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1.
Int J Mol Sci ; 22(6)2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33808970

RESUMEN

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.


Asunto(s)
Vesículas Extracelulares/genética , Proteoma/genética , Proteómica , Medicina Regenerativa/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/tendencias , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/genética , Regeneración/genética
2.
Gene ; 786: 145621, 2021 Jun 20.
Artículo en Inglés | MEDLINE | ID: mdl-33798680

RESUMEN

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.


Asunto(s)
Catarata/diagnóstico por imagen , Proteína p53 Supresora de Tumor/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Catarata/genética , Catarata/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Técnicas de Inactivación de Genes , Humanos , Peróxido de Hidrógeno/efectos adversos , Cristalino/citología , Cristalino/efectos de los fármacos , Cristalino/metabolismo , Microscopía Electrónica de Transmisión , Pez Cebra
3.
Sci Rep ; 11(1): 6621, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758289

RESUMEN

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.


Asunto(s)
Células Epiteliales/metabolismo , Proteoma/análisis , Proteómica/métodos , /metabolismo , Bronquios/citología , /virología , Técnicas de Cultivo de Célula , Línea Celular , Medios de Cultivo/química , Células Epiteliales/citología , Humanos , Espectrometría de Masas , Modelos Biológicos , Análisis de Componente Principal , /fisiología
4.
J Vis Exp ; (168)2021 02 10.
Artículo en Inglés | MEDLINE | ID: mdl-33645569

RESUMEN

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.


Asunto(s)
Disección , Cultivo Primario de Células/métodos , Epitelio Pigmentado de la Retina/citología , Animales , Bioensayo , Diferenciación Celular , Polaridad Celular , Separación Celular , Impedancia Eléctrica , Células Epiteliales/citología , Humanos , Ratones Endogámicos C57BL , Fagocitosis , Factores de Tiempo
5.
Methods Mol Biol ; 2212: 245-264, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33733360

RESUMEN

The changes in gene expression under microarray technology are valuable to recognize and study the evolution process of species or diseases. Among the existing methods of analyzing the changes in gene expression, statistical method is one of the common and accurate approaches. This paper presents a step-by-step protocol to use Biopeak, a statistical tool to identify and visualize any significant impulse-like change of the gene expression in genomic series data. Biopeak focuses on the temporal features of the gene expression as signals. Through the statistical approaches including finding the local maximum and subsequent filtering, the potential changes are detected as the peak of signals. To filter the outliers and mark the significant changes, the correlation heatmap and clustering approach can be applied. Biopeak also provides several clustering techniques with different cluster abilities for result comparison. The step-by-step application of Biopeak is carried out by running the dataset of human epithelial cells in response to heat. The results of the peak detection, the correlation heatmap, and the clustering are demonstrated.


Asunto(s)
Algoritmos , Análisis por Conglomerados , Biología Computacional/métodos , Epistasis Genética , Células Epiteliales/metabolismo , Células Cultivadas , Células Epiteliales/citología , Perfilación de la Expresión Génica , Calor , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de la Célula Individual , Programas Informáticos
6.
J Enzyme Inhib Med Chem ; 36(1): 659-668, 2021 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-33641565

RESUMEN

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.


Asunto(s)
Antivirales/farmacología , Células Epiteliales/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Fenilalanina/farmacología , Inhibidores de Proteasas/farmacología , Serina Endopeptidasas/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/enzimología , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepatocitos/enzimología , Humanos , Peróxido de Hidrógeno/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/enzimología , Ocludina/genética , Ocludina/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fenilalanina/análogos & derivados , Cultivo Primario de Células , Serina Endopeptidasas/genética
7.
J Vis Exp ; (168)2021 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-33616118

RESUMEN

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.


Asunto(s)
Diferenciación Celular , Colon/citología , Células Epiteliales/citología , Mucosa Intestinal/citología , Cultivo Primario de Células/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Ratones , Ratones Endogámicos C57BL
8.
Science ; 371(6531): 839-846, 2021 02 19.
Artículo en Inglés | MEDLINE | ID: mdl-33602855

RESUMEN

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.


Asunto(s)
Enfermedades de los Conductos Biliares/terapia , Conductos Biliares Intrahepáticos/fisiología , Conductos Biliares/citología , Tratamiento Basado en Trasplante de Células y Tejidos , Células Epiteliales/citología , Organoides/trasplante , Animales , Bilis , Conductos Biliares/fisiología , Conductos Biliares Intrahepáticos/citología , Conducto Colédoco/citología , Células Epiteliales/fisiología , Vesícula Biliar/citología , Regulación de la Expresión Génica , Humanos , Hígado/fisiología , Trasplante de Hígado , Trasplante de Células Madre Mesenquimatosas , Ratones , Organoides/fisiología , RNA-Seq , Obtención de Tejidos y Órganos , Transcriptoma
9.
Methods Mol Biol ; 2273: 131-138, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604849

RESUMEN

The current coronavirus disease-19 (COVID-19) pandemic, caused by "severe acute respiratory syndrome coronavirus 2" (SARS-CoV-2), underscores the threat posed by newly emerging viruses. The understanding of the mechanisms driving early infection events, that are crucial for the exponential spread of the disease, is mandatory and can be significantly implemented generating 3D in vitro models as experimental platforms to investigate the infection substrates and how the virus invades and ravages the tissues.We here describe a protocol for the creation of a synthetic hydrogel-based 3D culture system that mimics in vitro the complex architectures and mechanical cues distinctive of the upper airway epithelia. We then expose the in vitro generated 3D nasal and tracheal epithelia to gold nanoparticles (AuNPs) that display the typical shape and size distinctive of SARS-CoV-2 and of the majority of Coronaviridae presently known.The infection platform here described provides an efficient and highly physiological in vitro model that reproduces the host-pathogen early interactions, using virus-mimicking nanoparticles, and offers a flexible tool to study virus entry into the cell. At the same time, it reduces the risk of accidental infection/spillovers for researchers, which represents a crucial aspect when dealing with a virus that is highly contagious, virulent, and even deadly.


Asunto(s)
/metabolismo , Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Nanopartículas/metabolismo , Mucosa Respiratoria/citología , Animales , Línea Celular , Chlorocebus aethiops , Células Epiteliales/virología , Oro , Humanos , Nanopartículas del Metal/química , Imitación Molecular/inmunología , Nariz/virología , Mucosa Respiratoria/virología , /patogenicidad , Tráquea/virología , Células Vero , Internalización del Virus
10.
Nature ; 590(7847): 618-623, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33568811

RESUMEN

Errors in early embryogenesis are a cause of sporadic cell death and developmental failure1,2. Phagocytic activity has a central role in scavenging apoptotic cells in differentiated tissues3-6. However, how apoptotic cells are cleared in the blastula embryo in the absence of specialized immune cells remains unknown. Here we show that the surface epithelium of zebrafish and mouse embryos, which is the first tissue formed during vertebrate development, performs efficient phagocytic clearance of apoptotic cells through phosphatidylserine-mediated target recognition. Quantitative four-dimensional in vivo imaging analyses reveal a collective epithelial clearance mechanism that is based on mechanical cooperation by two types of Rac1-dependent basal epithelial protrusions. The first type of protrusion, phagocytic cups, mediates apoptotic target uptake. The second, a previously undescribed type of fast and extended actin-based protrusion that we call 'epithelial arms', promotes the rapid dispersal of apoptotic targets through Arp2/3-dependent mechanical pushing. On the basis of experimental data and modelling, we show that mechanical load-sharing enables the long-range cooperative uptake of apoptotic cells by multiple epithelial cells. This optimizes the efficiency of tissue clearance by extending the limited spatial exploration range and local uptake capacity of non-motile epithelial cells. Our findings show that epithelial tissue clearance facilitates error correction that is relevant to the developmental robustness and survival of the embryo, revealing the presence of an innate immune function in the earliest stages of embryonic development.


Asunto(s)
Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Células Epiteliales/citología , Fagocitos/citología , Fagocitosis , Pez Cebra/embriología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Apoptosis , Movimiento Celular , Forma de la Célula , Extensiones de la Superficie Celular , Inmunidad Innata , Ratones , Fosfatidilserinas/metabolismo , Proteína de Unión al GTP rac1/metabolismo
11.
Methods Mol Biol ; 2273: 103-110, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604847

RESUMEN

Efficient isolation, characterization, and culture of endometrial epithelial cells and stromal fibroblasts from calf uteri collected at the slaughterhouse is key to develop useful 3D culture tissue models to investigate uterine physiology and pathology without the need of performing invasive procedures to recover tissue samples.Here we provide a detail methodology that gives consistently pure and viable populations of distinct primary bovine endometrial cells.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Cultivo Primario de Células/métodos , Animales , Bovinos , Células Cultivadas , Endometrio/citología , Endometrio/crecimiento & desarrollo , Células Epiteliales/citología , Femenino , Fibroblastos , Modelos Biológicos , Células del Estroma/citología , Células del Estroma/metabolismo
13.
Cell Prolif ; 54(4): e13005, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33594777

RESUMEN

PURPOSE: We investigated the role of farnesoid X receptor (FXR), a ligand-dependent transcription factor, in renal ischaemia-reperfusion (I/R) injury. MATERIALS AND METHODS: We performed unilateral renal I/R model in FXR knockout (Fxr-/- ) and wild-type (WT) mice in vivo and a hypoxia-reoxygenation (H/R) model in vitro. The pathways by which FXR induces apoptosis were detected using a proteome profiler array. The effects of FXR on apoptosis were evaluated using immunoblotting, TUNEL assays and flow cytometry. RESULTS: Compared with WT mice, Fxr-/- mice showed improved renal function and reduced tubular injury scores and apoptosis. Consistent with the in vivo results, the silencing of FXR decreased the number of apoptotic HK-2 cells after H/R, while FXR overexpression aggravated apoptosis. Notably, bone marrow transplantation (BMT) and immunohistochemistry experiments revealed the involvement of FXR in the tubular epithelium rather than in inflammatory cells. Furthermore, in vivo and in vitro studies demonstrated that FXR deficiency increased phosphorylated Bcl-2 agonist of cell death (p-Bad) expression levels and the ratio of Bcl-2/Bcl-xL to Bax expression in the kidney. Treatment with wortmannin, which reduced p-Bad expression, inhibited the effects of FXR deficiency and eliminated the tolerance of Fxr-/- mouse kidneys to I/R injury. CONCLUSIONS: These results established the pivotal importance of FXR inactivation in tubular epithelial cells after I/R injury. FXR may promote the apoptosis of renal tubular epithelial cells by inhibiting PI3k/Akt-mediated Bad phosphorylation to cause renal I/R damage.


Asunto(s)
Apoptosis , Receptores Citoplasmáticos y Nucleares/metabolismo , Daño por Reperfusión/patología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/genética , Daño por Reperfusión/metabolismo , Transducción de Señal/efectos de los fármacos , Wortmanina/farmacología , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/metabolismo
14.
Methods Mol Biol ; 2273: 251-262, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604859

RESUMEN

Oviduct and uterus are key female reproductive organs lined by ciliated simple columnar epithelia, which are the first line of maternal contact with gametes and the developing embryo during reproduction and which warrant the optimal developmental environment for the conceptus. A major challenge for modeling these epithelia in vitro is the preservation of apical-basal polarization and cilia formation. The air-liquid interface (ALI) culture approach is a technology originally invented for modeling epidermal and airway epithelia. It has recently been shown that it also allows the establishment of highly differentiated in vitro models of epithelia that do not have access to ambient air in vivo. In this chapter, we present a comprehensive ALI procedure to model female reproductive tract (FRT) epithelia of different mammalian species in vitro over extended time periods. As a working example, the protocol focuses on primary oviductal epithelial cells (OEC) isolated from domestic pig. Hints on protocol variations for the culture of OEC from other species are provided in the Subheading 4.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Epiteliales/citología , Trompas Uterinas/citología , Animales , Diferenciación Celular , Separación Celular/métodos , Células Cultivadas , Femenino , Humanos , Microscopía Electrónica de Transmisión/métodos , Porcinos
15.
Methods Mol Biol ; 2273: 279-296, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604861

RESUMEN

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.


Asunto(s)
Células Epiteliales/citología , Poliestirenos/química , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Células CACO-2 , Línea Celular , Fibroblastos/citología , Humanos , Queratinocitos/citología , Porosidad , Piel/citología
16.
Nat Protoc ; 16(4): 1907-1935, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33627843

RESUMEN

Multiphoton intravital imaging is essential for understanding cellular behavior and function in vivo. The adipose-rich environment of the mammary gland poses a unique challenge to in vivo microscopy due to light scattering that impedes high-resolution imaging. Here we provide a protocol for high-quality, six-color 3D intravital imaging of regions across the entire mouse mammary gland and associated tissues for several hours while maintaining tissue access for microdissection and labeling. An incision at the ventral midline and along the right hind leg creates a skin flap that is then secured to a raised platform skin side down. This allows for fluorescence-guided microdissection of connective tissue to provide unimpeded imaging of mammary ducts. A sealed imaging chamber over the skin flap creates a stable environment while maintaining access to large tissue regions for imaging with an upright microscope. We provide a strategy for imaging single cells and the tissue microenvironment utilizing multicolor Confetti lineage-tracing and additional dyes using custom-designed filters and sequential excitation with dual multiphoton lasers. Furthermore, we describe a strategy for simultaneous imaging and photomanipulation of single cells using the Olympus SIM scanner and provide steps for 3D video processing, visualization and high-dimensional analysis of single-cell behavior. We then provide steps for multiplexing intravital imaging with fixation, immunostaining, tissue clearing and 3D confocal imaging to associate cell behavior with protein expression. The skin-flap surgery and chamber preparation take 1.5 h, followed by up to 12 h of imaging. Applications range from basic filming in 1 d to 5 d for multiplexing and complex analysis.


Asunto(s)
Microscopía Intravital/métodos , Glándulas Mamarias Animales/citología , Análisis de la Célula Individual , Anestesia , Animales , Células Epiteliales/citología , Femenino , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional , Glándulas Mamarias Animales/cirugía , Ratones Endogámicos C57BL , Ratones Transgénicos , Células del Estroma/citología
17.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33572938

RESUMEN

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global public health emergency. Periodontitis, the most prevalent disease that leads to tooth loss, is caused by infection by periodontopathic bacteria. Periodontitis is also a risk factor for pneumonia and the exacerbation of chronic obstructive pulmonary disease, presumably because of the aspiration of saliva contaminated with periodontopathic bacteria into the lower respiratory tract. Patients with these diseases have increased rates of COVID-19 aggravation and mortality. Because periodontopathic bacteria have been isolated from the bronchoalveolar lavage fluid of patients with COVID-19, periodontitis may be a risk factor for COVID-19 aggravation. However, the molecular links between periodontitis and COVID-19 have not been clarified. In this study, we found that the culture supernatant of the periodontopathic bacterium Fusobacterium nucleatum (CSF) upregulated the SARS-CoV-2 receptor angiotensin-converting enzyme 2 in A549 alveolar epithelial cells. In addition, CSF induced interleukin (IL)-6 and IL-8 production by both A549 and primary alveolar epithelial cells. CSF also strongly induced IL-6 and IL-8 expression by BEAS-2B bronchial epithelial cells and Detroit 562 pharyngeal epithelial cells. These results suggest that when patients with mild COVID-19 frequently aspirate periodontopathic bacteria, SARS-CoV-2 infection is promoted, and inflammation in the lower respiratory tract may become severe in the presence of viral pneumonia.


Asunto(s)
/metabolismo , Medios de Cultivo Condicionados/química , Citocinas/metabolismo , Fusobacterium nucleatum/metabolismo , /genética , /virología , Línea Celular , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Regulación hacia Arriba/efectos de los fármacos
18.
Int J Nanomedicine ; 16: 725-740, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33542627

RESUMEN

Purpose: As a dental material, polyetheretherketone (PEEK) is bioinert that does not induce cellular response and bone/gingival tissues regeneration. This study was to develop bioactive coating on PEEK and investigate the effects of coating on cellular response. Materials and Methods: Tantalum pentoxide (TP) coating was fabricated on PEEK surface by vacuum evaporation and responses of rat bone marrow mesenchymal stem (RBMS) cells/human gingival epithelial (HGE) were studied. Results: A dense coating (around 400 nm in thickness) of TP was closely combined with PEEK (PKTP). Moreover, the coating was non-crystalline TP, which contained many small humps (around 10 nm in size), exhibiting a nanostructured surface. In addition, the roughness, hydrophilicity, surface energy, and protein adsorption of PKTP were remarkably higher than that of PEEK. Furthermore, the responses (adhesion, proliferation, and osteogenic gene expression) of RBMS cells, and responses (adhesion and proliferation) of HGE cells to PKTP were remarkably improved in comparison with PEEK. It could be suggested that the nanostructured coating of TP on PEEK played crucial roles in inducing the responses of RBMS/HGE cells. Conclusion: PKTP with elevated surface performances and outstanding cytocompatibility might have enormous potential for dental implant application.


Asunto(s)
Células Epiteliales/citología , Encía/citología , Cetonas/farmacología , Células Madre Mesenquimatosas/citología , Nanoestructuras/química , Óxidos/farmacología , Polietilenglicoles/farmacología , Tantalio/farmacología , Adsorción , Fosfatasa Alcalina/metabolismo , Animales , Adhesión Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Nanoestructuras/ultraestructura , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Ratas Sprague-Dawley , Espectroscopía Infrarroja por Transformada de Fourier , Propiedades de Superficie , Difracción de Rayos X
19.
J Vis Exp ; (167)2021 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-33554964

RESUMEN

Cell dissociation has been an essential procedure for studies at the individual-cell level and/or at a cell-population level (e.g., single cell RNA sequencing and primary cell culture). Yielding viable, healthy cells in large quantities is critical, and the optimal conditions to do so are tissue dependent. Cell populations in the tongue epithelium and underlying mesenchyme/connective tissue are heterogeneous and tissue structures vary in different regions and at different developmental stages. We have tested protocols for isolating cells from the mouse tongue epithelium and mesenchyme/connective tissue in the early developmental [embryonic day 12.5 (E12.5)] and young adult (8-week) stages. A clean separation between the epithelium and underlying mesenchyme/connective tissue was easy to accomplish. However, to further process and isolate cells, yielding viable healthy cells in large quantities, and careful selection of enzymatic digestion buffer, incubation time, and centrifugation speed and time are critical. Incubation of separated epithelium or underlying mesenchyme/connective tissue in 0.25% Trypsin-EDTA for 30 min at 37 °C, followed by centrifugation at 200 x g for 8 min resulted in a high yield of cells at a high viability rate (>90%) regardless of the mouse stages and tongue regions. Moreover, we found that both dissociated epithelial and mesenchymal/connective tissue cells from embryonic and adult tongues could survive in the cell culture-based medium for at least 3 h without a significant decrease of cell viability. The protocols will be useful for studies that require the preparation of isolated cells from mouse tongues at early developmental (E12.5) and young adult (8-week) stages requiring cell dissociation from different tissue compartments.


Asunto(s)
Tejido Conectivo/embriología , Embrión de Mamíferos/citología , Células Epiteliales/citología , Epitelio/embriología , Mesodermo/citología , Lengua/embriología , Animales , Recuento de Células , Supervivencia Celular , Procesamiento de Imagen Asistido por Computador , Ratones Endogámicos C57BL
20.
Phys Rev Lett ; 126(4): 048101, 2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33576647

RESUMEN

Recent advances in microscopy techniques make it possible to study the growth, dynamics, and response of complex biophysical systems at single-cell resolution, from bacterial communities to tissues and organoids. In contrast to ordered crystals, it is less obvious how one can reliably distinguish two amorphous yet structurally different cellular materials. Here, we introduce a topological earth mover's (TEM) distance between disordered structures that compares local graph neighborhoods of the microscopic cell-centroid networks. Leveraging structural information contained in the neighborhood motif distributions, the TEM metric allows an interpretable reconstruction of equilibrium and nonequilibrium phase spaces and embedded pathways from static system snapshots alone. Applied to cell-resolution imaging data, the framework recovers time ordering without prior knowledge about the underlying dynamics, revealing that fly wing development solves a topological optimal transport problem. Extending our topological analysis to bacterial swarms, we find a universal neighborhood size distribution consistent with a Tracy-Widom law.


Asunto(s)
Modelos Teóricos , Reconocimiento de Normas Patrones Automatizadas/métodos , Algoritmos , Animales , Fenómenos Biofísicos , Coloides/química , Microscopía por Crioelectrón , Drosophila , Entropía , Células Epiteliales/citología , Interpretación de Imagen Asistida por Computador/métodos , Modelos Biológicos , Modelos Químicos , ARN/química
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