore surface. These findings provide a detailed account of the DNA clogging phenomenon and are of practical interest for DNA sensing based on solid-state nanopores.


Subject(s)
Nanopores , DNA , Hafnium , Oxides
8.
Biomater Sci ; 8(22): 6309-6321, 2020 Nov 21.
Article in English | MEDLINE | ID: mdl-33021608

ABSTRACT

To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti6Al4V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.


Subject(s)
Microfluidics , Titanium , Alloys , Biocompatible Materials/pharmacology , Cell Culture Techniques
9.
Annu Int Conf IEEE Eng Med Biol Soc ; 2020: 5033-5036, 2020 07.
Article in English | MEDLINE | ID: mdl-33019117

ABSTRACT

This article presents a method to plan BloodBrain Barrier (BBB) disruption with Focused Ultrasound, under neuronavigated robotic assistance. Robotic and acoustic constraints are defined to estimate brain target accessibility. The relevance of the proposed framework is illustrated in specific brain target examples.


Subject(s)
Blood-Brain Barrier , Robotics , Biological Transport , Blood-Brain Barrier/diagnostic imaging , Brain/diagnostic imaging , Ultrasonography
10.
Biomicrofluidics ; 13(6): 064123, 2019 Nov.
Article in English | MEDLINE | ID: mdl-31832121

ABSTRACT

Droplet microfluidics has shown great potential for on-chip biological and chemical assays. However, fluid exchange in droplet microfluidics with high particle recovery is still a major bottleneck. Here, using acoustophoresis, we present for the first time a label-free method to achieve continuous background dilution in droplets containing cells with high sample recovery. The system comprises droplet generation, acoustic focusing, droplet splitting, picoinjection, and serpentine mixing on the same chip. The capacities of the picoinjection and the droplet split to dilute the background fluorescent signal in the droplets have been characterized. The sample recovery at different droplet split ratios has also been characterized. The results show a maximum of 4.3-fold background dilution with 87.7% particle recovery. We also demonstrated that the system can be used to dilute background fluorescent signal in droplets containing either polystyrene particles or endothelial cells.

11.
J Biomed Mater Res B Appl Biomater ; 107(5): 1504-1515, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30267639

ABSTRACT

Vertebroplasty procedures provide a significant benefit for patients suffering from vertebral fractures. In order to address current issues of vertebroplasty procedures, an injection device able to control the bone cement viscosity has been developed. In addition, this device allows to protect the practitioner by removing him/her from the X-rays area. In this context, a study is first proposed to quantify the bone cement viscosity during its polymerization reaction on a rotational rheometer. These experimental measurements have led to the identification of a complete behavior law that takes into account the simultaneous effects of shear rate, time, and temperature. Based on this preliminary study, this article finally aims to prove the ability of estimating the viscosity of the flowing bone cement on the developed injection system. A final set of experiments validates that the injection device dedicated to vertebroplasty procedures can control the flowing bone cement viscosity by acting on the temperature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1504-1515, 2019.


Subject(s)
Bone Cements/chemistry , Spinal Fractures/therapy , Vertebroplasty , Polymethyl Methacrylate/chemistry , Viscosity
12.
Cardiovasc Intervent Radiol ; 42(2): 283-288, 2019 Feb.
Article in English | MEDLINE | ID: mdl-30306204

ABSTRACT

AIM: To compare robotic-assisted needle insertions performed under CBCT guidance to standard manual needle insertions. MATERIALS AND METHODS: A homemade robotic prototype was used by two operators to perform robotic and manual needle insertions on a custom-made phantom. Both the operators had no experience with the prototype before starting the trial. The primary endpoint was accuracy (i.e., the minimal distance between the needle tip and the center of the target) between robotic and manual insertions. Secondary endpoints included total procedure time and operators' radiation exposure. The Wilcoxon test was used. A p value less than 0.05 was considered statistically significant. RESULTS: Thirty-three (17 manual, 16 robotic) needle insertions were performed. Mean accuracy for robotic insertion was 2.3 ± 0.9 mm (median 2.1; range 0.8-4.2) versus 2.3 ± 1 mm (median 2.1; range 0.7-4.4) for manual insertion (p = 0.84). Mean procedure time was 683 ± 57 s (median 670; range 611-849) for the robotic group versus 552 ± 40 s (median 548; range 486-621) for the manual group (p = 0.0002). Mean radiation exposure was 3.25 times less for the robotic insertion on comparison to manual insertion for the operator 1 (0.4 vs 1.3 µGy); and 4.15 times less for the operator 2 (1.9 vs 7.9 µGy). CONCLUSION: The tested robotic prototype showed accuracy comparable to that achieved with manual punctures coupled to a significant reduction of operators' radiation exposure. Further, in vivo studies are necessary to confirm the efficiency of the system.


Subject(s)
Cone-Beam Computed Tomography/methods , Image-Guided Biopsy/instrumentation , Robotics/methods , Needles , Phantoms, Imaging , Punctures , Radiation Exposure , Time Factors
13.
Annu Int Conf IEEE Eng Med Biol Soc ; 2018: 1708-1712, 2018 Jul.
Article in English | MEDLINE | ID: mdl-30440724

ABSTRACT

In this paper, we present the work achieved to define the robotic functionalities of interest for percutaneous procedures as performed in interventional radiology. Our contributions are twofold. First, a detailed task analysis is performed with workflow analysis of biopsies, one of the most frequent tasks, under three imaging modalities, namely CT, CBCT and MRI. Second, the functionalities of a robotic assistant are identified, and we analyze whether a single device can bring an added value during procedures in the three modalities while keeping the robotized workflow close to manual tasks, to minimize learning time and difficulty of use. Experimental analysis on CBCT is notably used to confirm the interest of the determined robotic functionalities.


Subject(s)
Magnetic Resonance Imaging , Radiology , Robotic Surgical Procedures , Robotics , Spiral Cone-Beam Computed Tomography , Humans , Magnetic Resonance Imaging/instrumentation , Radiology/instrumentation , Radiology/methods , Robotic Surgical Procedures/instrumentation , Robotics/instrumentation , Robotics/standards , Spiral Cone-Beam Computed Tomography/instrumentation , Workflow
14.
Front Robot AI ; 5: 84, 2018.
Article in English | MEDLINE | ID: mdl-33500963

ABSTRACT

The growing interest in soft robots comes from the new possibilities offered by these systems to cope with problems that cannot be addressed by robots built from rigid bodies. Many innovative solutions have been developed in recent years to design soft components and systems. They all demonstrate how soft robotics development is closely dependent on advanced manufacturing processes. This review aims at giving an insight on the current state of the art in soft robotics manufacturing. It first puts in light the elementary components that can be used to develop soft actuators, whether they use fluids, shape memory alloys, electro-active polymers or stimuli-responsive materials. Other types of elementary components, such as soft smart structures or soft-rigid hybrid systems, are then presented. The second part of this review deals with the manufacturing methods used to build complete soft structures. It includes molding, with possibly reinforcements and inclusions, additive manufacturing, thin-film manufacturing, shape deposition manufacturing, and bonding. The paper conclusions sums up the pros and cons of the presented techniques, and open to developing topics such as design methods for soft robotics and sensing technologies.

15.
J Vis Exp ; (107)2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26779607

ABSTRACT

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17ß-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 µl of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Estradiol/isolation & purification , Solid Phase Extraction/methods , Water Pollutants, Chemical/isolation & purification , Water/chemistry , Endocrine Disruptors/analysis , Endocrine Disruptors/isolation & purification , Estradiol/analysis , Organic Chemicals/analysis , Organic Chemicals/isolation & purification , Seawater/chemistry , Silicon Dioxide/chemistry , Solvents/analysis , Solvents/isolation & purification , Water Pollutants, Chemical/analysis
16.
Magn Reson Med ; 75(3): 1110-8, 2016 Mar.
Article in English | MEDLINE | ID: mdl-25846380

ABSTRACT

PURPOSE: MRI-guided thermal ablations require reliable monitoring methods to ensure complete destruction of the diseased tissue while avoiding damage to the surrounding healthy tissue. Based on the fact that thermal ablations result in substantial changes in biomechanical properties, interventional MR elastography (MRE) dedicated to the monitoring of MR-guided thermal therapies is proposed here. METHODS: Interventional MRE consists of a needle MRE driver, a fast and interactive gradient echo pulse sequence with motion encoding, and an inverse problem solver in real-time. This complete protocol was tested in vivo on swine and the ability to monitor elasticity changes in real-time was assessed in phantom. RESULTS: Thanks to a short repetition time, a reduction of the number of phase-offsets and the use of a sliding window, one refreshed elastogram was provided every 2.56 s for an excitation frequency of 100 Hz. In vivo elastograms of swine liver were successfully provided in real-time during one breath-hold. Changes of elasticity were successfully monitored in a phantom during its gelation with the same elastogram frame rate. CONCLUSION: This study demonstrates the ability of detecting elasticity changes in real-time and providing elastograms in vivo with interventional MRE that could be used for the monitoring of thermal ablations.


Subject(s)
Elasticity Imaging Techniques/methods , Endovascular Procedures/methods , Image Processing, Computer-Assisted/methods , Magnetic Resonance Imaging/methods , Algorithms , Animals , Feasibility Studies , Liver/blood supply , Liver/surgery , Models, Biological , Phantoms, Imaging , Swine
18.
J Chromatogr A ; 1381: 22-8, 2015 Feb 13.
Article in English | MEDLINE | ID: mdl-25604269

ABSTRACT

A fully automated and portable system for solid phase extraction (SPE) has been developed for the analysis of the natural hormone 17ß-estradiol (E2) in environmental water by enzyme linked immuno-sorbent assay (ELISA). The system has been validated with de-ionized and artificial sea water as model samples and allowed for pre-concentration of E2 at levels of 1, 10 and 100 ng/L with only 100 ml of sample. Recoveries ranged from 24±3% to 107±6% depending on the concentration and sample matrix. The method successfully allowed us to determine the concentration of two seawater samples. A concentration of 15.1±0.3 ng/L of E2 was measured in a sample obtained from a food production process, and 8.8±0.7 ng/L in a sample from the Adriatic Sea. The system would be suitable for continuous monitoring of water quality as it is user friendly, and as the method is reproducible and totally compatible with the analysis of water sample by simple immunoassays and other detection methods such as biosensors.


Subject(s)
Estradiol/analysis , Estrogens/analysis , Water Pollutants, Chemical/analysis , Water/analysis , Immunoassay , Seawater/analysis , Solid Phase Extraction/methods
19.
Biomicrofluidics ; 7(1): 11805, 2013.
Article in English | MEDLINE | ID: mdl-24403987

ABSTRACT

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

20.
Lab Chip ; 12(3): 640-6, 2012 Feb 07.
Article in English | MEDLINE | ID: mdl-22146948

ABSTRACT

We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 µm h(-1) healing rate).


Subject(s)
Epithelium/drug effects , Hepatocyte Growth Factor/pharmacology , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Microfluidics/instrumentation , Wound Healing/drug effects , Cell Line , Epithelial Cells/drug effects , Equipment Design , Humans , Phenotype , Pulmonary Alveoli/pathology , Regeneration/drug effects , Respiratory Mucosa/pathology
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