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1.
Med Sci (Paris) ; 36(10): 879-885, 2020 Oct.
Artigo em Francês | MEDLINE | ID: mdl-33026330

RESUMO

Pancreatic islet transplantation is a valid cure for selected type-1 diabetic patients. It offers a minimally invasive ß-cell replacement approach and has proven its capacity to significantly enhance patients quality of life. However, these insulin-secreting mini-organs suffer from the loss of intrinsic vascularization and extra-cellular matrix occurring during isolation, resulting in hypoxic stress and necrosis. In addition, they have to face inflammatory and immune destruction once transplanted in the liver. Organoid generation represents a strategy to overcome these obstacles by allowing size and shape control as well as composition. It does offer the possibility to add supporting cells such as endothelial cells, in order to facilitate revascularization or cells releasing anti-inflammatory and/or immunomodulatory factors. This review describes the limitations of pancreatic islet transplantation and details the benefits offered by organoids as a cornerstone toward the generation of a bioartificial pancreas.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Organoides/metabolismo , Pâncreas Artificial , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/terapia , Humanos , Secreção de Insulina/fisiologia , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas/métodos , Organoides/citologia , Pâncreas Artificial/provisão & distribução , Técnicas de Cultura de Tecidos/métodos
2.
Nature ; 585(7826): 574-578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939089

RESUMO

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.


Assuntos
Homeostase , Intestinos/embriologia , Morfogênese , Organoides/embriologia , Tecidos Suporte , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Cryptosporidium parvum/patogenicidade , Células-Tronco Embrionárias Humanas/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Intestinos/citologia , Intestinos/parasitologia , Intestinos/patologia , Camundongos , Modelos Biológicos , Organoides/citologia , Organoides/parasitologia , Organoides/patologia , Regeneração , Medicina Regenerativa , Células-Tronco , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual
3.
Methods Mol Biol ; 2203: 77-88, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833205

RESUMO

Porcine deltacoronavirus (PDCoV) has emerged as a novel, contagious swine enteric coronavirus that causes watery diarrhea and/or vomiting and intestinal villous atrophy in nursing piglets. PDCoV-related diarrhea first occurred in the USA in 2014 and was subsequently reported in South Korea, China, Thailand, Vietnam, and Lao People's Democratic Republic, leading to massive economic losses and posing a threat to the swine industry worldwide. Currently, no treatments or vaccines for PDCoV are available. The critical step in the development of potential vaccines against PDCoV infection is the isolation and propagation of PDCoV in cell culture. This chapter provides a detailed protocol for isolation and propagation of PDCoV in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures supplemented with pancreatin and trypsin, respectively. Filtered clinical samples (swine intestinal contents or feces) applied to ST or LLC-PK cells produce cytopathic effects characterized by rounding, clumping, and detachment of cells. PDCoV replication in cells can be quantifiably monitored by qRT-PCR, immunofluorescence assays, and immune-electron microscopy. Infectious viral titers can be evaluated by using plaque assays or 50% tissue culture infectious dose (TCID50) assays. The ST or LLC-PK cells efficiently supported serial passage and propagation of PDCoV. After serial passage of PDCoV in either ST or LLC-PK cells, the virus can be purified further in ST cells by plaque assays.


Assuntos
Coronavirus/isolamento & purificação , Doenças dos Suínos/virologia , Técnicas de Cultura de Tecidos/métodos , Animais , Células Cultivadas , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Fezes/virologia , Inoculações Seriadas , Suínos
4.
Nat Protoc ; 15(8): 2413-2442, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32690957

RESUMO

Cancer invasion and metastasis are challenging to study in vivo since they occur deep inside the body over extended time periods. Organotypic 3D culture of fresh tumor tissue enables convenient real-time imaging, genetic and microenvironmental manipulation and molecular analysis. Here, we provide detailed protocols to isolate and culture heterogenous organoids from murine and human primary and metastatic site tumors. The time required to isolate organoids can vary based on the tissue and organ type but typically takes <7 h. We describe a suite of assays that model specific aspects of metastasis, including proliferation, survival, invasion, dissemination and colony formation. We also specify comprehensive protocols for downstream applications of organotypic cultures that will allow users to (i) test the role of specific genes in regulating various cellular processes, (ii) distinguish the contributions of several microenvironmental factors and (iii) test the effects of novel therapeutics.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Técnicas de Cultura de Tecidos/métodos , Animais , Humanos , Camundongos , Metástase Neoplásica
5.
Med Sci (Paris) ; 36(6-7): 626-632, 2020.
Artigo em Francês | MEDLINE | ID: mdl-32614314

RESUMO

Generation of retinal organoids from pluripotent stem cells represents an important advance in the study of retinal development and offer new perspectives for the study of retinal diseases missing suitable animal models. Understanding the key stages of retinal development in vertebrates enabled to design protocols to generate self-organized three-dimensional structures derived from pluripotent stem cells and containing all retinal cell types. In addition to their application in basic research, such as the characterization of molecular and cellular mechanisms in retinal pathophysiology, these miniature organs also open up encouraging prospects in the field of cell therapy or the screening of therapeutic molecules, although some obstacles remain to be overcome.


Assuntos
Organoides/citologia , Retina/citologia , Doenças Retinianas/etiologia , Doenças Retinianas/patologia , Doenças Retinianas/terapia , Animais , Células Cultivadas , Humanos , Modelos Biológicos , Organoides/fisiologia , Retina/patologia , Retina/fisiologia , Terapias em Estudo/métodos , Terapias em Estudo/tendências , Técnicas de Cultura de Tecidos/métodos , Técnicas de Cultura de Tecidos/tendências
6.
Nat Commun ; 11(1): 3265, 2020 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-32601271

RESUMO

The culture of live pancreatic tissue slices is a powerful tool for the interrogation of physiology and pathology in an in vitro setting that retains near-intact cytoarchitecture. However, current culture conditions for human pancreatic slices (HPSs) have only been tested for short-term applications, which are not permissive for the long-term, longitudinal study of pancreatic endocrine regeneration. Using a culture system designed to mimic the physiological oxygenation of the pancreas, we demonstrate high viability and preserved endocrine and exocrine function in HPS for at least 10 days after sectioning. This extended lifespan allowed us to dynamically lineage trace and quantify the formation of insulin-producing cells in HPS from both non-diabetic and type 2 diabetic donors. This technology is expected to be of great impact for the conduct of real-time regeneration/developmental studies in the human pancreas.


Assuntos
Ilhotas Pancreáticas/citologia , Pâncreas/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Humanos , Estudos Longitudinais , Camundongos , Modelos Biológicos , Regeneração , Células-Tronco/citologia
7.
J Fr Ophtalmol ; 43(6): 477-483, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32444133

RESUMO

BACKGROUND AND PURPOSE: The purpose of this study is to compare two alternative methods of collecting and transporting media for the diagnosis of corneal ulcers, as not all clinical settings have conventional culture materials and transport media available. METHODS: In this open-label, prospective, comparative, and randomized study, patients with clinical suspicion of infectious keratitis with high risk of loss of vision had corneal specimens collected using two methods and transport media: Eswab scraping with Amies transport medium and 23-gauge needle scraping in BACTEC Peds broth. The order of each collection method was randomized. The samples were processed by standard methods, comparing the positivity frequencies for both by parametric and nonparametric tests, according to normality criteria. RESULTS: Corneal infiltrates from 40 eyes of 40 patients were analyzed. Culture positivity rate was 50% for Eswab and 35% for 23-gauge needle (P=0.258). The overall growth rate of the two methods combined was not higher than with the swab alone. The results obtained with a swab were not influenced by the collection sequence (P=0.112); however, the positivity rate was significantly higher when the sample taken with the needle was performed first (P=0.046). CONCLUSIONS: The single sample Eswab method of collection and transportation for the diagnosis of high risk corneal ulcers is a valid alternative and can be used in cases in which, for various reasons, there is no access to the full set of traditional culture materials.


Assuntos
Córnea , Úlcera da Córnea/patologia , Infecções Oculares Bacterianas/patologia , Ceratite/patologia , Manejo de Espécimes/métodos , Coleta de Tecidos e Órgãos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Córnea/patologia , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/microbiologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Infecções Oculares Bacterianas/diagnóstico , Infecções Oculares Bacterianas/microbiologia , Feminino , Humanos , Ceratite/diagnóstico , Ceratite/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Cultura de Tecidos/métodos , Transportes , Adulto Jovem
8.
Development ; 147(7)2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32253255

RESUMO

Organoids are three-dimensional multicellular structures grown in vitro from stem cells and which recapitulate some organ function. They are derivatives of living tissue that can be stored in biobanks for a multitude of research purposes. Biobank research on organoids derived from patients is highly promising for precision medicine, which aims to target treatment to individual patients. The dominant approach for protecting the interests of biobank participants emphasizes broad consent in combination with privacy protection and ex ante (predictive) ethics review. In this paradigm, participants are positioned as passive donors; however, organoid biobanking for precision medicine purposes raises challenges that we believe cannot be adequately addressed without more ongoing involvement of patient-participants. In this Spotlight, we argue why a shift from passive donation towards more active involvement is particularly crucial for biobank research on organoids aimed at precision medicine, and suggest some approaches appropriate to this context.


Assuntos
Organoides/citologia , Medicina de Precisão/ética , Medicina de Precisão/métodos , Bancos de Espécimes Biológicos/ética , Participação da Comunidade , Doação Dirigida de Tecido/ética , Doação Dirigida de Tecido/tendências , Necessidades e Demandas de Serviços de Saúde , Humanos , Técnicas de Cultura de Tecidos/ética , Técnicas de Cultura de Tecidos/métodos
9.
Nat Biomed Eng ; 4(5): 544-559, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32341538

RESUMO

Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman's correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.


Assuntos
Composição de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Robótica , Técnicas de Cultura de Tecidos/métodos , Administração Oral , Animais , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Humanos , Absorção Intestinal , Jejuno/fisiologia , Ocitocina/administração & dosagem , Ocitocina/farmacocinética , Ocitocina/farmacologia , Permeabilidade , Reprodutibilidade dos Testes , Suínos , Interface Usuário-Computador
10.
Spine (Phila Pa 1976) ; 45(9): E525-E532, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32282655

RESUMO

MINI: We compared the sensitivity and specificity of peri-implant tissue culture to the vortexing-sonication technique for the diagnosis of spinal implant infection (SII). Lower thresholds of sonicate fluid culture positivity showed increased sensitivity with maintained specificity. We recommend a threshold of 20 CFU/10 mL for sonicate culture positivity for the diagnosis of SII. STUDY DESIGN: This is a retrospective study comparing the diagnosis of spinal implant infection (SII) by peri-implant tissue culture to vortexing-sonication of retrieved spinal implants. OBJECTIVE: We hypothesized that vortexing-sonication would be more sensitive than peri-implant tissue culture. SUMMARY OF BACKGROUND DATA: We previously showed implant vortexing-sonication followed by culture to be more sensitive than standard peri-implant tissue culture for diagnosing of SII. In this follow-up study, we analyzed the largest sample size available in the literature to compare these two culture methods and evaluated thresholds for positivity for sonicate fluid for SII diagnosis. METHODS: We compared peri-implant tissue culture to the vortexing-sonication technique which samples bacterial biofilm on the surface of retrieved spinal implants. We evaluated different thresholds for sonicate fluid positivity and assessed the sensitivity and specificity of the two culture methods for the diagnosis of SII. RESULTS: A total of 152 patients were studied. With more than 100 colony forming units (CFU)/10 mL as a threshold for sonicate fluid culture positivity, there were 46 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 65.2% and 79.6%; the specificities were 88.7% and 93.4%, respectively. With more than 50 CFU/10 mL as a threshold, there were 50 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 68.0% and 76.0%; the specificities were 92.2% for both methods. Finally, with more than or equal to 20 CFU/10 mL as a threshold, there were 52 patients with SII. The sensitivities of peri-implant tissue and sonicate fluid culture were 69.2% and 82.7%; the specificities were 94.0% and 92.0%, respectively. CONCLUSION: Implant sonication followed by culture is a sensitive and specific method for the diagnosis of SII. Lower thresholds for defining sonicate fluid culture positivity allow for increased sensitivity with a minimal decrease in specificity, enhancing the clinical utility of implant sonication. LEVEL OF EVIDENCE: 4.


Assuntos
Biofilmes/crescimento & desenvolvimento , Próteses e Implantes/microbiologia , Próteses e Implantes/normas , Infecções Relacionadas à Prótese/diagnóstico , Sonicação/normas , Técnicas de Cultura de Tecidos/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Enterobacter cloacae/isolamento & purificação , Enterobacter cloacae/fisiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mycobacterium chelonae/isolamento & purificação , Mycobacterium chelonae/fisiologia , Estudos Retrospectivos , Sonicação/métodos , Técnicas de Cultura de Tecidos/métodos , Adulto Jovem
11.
Med Sci (Paris) ; 36(3): 261-263, 2020 Mar.
Artigo em Francês | MEDLINE | ID: mdl-32228845

RESUMO

Organoids offer an elegant approach to model human diseases and test new drugs. Nonalcoholic fatty liver disease (NAFLD) whose incidence has dramatically increased in recent years with the rise of obesity, is defined by triglyceride accumulation in hepatocytes, inflammation, liver injury, and progression to fibrosis. There is currently no approved therapy but many pathways are being explored. Two American teams have created mini-steatotic livers using different approaches, both using induced pluripotent stem cells (iPS), thus offering new tools to test developing drugs.


Assuntos
Bioengenharia/tendências , Ciência de Laboratório Médico/tendências , Hepatopatia Gordurosa não Alcoólica/patologia , Técnicas de Cultura de Tecidos/tendências , Animais , Bioengenharia/métodos , Células Cultivadas , Progressão da Doença , Hepatócitos/citologia , Hepatócitos/patologia , Hepatócitos/fisiologia , Humanos , Fígado/patologia , Fígado/fisiologia , Cirrose Hepática/patologia , Ciência de Laboratório Médico/métodos , Modelos Biológicos , Técnicas de Cultura de Tecidos/métodos , Tecidos Suporte
12.
PLoS One ; 15(4): e0231176, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298291

RESUMO

Cultured meat, in particular beef, is an emerging food technology potentially challenged by issues of consumer acceptance. To understand drivers of consumer acceptance as well as sensory perception of cultured meat, we investigated the effect of information content on participants' acceptance of cultured meat in a tasting context. Hundred ninety-three citizens from the Netherlands participated, divided across three age and sex-matched groups which each received information on either societal benefits, personal benefits or information on the quality and taste of cultured meat. They filled out a questionnaire and tasted two pieces of hamburger, labeled 'conventional' or 'cultured', although both pieces were in fact conventional. Sensory analysis of both hamburgers was performed. We observed that provision of information and the tasting experience increased acceptance of cultured meat and that information on personal benefits of cultured meat increased acceptance more than information on quality and taste but not than societal benefits of cultured meat. Previous awareness of cultured meat was the best predictor of its acceptance. In contrast to previous studies, sex and social economic status were not associated with different acceptance rates. Surprisingly, 58% of the respondents were willing to pay a premium for cultured meat of, on average, 37% above the price of regular meat. All participants tasted the 'cultured' hamburger and evaluated its taste to be better than the conventional one in spite of the absence of an objective difference. This is the first acceptance study of cultured meat where participants were offered to eat and evaluate meat that was labeled 'cultured'. We conclude that having positive information importantly improves acceptance and willingness to taste and that the specific content of the information is of subordinate importance. Awareness of cultured meat is the best predictor of acceptance.


Assuntos
Comportamento do Consumidor , Rotulagem de Alimentos , Preferências Alimentares/psicologia , Tecnologia de Alimentos/métodos , Carne Vermelha , Adulto , Idoso , Animais , Bovinos , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Fatores Sexuais , Classe Social , Inquéritos e Questionários/estatística & dados numéricos , Paladar , Técnicas de Cultura de Tecidos/métodos
13.
Int J Mol Sci ; 21(3)2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32033195

RESUMO

Brachypodium distachyon has become an excellent model for plant breeding and bioenergy grasses that permits many fundamental questions in grass biology to be addressed. One of the constraints to performing research in many grasses has been the difficulty with which they can be genetically transformed and the generally low frequency of such transformations. In this review, we discuss the contribution that transformation techniques have made in Brachypodium biology as well as how Brachypodium could be used to determine the factors that might contribute to transformation efficiency. In particular, we highlight the latest research on the mechanisms that govern the gradual loss of embryogenic potential in a tissue culture and propose using B. distachyon as a model for other recalcitrant monocots.


Assuntos
Brachypodium/genética , Técnicas de Cultura de Tecidos/métodos , Proteínas de Plantas/genética , Técnicas de Embriogênese Somática de Plantas/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética/genética
14.
Theriogenology ; 144: 33-40, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31895996

RESUMO

To establish a protocol of optimized three-dimensional (3D) culture of ovarian follicles, various biomaterials have been investigated with regard to their properties and functions on in vitro follicle growth. The present study aims to compare the new biomaterial, extracellular matrix-derived soft hydrogel (ES-hydrogel) and alginate, and evaluate the effects of biomaterials on further in vitro 3D culture growth of ovarian follicle and oocyte maturation. The isolated follicles from mouse ovaries were randomly divided into two-dimensional (2D) culture, alginate and ES-hydrogel, and just seeded on culture wells (2D culture) or encapsulated with alginate or ES-hydrogel (3D culture). Culture media from each group were collected on days 4, 8 and 10 or 11 for 17ß-oestradiol (E2) and progesterone (P4) measurement. On day 10 of in vitro culture, follicular survival and pseudo-antrum formation rate were examined, and oocyte maturation was induced by adding human chorionic gonadotropin and epidermal growth factor. After 17 h, ovulated mature oocytes collected and analyzed for oocyte diameter, normal spindle and chromosome alignment configuration, reactive oxygen species (ROS) level, and mitochondrial membrane potential level. To compare mechanical properties of two biomaterials, storage modulus was measured with the advanced rheometric expansion system. Our results showed that follicles cultured in ES-hydrogel, were significantly superior to those cultured 2D or alginate in the pseudo-antrum formation rate, cumulus-oocyte complexes (COCs) rate, MII oocyte rate, normal spindle rate, and E2 production. The ES-hydrogel and alginate groups were not significantly different in follicle survival rate, oocyte diameter, P4 production, ROS, and mitochondrial membrane potential levels. The storage modulus of ES-hydrogel was lower than that of alginate, suggesting that the improved follicular physiology and oocyte maturation in the ES-hydrogel group was due to better hormone exchange through a less stiff encapsulating material. This study shows that 3D culture system using ES-hydrogel effectively improve the outcome of in vitro ovarian follicle culture, supporting follicle morphology and growth and enhancing oocyte maturation. It means one of the most important factors for 3D culture of ovarian follicle was the selection of appropriate and effective biomaterial that can preserve the structure and morphology of ovarian follicle and facilitate nutrition and hormone exchange.


Assuntos
Materiais Biocompatíveis , Folículo Ovariano/citologia , Folículo Ovariano/fisiologia , Técnicas de Cultura de Tecidos/instrumentação , Animais , Estradiol/metabolismo , Feminino , Hidrogéis , Concentração de Íons de Hidrogênio , Camundongos , Progesterona/metabolismo , Técnicas de Cultura de Tecidos/métodos
15.
Oncol Rep ; 43(2): 405-414, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31894341

RESUMO

Living tumors are of great scientific value for clinical medicine and basic research, especially for drug testing. An increasing number of drug tests fail due to the use of imperfect models. The aim of the present study was to develop a novel method combining vitrification­based cryopreservation of tumor biopsies and precision­cut slice cultivation for the assessment of anticancer drug responses. Biological characteristics of rectal cancer liver metastasis biopsies could be retained by vitrification­based cryopreservation. The patient­derived xenograft models were successfully established using both fresh and warmed biopsy tissues. Precision­cut slicing provided a similar three­dimensional architecture and heterogeneity to the original tumor. The positive drug responses in the xenograft model were consistent with those in precision­cut slice cultures in vitro. The present study demonstrated that live tumor biopsies could be preserved using vitrification­based cryopreservation. The warmed tissues developed xenograft tumors, which were also useful for either in vivo or in vitro anticancer drug testing. Precision­cut slices derived from the warmed tissues provided an efficient tool to assess anticancer drug response in vitro.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/patologia , Técnicas de Cultura de Tecidos/métodos , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biópsia , Criopreservação , Feminino , Humanos , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Pessoa de Meia-Idade , Resultado do Tratamento , Vitrificação , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Acta Histochem ; 122(2): 151484, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31902536

RESUMO

The aim of this study was to evaluate whether the addition of synthetic polymers to the vitrification solution affected follicular morphology and development and the expression of Ki-67, Aquaporin 3 (AQP3) and cleaved Caspase-3 proteins in ovarian tissue of the caprine species. Caprine ovaries were fragmented and two fragments were immediately fixed (Fresh Control) for morphological evaluation, while other two were in vitro cultured for 7 days (Cultured Control) and fixed as well. The remaining fragments were distributed in two different vitrification groups: Vitrified and Vitrified/Cultured. Each group was composed of 4 different treatments: 1) Sucrose (SUC); 2) SuperCool X-1000 0.2 % (X-1000); 3) SuperCool Z-1000 0.4 % (Z-1000) or 4) with polyvinylpyrrolidone K-12 0.2 % (PVP). Also, Fresh Control, Cultured Control, SUC and X-1000 were destined to immunohistochemical detection of Ki-67, AQP3 and cleaved Caspase-3 proteins. Morphologically, the treatment with X-1000 showed no significant difference with the Fresh Control group and was superior to the other treatments. After the cleaved caspase-3 analysis, X-1000 showed the lowest percentages of strong immunostaining while Cultured Control showed the highest. Also, a positive correlation was found between the percentages of degenerated follicles and the percentages of strong staining intensity follicles. Regarding the AQP3 analysis, the highest percentages of strong AQP3 staining intensity were found in X-1000. In conclusion, we have demonstrated that the addition of the synthetic polymer SuperCool X-1000 to the vitrification solution improved the current vitrification protocol of caprine ovarian tissue.


Assuntos
Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Polímeros/farmacologia , Animais , Criopreservação/métodos , Feminino , Cabras , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Técnicas de Cultura de Tecidos/métodos , Vitrificação
17.
Nat Prod Res ; 34(3): 434-440, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30585087

RESUMO

Carbazole alkaloids are major constituents in Clausena spp. and exhibit a wide range of biological activities. The roots of Clausena harmandiana are a rich source of active carbazole alkaloids. However, its roots take several years to grow to be able to harvest. To obtain an alternative source of carbazole alkaloids, in vitro callus cultures of C. harmandiana were induced, and the formation of two active carbazole alkaloids was investigated. The effects of precursor, concentrations of sucrose, elicitors and light were studied to improve carbazole alkaloids formation. In this study, light had a strong effect on the formation of both carbazole alkaloids. The highest yields of clausine K and 7-methoxymukonal were 4.74 ± 0.26 and 0.92 ± 0.04 mg/g DW, respectively, which have more than 10-fold found in intact roots. According to the results of this study, C. harmandiana callus cultures can be used as an alternative source of carbazole alkaloids for additional biological studies.


Assuntos
Clausena/química , Técnicas de Cultura de Tecidos/métodos , Alcaloides/metabolismo , Carbazóis/metabolismo , Clausena/citologia , Raízes de Plantas/química
18.
Proc Natl Acad Sci U S A ; 116(51): 25932-25940, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31796592

RESUMO

Formation of tissue models in 3 dimensions is more effective in recapitulating structure and function compared to their 2-dimensional (2D) counterparts. Formation of 3D engineered tissue to control shape and size can have important implications in biomedical research and in engineering applications such as biological soft robotics. While neural spheroids routinely are created during differentiation processes, further geometric control of in vitro neural models has not been demonstrated. Here, we present an approach to form functional in vitro neural tissue mimic (NTM) of different shapes using stem cells, a fibrin matrix, and 3D printed molds. We used murine-derived embryonic stem cells for optimizing cell-seeding protocols, characterization of the resulting internal structure of the construct, and remodeling of the extracellular matrix, as well as validation of electrophysiological activity. Then, we used these findings to biofabricate these constructs using neurons derived from human embryonic stem cells. This method can provide a large degree of design flexibility for development of in vitro functional neural tissue models of varying forms for therapeutic biomedical research, drug discovery, and disease modeling, and engineering applications.


Assuntos
Tecido Nervoso/citologia , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual/métodos , Tecidos Suporte , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Esferoides Celulares/citologia
19.
Sci Rep ; 9(1): 20239, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882926

RESUMO

Elucidation of the molecular mechanism related to the dedifferentiation and redifferentiation during tissue culture will be useful for optimizing regeneration system of tea plant. In this study, an integrated sRNAome and transcriptome analyses were carried out during phase changes of the stem explant culture. Among 198 miRNAs and 8001 predicted target genes, 178 differentially expressed miRNAs and 4264 potential targets were screened out from explants, primary calli, as well as regenerated roots and shoots. According to KEGG analysis of the potential targets, pathway of "aminoacyl-tRNA biosynthesis", "proteasome" and "glutathione metabolism" was of great significance during the dedifferentiation, and pathway of "porphyrin and chlorophyll metabolism", "mRNA surveillance pathway", "nucleotide excision repair" was indispensable for redifferentiation of the calli. Expression pattern of 12 miRNAs, including csn-micR390e, csn-miR156b-5p, csn-miR157d-5p, csn-miR156, csn-miR166a-3p, csn-miR166e, csn-miR167d, csn-miR393c-3p, csn-miR394, csn-miR396a-3p, csn-miR396 and csn-miR396e-3p, was validated by qRT-PCR among 57 differentially expressed phase-specific miRNAs. Validation also confirmed that regulatory module of csn-miR167d/ERF3, csn-miR156/SPB1, csn-miR166a-3p/ATHB15, csn-miR396/AIP15A, csn-miR157d-5p/GST and csn-miR393c-3p/ATG18b might play important roles in regulating the phase changes during tissue culture of stem explants.


Assuntos
Camellia sinensis/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , RNA de Plantas/genética , Chá , Desdiferenciação Celular/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Cultura de Tecidos/métodos
20.
Int J Mol Sci ; 20(21)2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683725

RESUMO

Agrobacterium-mediated genetic transformation is well established in the model grass Brachypodium distachyon. However, most protocols employ immature embryos because of their better regenerative capacity. A major problem associated with the immature embryo system is that they are available only during a limited time window of growing plants. In this study, we have developed an optimized Agrobacterium-mediated genetic transformation protocol that utilizes mature embryos. We have adopted seed shearing and photoautotrophic rooting (PR) in callus induction and root regeneration, respectively, with evident significant improvement in these aspects. We have also revealed that the newly developed chemical inducer Fipexide (FPX) had the ability to induce callus, shoots, and roots. By comparison, we have demonstrated that FPX shows higher efficiency in shoot generation than other frequently used chemicals in our mature embryo-based system. In addition, we demonstrated that the age of embryogenetic callus severely affects the transformation efficiency (TE), with the seven-week-old embryogenetic callus having the highest TE reaching 52.6%, which is comparable with that in immature embryo transformation. The new methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.


Assuntos
Brachypodium/genética , Sementes/genética , Técnicas de Cultura de Tecidos/métodos , Agrobacterium/fisiologia , Brachypodium/efeitos dos fármacos , Brachypodium/embriologia , Piperazinas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/embriologia , Raízes de Plantas/genética , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/embriologia , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Regeneração/efeitos dos fármacos , Regeneração/genética , Sementes/efeitos dos fármacos , Sementes/embriologia , Transformação Genética
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