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1.
Altern Lab Anim ; 47(1): 19-29, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31237165

RESUMO

Ex vivo organ cultures represent unique research models, as they combine the advantages of cell cultures with those of animal models. Being able to mimic in vivo situations through the use of organ cultures provides an excellent opportunity to investigate cellular processes, molecular pathways and cell-cell interactions, as well as structural and synaptic organisation. Human and animal organ cultures are now well established and comprise sensitive, easy-to-manipulate experimental systems that raise minimal ethical concerns. The eye, in particular, is a very complex organ that is not easy to reproduce in vitro. However, a lot of research has been dedicated to the development of suitable ocular organ cultures. This review covers the various ex vivo retinal organ culture systems available for use in ophthalmology research and compares them with commonly used animal models. In particular, bovine and porcine retinal organ culture systems are described, because the size, anatomy, physiology and vessel morphology of bovine and porcine eyes are similar to the human eye in an undisputed way, thus making them good models. In addition, these animals are widely used by the food industry and the eyes are considered surplus material. A short overview of murine, rat, rabbit, cat, canine and simian retinal organ cultures is also provided.


Assuntos
Técnicas de Cultura de Órgãos , Retina , Animais , Técnicas de Cultura de Células , Humanos , Modelos Animais , Modelos Biológicos , Retina/citologia , Suínos
2.
Stud Health Technol Inform ; 261: 274-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156129

RESUMO

The main goal of this research is to design, develop and implement an efficient protocol to generate 3D neural cultures derived from human induced Pluripotent Stem Cells (hiPSCs) coupled to Micro Electrode Arrays (MEA) in order to obtain an engineered and controlled brain-on-a-chip model. The use of patient specific iPSCs may offer novel insights into the pathophysiology of a large variety of disorders, including numerous neurodevelopmental and late-onset neurodegenerative conditions. With these in vitro patient specific models, we may have the possibility to test drugs and find ad hoc therapies in the direction of precision medicine.


Assuntos
Encéfalo , Células-Tronco Pluripotentes Induzidas , Dispositivos Lab-On-A-Chip , Técnicas de Cultura de Órgãos , Encéfalo/fisiologia , Humanos , Doenças Neurodegenerativas , Transtornos do Neurodesenvolvimento , Organoides
3.
Vet Microbiol ; 234: 119-127, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213267

RESUMO

Ex vivo organ cultures (EVOCs) are extensively used to study the cellular tropism and infectivity of different pathogens. In this study, we used ovine and porcine respiratory EVOCs to investigate the replication kinetics and cellular tropism of selected emerging reoviruses namely Pteropine orthoreovirus, an emerging bat-borne zoonotic respiratory virus, and atypical Bluetongue virus (BTV) serotypes which, unlike classical serotypes, do not cause Bluetongue, a major OIE-listed disease of ruminants. BTV failed to replicate in ovine EVOCs. Instead, PRV showed slight replication in porcine lower respiratory EVOCs and a more sustained replication in all ovine respiratory tissues. By confocal laser scanning microscopy, PRV was demonstrated to infect bronchiolar and type I pneumocytes of ovine tissues. Overall, respiratory EVOCs from different animal species, eventually obtained at slaughterhouse, are a useful tool for testing and preliminarily characterize novel and emerging viruses addressing the essential in vivo animal work. Further experiments are, indeed, warranted in order to characterize the pathogenesis and transmission of these emerging reoviruses.


Assuntos
Orthoreovirus/fisiologia , Tropismo Viral , Replicação Viral , Células Epiteliais Alveolares/virologia , Animais , Vírus Bluetongue/fisiologia , Brônquios/citologia , Brônquios/virologia , Cinética , Técnicas de Cultura de Órgãos , Ovinos , Suínos
4.
Cornea ; 38(7): 829-835, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31170101

RESUMO

PURPOSE: To report the rate of microbial contamination and analyze possible risk factors for contamination of banked corneas stored using the organ culture method. METHODS: Data from the New South Wales Tissue Banks incorporating the Lions NSW Eye Bank, between September 1, 2011, and November 30, 2017, were reviewed retrospectively. All corneas collected during this period and stored in organ culture storage media were tested for microbial contamination. The influence of potential factors on the rate of contamination was analyzed using the χ test and logistic regression using generalized estimating equations. RESULTS: A total of 4410 corneas were included in this study, of which 110 were medium culture positive, representing a microbial contamination rate of 2.5%. The main contaminants were Candida species followed by Staphylococcus species. Corneal tissue collected in summer and autumn had a significantly higher contamination rate (P = 0.006). All other factors studied were not shown to have a statistically significant association with contamination after accounting for within-pair correlation and confounders. CONCLUSIONS: A relatively low contamination rate of 2.5% observed in our study reflects the stringent laboratory protocols, strict donor selection criteria, and high level of experience among staff at the Lions NSW Eye Bank. Our study demonstrated that the season of collection had a strong association with the rate of organ culture contamination. Because Candida species contributed the largest percentage of contamination, specific measures to reduce and eliminate fungal proliferation should be considered by eye banks particularly in warm seasons.


Assuntos
Córnea/microbiologia , Técnicas de Cultura de Órgãos , Preservação de Órgãos/métodos , Adulto , Idoso , Bactérias/isolamento & purificação , Bancos de Olhos/estatística & dados numéricos , Infecções Oculares Bacterianas/microbiologia , Feminino , Fungos/isolamento & purificação , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , New South Wales , Estudos Retrospectivos , Fatores de Risco , Doadores de Tecidos
5.
Invest Ophthalmol Vis Sci ; 60(6): 1914-1927, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31042799

RESUMO

Purpose: Retinal explant cultures provide simplified systems where the functions of the retina and the effects of ocular therapies can be studied in an isolated environment. The purpose of this study was to provide insight into long-term preservation of retinal tissue in culture conditions, enable a deeper understanding of the interdependence of retinal morphology and function, and ensure the reliability of the explant technique for prolonged experiments. Methods: Retinal explants from adult mice were cultured as organotypic culture at the air-medium interface for 14 days in vitro (DIV). Retinal functionality was assessed by multielectrode array technique and morphology by immunohistochemical methods at several time points during culture. Results: Retinal explants retained viability for 14 DIV, although with diminishing neuronal activity, progressing neuronal loss, and increasing reactive gliosis. We recorded spontaneous retinal ganglion cell (RGC) activity up to 14 DIV with temporally changing distribution of RGC firing rates. Light responsiveness was measurable from RGCs for 7 DIV and from photoreceptors for 2 DIV. Apoptotic cells were detected beginning at 3 DIV with their density peaking at 7 DIV. The number of RGCs gradually decreased by 70% during 14 DIV. The change was accompanied by the loss of RGC functionality, resulting in 84% loss of electrically active RGCs. Conclusions: Retinal explants provide a valuable tool for studies of retinal functions and development of ocular therapies. However, critical for long-term use, retinal functionality was lost before structural loss, emphasizing a need for both functional and morphologic readouts to determine the overall state of the cultured retina.


Assuntos
Eletrorretinografia/métodos , Células Ganglionares da Retina/citologia , Preservação de Tecido/métodos , Animais , Sobrevivência Celular , Células Cultivadas , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Técnicas de Cultura de Órgãos , Reprodutibilidade dos Testes , Células Ganglionares da Retina/fisiologia
6.
Nat Mater ; 18(6): 627-637, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31114073

RESUMO

Cells are transplanted to regenerate an organs' parenchyma, but how transplanted parenchymal cells induce stromal regeneration is elusive. Despite the common use of a decellularized matrix, little is known as to the pivotal signals that must be restored for tissue or organ regeneration. We report that Alx3, a developmentally important gene, orchestrated adult parenchymal and stromal regeneration by directly transactivating Wnt3a and vascular endothelial growth factor. In contrast to the modest parenchyma formed by native adult progenitors, Alx3-restored cells in decellularized scaffolds not only produced vascularized stroma that involved vascular endothelial growth factor signalling, but also parenchymal dentin via the Wnt/ß-catenin pathway. In an orthotopic large-animal model following parenchyma and stroma ablation, Wnt3a-recruited endogenous cells regenerated neurovascular stroma and differentiated into parenchymal odontoblast-like cells that extended the processes into newly formed dentin with a structure-mechanical equivalency to native dentin. Thus, the Alx3-Wnt3a axis enables postnatal progenitors with a modest innate regenerative capacity to regenerate adult tissues. Depleted signals in the decellularized matrix may be reinstated by a developmentally pivotal gene or corresponding protein.


Assuntos
Proteínas de Homeodomínio/metabolismo , Tecido Parenquimatoso/fisiologia , Dente/citologia , Dente/embriologia , Adolescente , Animais , Feminino , Proteínas de Homeodomínio/genética , Humanos , Incisivo/citologia , Incisivo/embriologia , Camundongos Endogâmicos , Dente Serotino/citologia , Técnicas de Cultura de Órgãos , Tecido Parenquimatoso/citologia , Gravidez , Regiões Promotoras Genéticas , Regeneração , Células Estromais/fisiologia , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo
7.
Mar Drugs ; 17(5)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035725

RESUMO

Melanoma is one of the most malignant and aggressive types of cancer worldwide. Fibroblast growth factor 2 (FGF2) is one of the critical regulators of melanoma angiogenesis and metastasis; thus, it might be an effective anti-cancer strategy to explore FGF2-targeting drug candidates from existing drugs. In this study, we evaluate the effect of the marine drug propylene glycol alginate sodium sulfate (PSS) on FGF2-mediated angiogenesis and invasion. The data shows that FGF2 selectively bound to PSS with high affinity. PSS inhibited FGF2-mediated angiogenesis in a rat aortic ring model and suppressed FGF2-mediated invasion, but not the migration of murine melanoma B16-F10 cells. The further mechanism study indicates that PSS decreased the expression of activated matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9), and also suppressed their activity. In addition, PSS was found to decrease the level of Vimentin in B16-F10 cells, which is known to participate in the epithelial-mesenchymal transition. Notably, PSS did not elicit any changes in cancer cell viability. Based on the results above, we conclude that PSS might be a potential drug to regulate the tumor microenvironment in order to facilitate the recovery of melanoma patients.


Assuntos
Alginatos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Alginatos/uso terapêutico , Animais , Aorta/efeitos dos fármacos , Organismos Aquáticos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide , Avaliação Pré-Clínica de Medicamentos , Transição Epitelial-Mesenquimal , Humanos , Laminaria/química , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Camundongos , Invasividade Neoplásica/patologia , Invasividade Neoplásica/prevenção & controle , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/patologia , Microambiente Tumoral/efeitos dos fármacos
8.
Folia Neuropathol ; 57(1): 24-35, 2019.
Artigo em Polonês | MEDLINE | ID: mdl-31038185

RESUMO

INTRODUCTION: This study was performed to determine whether the disturbed maturation of oligodendrocyte (OL) progenitors might be related to lipopolysaccharide (LPS)-induced hypomyelination. MATERIAL AND METHODS: We created organotypic cultures of forebrain slices from neonatal rats and explored the morphological changes of glial cells expressing tumour necrosis factor  (TNF-) following LPS exposure. RESULTS: We observed marked activation of glial fibrillary acidic protein-positive astrocytes and OX42-positive microglia co-labelled with TNF- four days following LPS exposure. Our results further demonstrated a reduced expression of O4-positive and O1-positive OL progenitors; moreover, we found that their morphologies were suggestive of degeneration (e.g., scanty, rounded bodies with short, fragmented processes and/or cytoplasmic condensation). At seven days following LPS exposure, astrocytes and microglia were still co-labelled for TNF-; however, the expression of O4-positive and O1-positive cells somewhat increased compared to the number observed at 4 days; despite remaining undifferentiated and exhibiting immature morphologies, the cells were likely indicative of regeneration. In contrast, O4-positive and O1-positive cells in controls were well-differentiated, displaying round, thick cell bodies and long, branching processes. CONCLUSIONS: In conclusion, maturation arrest and/or under-differentiation of OL progenitors commonly occur during regeneration: they may underlie the degeneration and consequent hypomyelination occurring late after injury, or apoptosis during the acute stage post-injury. Microglia and astrocytes expressing TNF- may also contribute to later myelination failure.


Assuntos
Inflamação/patologia , Fibras Nervosas Mielinizadas , Neurogênese/fisiologia , Células Precursoras de Oligodendrócitos/patologia , Animais , Animais Recém-Nascidos , Lipopolissacarídeos/toxicidade , Técnicas de Cultura de Órgãos , Prosencéfalo/embriologia , Ratos , Ratos Sprague-Dawley
9.
J Headache Pain ; 20(1): 47, 2019 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053059

RESUMO

BACKGROUND: Racemic isometheptene [(RS)-isometheptene] is an antimigraine drug that due to its cardiovascular side-effects was separated into its enantiomers, (R)- and (S)-isometheptene. This study set out to characterize the contribution of each enantiomer to its vasoactive profile. Moreover, rat neurogenic dural vasodilatation was used to explore their antimigraine mechanism of action. METHODS: Human blood vessel segments (middle meningeal artery, proximal and distal coronary arteries, and saphenous vein) were mounted in organ baths and concentration response curves to isometheptene were constructed. Calcitonin gene-related peptide (CGRP)-induced neurogenic dural vasodilation was elicited in the presence of the enantiomers using a rat closed cranial window model. RESULTS: The isometheptene enantiomers did not induce any significant contraction in human blood vessels, except in the middle meningeal artery, when they were administered at the highest concentration (100 µM). Interestingly in rats, (S)-isometheptene induced more pronounced vasopressor responses than (R)-isometheptene. However, none of these compounds affected the CGRP-induced vasodilator responses. CONCLUSION: The isometheptene enantiomers displayed a relatively safe peripheral vascular profile, as they failed to constrict the human coronary artery. These compounds do not appear to modulate neurogenic dural CGRP release, therefore, their antimigraine site of action remains to be determined.


Assuntos
Vasos Coronários/efeitos dos fármacos , Artérias Meníngeas/efeitos dos fármacos , Metilaminas/farmacologia , Transtornos de Enxaqueca , Veia Safena/efeitos dos fármacos , Adulto , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Vasos Coronários/fisiologia , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Artérias Meníngeas/fisiologia , Metilaminas/química , Metilaminas/uso terapêutico , Pessoa de Meia-Idade , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/fisiopatologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Veia Safena/fisiologia , Estereoisomerismo , Vasoconstritores/química , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Vasodilatadores/química , Vasodilatadores/farmacologia
10.
Invest Ophthalmol Vis Sci ; 60(5): 1776-1788, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31022732

RESUMO

Purpose: To determine the effects of αvß3 integrin expression and activation on intraocular pressure (IOP). Methods: Cre+/-ß3flox/flox mice were treated with topical tamoxifen eye drops for 5 days to activate Cre and excise the ß3 integrin gene from the anterior segment. IOP was measured weekly for 11 weeks using rebound tonometry. Mice were then killed and changes in expression of the ß3 integrin subunit in Cre+/- ß3flox/flox mice were determined using Western blotting analysis and immunofluorescence microscopy. To determine the effect of αvß3 integrin activation on outflow facility, porcine organ culture anterior segments (POCAS) were perfused with the αvß3 integrin-activating antibody AP5 or an isotype IgG control for 21 hours. The effect of αvß3 integrin activation on IOP was measured over 7 days in C57BL/6J mice intracamerally infused with AP5, AP3, IgG, or PBS. Results: Deletion of the ß3 integrin subunit using the tamoxifen-inducible Cre-loxP system resulted in a decrease in expression of the ß3 integrin subunit in the trabecular meshwork and ciliary muscle. Morphologically no gross changes in the anterior segment were detected. Deletion of the ß3 integrin subunit resulted in a significantly (P < 0.05) lower IOP in mice within 2 weeks following the tamoxifen treatment and persisted for 11 weeks. Activating the αvß3 integrin with the AP5 antibody resulted in a significant (P < 0.05) increase in IOP in C57BL/6J mice and a decrease in outflow facility in 42% of the POCAS. Conclusions: These studies demonstrate a role for αvß3 integrin signaling in the regulation of IOP.


Assuntos
Segmento Anterior do Olho/metabolismo , Regulação da Expressão Gênica/fisiologia , Integrina alfaVbeta3/genética , Pressão Intraocular/fisiologia , Idoso de 80 Anos ou mais , Animais , Segmento Anterior do Olho/efeitos dos fármacos , Western Blotting , Feminino , Humanos , Integrases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Técnicas de Cultura de Órgãos , Reação em Cadeia da Polimerase em Tempo Real , Suínos , Tamoxifeno/farmacologia , Tonometria Ocular
11.
J Dermatol Sci ; 93(3): 144-149, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30904351

RESUMO

Androgenetic alopecia (AGA) is the most common form of hair loss disorder. As the prevalence of AGA rises, the demand for AGA treatments is rising accordingly, prompting research to identify therapeutic candidates to treat AGA. Because AGA is caused by crosstalk among multiple hair follicle (HF) cell components, understanding the effects of candidate molecules on HF cells is essential to determining therapeutic candidates for treatment. To date, research has centered on HF dermal papilla and outer root sheath cells and has indicated that the hair growth effects of candidate substances may be mediated via alterations in several signaling pathways and signature genes in these HF cells. In more integrative evaluations, the HF unit is used as an ex vivo organ culture model to verify the effects of therapeutic candidates. Animal models have also been used to evaluate the effects of candidate substances. The main outcomes used to evaluate the effects of candidate substances are 1) changes in HF growth rates in vitro, 2) anagen induction capabilities, and 3) the effects of androgen modulation. This article reviews a series of methods used to evaluate the hair growth-promoting effects of candidate substances, providing an overview of cell assays, organs, and animal models used in AGA research in order to facilitate AGA research moving forward.


Assuntos
Alopecia/tratamento farmacológico , Fármacos Dermatológicos/farmacologia , Folículo Piloso/efeitos dos fármacos , Modelos Animais , Técnicas de Cultura de Órgãos/métodos , Alopecia/patologia , Animais , Fármacos Dermatológicos/uso terapêutico , Avaliação Pré-Clínica de Medicamentos/métodos , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/patologia , Humanos , Transdução de Sinais/efeitos dos fármacos
12.
Pak J Pharm Sci ; 32(1(Supplementary)): 261-268, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30829202

RESUMO

Using rat thoracic aortic rings to test the relaxing effects of the 95% ethanol extract and aqueous extract of Taohong Siwu decoction (THSW) on endothelium intact or endothelium removed aortic rings. Results showed that the 95% ethanol extract (0.1, 1, 10, 100, 1000 mg•L-1) and aqueous extract (0.1, 1, 10, 100, 1000 mg•L-1) of THSW were able to relax the intact endothelium aortic rings pre-contracted by 10-6 mol•L-1 PE. 10-4 mol•L-1 L-NAME and 10-5 mol•L-1 methylene blue both were able to inhibit the relaxation other than indomethacin. For the endothelium removed aortic rings, potassium channel blocker 3×10-3mol•L-1 tetraethylammonium chloride and 10-5 mol•L-1 glibenclamide had no effect on the relaxation effects caused by the 95% ethanol extract and aqueous extract of THSW. It could be concluded that the 95% ethanol extract and aqueous extract of THSW relax blood vessel by endothelium-dependent way.


Assuntos
Aorta/efeitos dos fármacos , GMP Cíclico/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Óxido Nítrico/metabolismo , Vasodilatadores/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Glibureto/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Técnicas de Cultura de Órgãos , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Tetraetilamônio/farmacologia
13.
Zygote ; 27(2): 55-63, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30871647

RESUMO

SummaryStudies have shown that daily exposure to different products, whether chemical or natural, can cause irreversible damage to women's reproductive health. Therefore it is necessary to use tests that evaluate the safety and efficacy of these products. Most reproductive toxicology tests are performed in vivo. However, in recent years, various cell culture methods, including embryonic stem cells and tissues have been developed with the aim of reducing the use of animals in toxicological tests. This is a major advance in the area of toxicology, as these systems have the potential to become a widely used tool compared with in vivo tests routinely used in reproductive biology and toxicology. The present review describes and highlights data on in vitro culture processes used to evaluate reproductive toxicity as an alternative to traditional methods using in vivo tests.


Assuntos
Técnicas de Cultura de Órgãos/métodos , Folículo Ovariano/citologia , Ovário/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodos , Animais , Linhagem Celular , Feminino , Humanos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/fisiologia
14.
AAPS PharmSciTech ; 20(4): 156, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30927154

RESUMO

The present research work explored the possibility of harnessing the benefits of vesicular carriers for overcoming imiquimod-associated complaints or side effects. Hybrid vesicles were prepared by the most common and easily scalable method, i.e., thin film hydration. The chaffing of myriad of factors, both process and material related, affecting the desirable attributes of conceived vesicles, was performed through Taguchi design. Based upon the analysis of Pareto chart and prior experiences, concentration of phospholipid and poloxamer 407 was selected for optimization by 2 levels, 13 run central composite design (CCD). The optimized hybrid vesicles contained 1% w/v phospholipid and 3% w/v poloxamer 407. The optimized hybrid vesicles were incorporated into the 3% w/v carbopol 940 gel and characterized for morphology, physicochemical properties, and rheological behavior. The release (%) and skin retention (% of total dose) across rat skin from gel at same amount of formulation was more than Imiquad®. The gel delivered the loaded cargo, preferably, in the viable region of skin and formed local depot in confocal microscopic studies. The gel followed one compartment open body dermatokinetic model in rat skin. There was not any harmful effect on the mice skin after repeated applications. The gel was stable at room conditions for 1 year.


Assuntos
Portadores de Fármacos/síntese química , Portadores de Fármacos/farmacocinética , Imiquimode/síntese química , Imiquimode/farmacocinética , Absorção Cutânea/efeitos dos fármacos , Adjuvantes Imunológicos , Animais , Estabilidade de Medicamentos , Feminino , Géis/química , Masculino , Camundongos , Técnicas de Cultura de Órgãos , Tamanho da Partícula , Fosfolipídeos/química , Fosfolipídeos/farmacocinética , Ratos , Reologia , Pele/efeitos dos fármacos , Pele/metabolismo , Absorção Cutânea/fisiologia
15.
Ann Otol Rhinol Laryngol ; 128(7): 585-594, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30832485

RESUMO

OBJECTIVES: In tracheal regeneration, the slow process of epithelialization is often a barrier to the stability and safety of the transplanted trachea. The aim of this study was to examine a new tracheal regeneration technique using organotypically cultured tissue composed of autologous cells. METHODS: Nine beagles were prepared. Chondrocytes from auricular cartilage and epithelial cells from buccal mucosa were isolated and cultured. Tissue-engineered cartilages were fabricated with chondrocytes at a density of 1 × 107 cells/mL (high-density group) and 1 × 106 cells/mL (low-density group). A fabricated epithelial cell sheet was laid on a poly(lactic-co-glycolic acid) block in atelocollagen gel containing the chondrocytes, and the organotypically cultured tissues were transplanted into a partially resected trachea. The control group had only the block transplanted. RESULTS: The tissue-engineered cartilages in the high-density group contained many viable chondrocytes and many cartilage matrices. The low-density group had abundant collagen fibers and no chondrocytes. Tracheal endoscopy revealed no deformation or atrophy at the transplant site in the high-density group. Histologically, partially hyaline cartilages covered with epithelium and lamina propria were found in the high-density group but not in the low-density and control groups. CONCLUSIONS: Stable tracheal regeneration was achieved using organotypically cultured tissue fabricated with autologous high-density chondrocytes and epithelial cells.


Assuntos
Condrócitos/citologia , Células Epiteliais/citologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos , Traqueia/citologia , Animais , Materiais Biocompatíveis , Condrócitos/transplante , Colágeno , Cães , Cartilagem da Orelha/citologia , Células Epiteliais/transplante , Mucosa Bucal/citologia , Técnicas de Cultura de Órgãos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Regeneração , Tecidos Suporte , Traqueia/transplante , Transplante Autólogo
16.
Microbiome ; 7(1): 43, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30890187

RESUMO

BACKGROUND: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. RESULTS: We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites-4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid-that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. CONCLUSION: Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.


Assuntos
Bactérias/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/patologia , Intestinos/citologia , Técnicas de Cultura de Órgãos/métodos , Animais , Benzoatos/farmacologia , Caproatos/farmacologia , Células Cultivadas , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Microbioma Gastrointestinal , Ácidos Heptanoicos/farmacologia , Humanos , Intestinos/microbiologia , Masculino , Camundongos , Procedimentos Analíticos em Microchip , Especificidade da Espécie
17.
Methods Mol Biol ; 1940: 109-127, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788821

RESUMO

Research on lung development and disease frequently utilizes mouse models to conduct in vitro experiments. Such experiments involve multiple methodologically distinct stages, from careful consideration of mouse models used to obtain biological samples, to the culturing and imaging of those samples, and finally, to post-imaging analysis. Here, we detail basic protocols to assist with each of these stages. First, we discuss harvesting and preparing biological samples; second, we focus on culturing embryonic whole lung explants and isolated mesenchyme and epithelium; third, we specify the basics of obtaining still and live images; and finally, we bring these methods together by considering and briefly analyzing a lineage-labelling experiment.


Assuntos
Desenvolvimento Embrionário/fisiologia , Epitélio/crescimento & desenvolvimento , Pulmão/embriologia , Mesoderma/crescimento & desenvolvimento , Imagem Óptica/métodos , Técnicas de Cultura de Órgãos/métodos , Animais , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/embriologia , Proteínas Luminescentes/química , Pulmão/citologia , Camundongos , Camundongos Endogâmicos C57BL
18.
Methods Mol Biol ; 1940: 181-191, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788826

RESUMO

Retinal explant culture systems have the potential to mimic the functional dynamics of the organ beyond those of the dissociated cells, thus making this technique a very powerful intermediate model system between in vitro cell cultures and in vivo animal models. The different retinal layers made of highly specialized cell types remain intact, while glia cell reactions and/or intercellular interactions can be evaluated under well-defined conditions in the lab.In retinal disorders neurodegeneration of mature retinal cells takes place. Therefore, we investigated the adult murine neuroretina in organ culture to test its suitability for use in preclinical therapeutic applications. Here we describe a method for the organ culture of adult murine retina (>12 weeks) used to establish survival, cellular changes and early degeneration patterns of neuronal and glial cells. After enucleation of the eyeball and careful dissection of the retina from the sclera and retinal pigment epithelium, the detached retina is cultured with photoreceptor facing down on a supporting track-etched polycarbonate membrane in a 6-well culture plate maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. After 1, 2, 3, 4, 6, 8, or 10 days retinal explants can be harvested and immediately processed for RNA isolation or fixed in paraformaldehyde for histological analysis.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Técnicas de Cultura de Órgãos/métodos , Retina/crescimento & desenvolvimento , Retina/metabolismo , Doenças Retinianas/terapia , Animais , Técnicas de Cultura de Células , Células Cultivadas , Modelos Animais de Doenças , Gliose/terapia , Camundongos , Neuroglia/citologia , Neuroglia/patologia , Neurônios/citologia , Neurônios/patologia , Retina/transplante , Degeneração Retiniana/terapia , Descolamento Retiniano/terapia , Doenças Retinianas/patologia
19.
Methods Mol Biol ; 1940: 255-265, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788831

RESUMO

Pancreatic islets constitute an important tool for research and clinical applications in the field of diabetes. They are used for transplantation, unraveling new mechanisms in insulin secretion, studying pathophysiological pathways in diseased cells, and pharmacological research aimed at developing improved therapeutic strategies. Therefore, fine-tuning islet isolation protocols remains an important objective for reliable investigations. Here we describe a relatively simple mouse islet isolation protocol that relies on enzymatic digestion using low-activity collagenase and several sedimentation and Percoll gradient steps.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Ilhotas Pancreáticas/citologia , Animais , Células Cultivadas , Colagenases/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos
20.
Methods Mol Biol ; 1940: 297-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788834

RESUMO

Precision-cut lung slices (PCLS) represent an ex vivo model widely used in visualizing interactions between lung structure and function. The major advantage of this technique is that the presence, differentiation state, and localization of the more than 40 cell types that make up the lung are in accordance with the physiological situation found in lung tissue, including the right localization and patterning of extracellular matrix elements. Here we describe the methodology involved in preparing and culturing PCLS followed by detailed practical information about their possible applications.


Assuntos
Pulmão/citologia , Técnicas de Cultura de Órgãos/métodos , Animais , Células Cultivadas , Meios de Cultura/química , Matriz Extracelular/fisiologia , Camundongos , Técnicas de Cultura de Tecidos
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