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1.
J Vis Exp ; (168)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33645552

RESUMEN

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.


Asunto(s)
Movimiento Celular , Separación Celular/métodos , Embrión de Mamíferos/citología , Mesodermo/citología , Paladar (Hueso)/citología , Imagen de Lapso de Tiempo , Animales , Rastreo Celular , Células Cultivadas , Criopreservación , Modelos Animales de Enfermedad , Disección , Femenino , Ratones , Cicatrización de Heridas
2.
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
3.
J Vis Exp ; (168)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33645553

RESUMEN

Many human neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.


Asunto(s)
Células Sanguíneas/citología , Neuronas/citología , Anticuerpos/química , Técnicas de Cultivo de Célula , Desdiferenciación Celular , Diferenciación Celular/fisiología , Separación Celular , Células Cultivadas , Reactivos de Enlaces Cruzados/química , Glicoproteínas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Humanos , Inmunofenotipificación , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuronas/ultraestructura
4.
Methods Mol Biol ; 2269: 221-231, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687682

RESUMEN

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) offer great therapeutic potential for osteoarthritis (OA) treatment. Recent investigations have revealed the contribution of extracellular vesicles (EVs) to AD-MSC actions. Here, we describe a method to study the in vitro effects of EVs from AD-MSCs in OA chondrocytes. This chapter includes the isolation and analysis of human AD-MSCs and their EVs as well as the isolation and treatment of OA chondrocytes.


Asunto(s)
Tejido Adiposo/metabolismo , Condrocitos/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteoartritis/metabolismo , Tejido Adiposo/patología , Separación Celular , Condrocitos/patología , Técnicas de Cocultivo , Humanos , Células Madre Mesenquimatosas/patología , Osteoartritis/patología
5.
Medicine (Baltimore) ; 100(9): e24777, 2021 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-33655941

RESUMEN

INTRODUCTION: Knee osteoarthritis is a common condition that affects daily functioning and decreases the quality of life. There are many ways of treatment depending on the stage of the disease. Advanced cases are qualified for arthroplasty, which is an extensive and demanding surgical procedure. Less advanced stages are treated in various ways: from rehabilitation, through oral and intra-articular pharmacotherapy, to surgical treatment (arthroscopy, osteotomy). Because surgical treatment is risky, scientists focus on less invasive therapeutic methods. The most valuable management is based on regeneration. Mesenchymal stromal cells (MSC) derived from the adipose tissue have a great regenerative and anti-inflammatory potential, therefore an attempt is being made to take advantage of them in knee osteoarthritis treatment.The study aims to compare the clinical effects of treatment of knee osteoarthritis using adipose tissue MSC obtained by an enzymatic method with the outcomes of the therapy with the mechanically fragmented adipose tissue. METHODS: One hundred adults with primary knee osteoarthritis will undergo lipoaspiration under sterile conditions. The collected lipoaspirates will be further processed, depending on the randomly assigned group-enzymatically with the use of collagenase or mechanically using the Lipogems system. The preparations will be administered to the patients' knee joints in the operating room under ultrasound control.The results of treatment will be assessed using Knee Injury and Osteoarthritis Outcome Score, measuring the flexibility of the knee joint, evaluating joint gap in X-ray and the quality of cartilage in magnetic resonance T2-mapping during 1 year after treatment. DISCUSSION/CONCLUSION: Identification and functional analysis of the regenerative capacity of adipose-derived MSC depending on three variables (body weight, sex, and age) will help to develop a targeted therapy for different groups of patients and will determine the effectiveness of both methods of treatment. An attempt will be made to identify groups of patients with the greatest regenerative potential of the adipose tissue, and thus indicate those with the most probable improvement of the joint condition. TRIAL REGISTRATION: This study protocol has been approved by the Ethics Committee of Medical University of Warsaw and registered on www.clinicaltrials.gov: NCT04675359 (06 Jan 2021).


Asunto(s)
Tejido Adiposo/citología , Separación Celular/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Osteoartritis de la Rodilla/cirugía , Adulto , Técnicas de Cultivo de Célula , Colagenasas , Digestión/fisiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Medición de Resultados Informados por el Paciente , Ensayos Clínicos Controlados Aleatorios como Asunto , Estrés Mecánico , Resultado del Tratamiento
6.
J Vis Exp ; (168)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33645564

RESUMEN

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iVα14-Jα18 transgenic mice (iVα14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iVα14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.


Asunto(s)
Separación Celular/métodos , Células T Asesinas Naturales/inmunología , Animales , Antígenos CD4/metabolismo , Proliferación Celular , Activación de Linfocitos/inmunología , Ratones , Células T Asesinas Naturales/citología , Bazo/citología
7.
Sci Rep ; 11(1): 4904, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649400

RESUMEN

SARS-CoV-2 virus infection is responsible for coronavirus disease (COVID-19), which is characterised by a hyperinflammatory response that plays a major role in determining the respiratory and immune-mediated complications of this condition. While isolating peripheral blood mononuclear cells (PBMCs) from whole blood of COVID-19 patients by density gradient centrifugation, we noticed some changes in the floating properties and in the sedimentation of the cells on density medium. Investigating this further, we found that in early phase COVID-19 patients, characterised by reduced circulating lymphocytes and monocytes, the PBMC fraction contained surprisingly high levels of neutrophils. Furthermore, the neutrophil population exhibited alterations in the cell size and in the internal complexity, consistent with the presence of low density neutrophils (LDNs) and immature forms, which may explain the shift seen in the floating abilities and that may be predictive of the severity of the disease. The percentage of this subset of neutrophils found in the PBMC band was rather spread (35.4 ± 27.2%, with a median 28.8% and IQR 11.6-56.1, Welch's t-test early phase COVID-19 versus blood donor healthy controls P < 0.0001). Results confirm the presence of an increased number of LDNs in patients with early stage COVID-19, which correlates with disease severity and may be recovered by centrifugation on a density gradient together with PBMCs.


Asunto(s)
/sangre , Separación Celular , Leucocitos Mononucleares/metabolismo , /metabolismo , Adulto , Centrifugación por Gradiente de Densidad , Femenino , Humanos , Leucocitos Mononucleares/patología , Masculino , Persona de Mediana Edad
8.
Int J Nanomedicine ; 16: 1231-1244, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33633448

RESUMEN

Background and Aim: Acute myeloid leukemia (AML), initiated and maintained by leukemia stem cells (LSCs), is often relapsed or refractory to therapy. The present study aimed at assessing the effects of nanozyme-like Fe3O4 nanoparticles (IONPs) combined with cytosine arabinoside (Ara-C) on LSCs in vitro and in vivo. Methods: The CD34+CD38-LSCs, isolated from human AML cell line KG1a by a magnetic activated cell sorting method, were treated with Ara-C, IONPs, and Ara-C+ IONPs respectively in vitro. The cellular proliferation, apoptosis, reactive oxygen species (ROS), and the related molecular expression levels in LSCs were analyzed using flow cytometry, RT-qPCR, and Western blot. The nonobese diabetic/severe combined immune deficiency mice were transplanted with LSCs or non-LSCs via tail vein, and then the mice were treated with Ara-C, IONPs and IONPs plus Ara-C, respectively. The therapeutic effects on the AML bearing mice were further evaluated. Results: LSCs indicated stronger cellular proliferation, more clone formation, and more robust resistance to Ara-C than non-LSCs. Compared with LSCs treated with Ara-C alone, LSCs treated with IONPs plus Ara-C showed a significant increase in apoptosis and ROS levels that might be regulated by nanozyme-like IONPs via improving the expression of pro-oxidation molecule gp91-phox but decreasing the expression of antioxidation molecule superoxide dismutase 1. The in vivo results suggested that, compared with the AML bearing mice treated with Ara-C alone, the mice treated with IONPs plus Ara-C markedly reduced the abnormal leukocyte numbers in peripheral blood and bone marrow and significantly extended the survival of AML bearing mice. Conclusion: IONPs combined with Ara-C showed the effectiveness on reducing AML burden in the mice engrafted with LSCs and extending mouse survival by increasing LSC's ROS level to induce LSC apoptosis. Our findings suggest that targeting LSCs could control the AML relapse by using IONPs plus Ara-C.


Asunto(s)
Citarabina/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/patología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Separación Celular , Citarabina/farmacología , Hemoglobinas/metabolismo , Humanos , Leucemia Mieloide Aguda/sangre , Recuento de Leucocitos , Ratones Endogámicos NOD , Ratones SCID , NADPH Oxidasa 2/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
9.
J Vis Exp ; (167)2021 01 22.
Artículo en Inglés | MEDLINE | ID: mdl-33554965

RESUMEN

Extensive studies have characterized the development and differentiation of murine B cells in secondary lymphoid organs. Antibodies secreted by B cells have been isolated and developed into well-established therapeutics. Validation of murine B cell development, in the context of autoimmune prone mice, or in mice with modified immune systems, is a crucial component of developing or testing therapeutic agents in mice and is an appropriate use of flow cytometry. Well established B cell flow cytometric parameters can be used to evaluate B cell development in the murine peritoneum, bone marrow, and spleen, but a number of best practices must be adhered to. In addition, flow cytometric analysis of B cell compartments should also complement additional readouts of B cell development. Data generated using this technique can further our understanding of wild type, autoimmune prone mouse models as well as humanized mice that can be used to generate antibody or antibody-like molecules as therapeutics.


Asunto(s)
Linfocitos B/citología , Citometría de Flujo/métodos , Animales , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Recuento de Células , Diferenciación Celular , Separación Celular , Análisis de Datos , Femenino , Cadenas lambda de Inmunoglobulina/metabolismo , Inmunoglobulinas/metabolismo , Activación de Linfocitos , Subgrupos Linfocitarios/citología , Ratones Endogámicos C57BL , Peritoneo/citología , Bazo/citología , Coloración y Etiquetado
10.
Nat Commun ; 12(1): 1029, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33589635

RESUMEN

A primary challenge in single-cell RNA sequencing (scRNA-seq) studies comes from the massive amount of data and the excess noise level. To address this challenge, we introduce an analysis framework, named single-cell Decomposition using Hierarchical Autoencoder (scDHA), that reliably extracts representative information of each cell. The scDHA pipeline consists of two core modules. The first module is a non-negative kernel autoencoder able to remove genes or components that have insignificant contributions to the part-based representation of the data. The second module is a stacked Bayesian autoencoder that projects the data onto a low-dimensional space (compressed). To diminish the tendency to overfit of neural networks, we repeatedly perturb the compressed space to learn a more generalized representation of the data. In an extensive analysis, we demonstrate that scDHA outperforms state-of-the-art techniques in many research sub-fields of scRNA-seq analysis, including cell segregation through unsupervised learning, visualization of transcriptome landscape, cell classification, and pseudo-time inference.


Asunto(s)
Redes Neurales de la Computación , Análisis de Secuencia de ARN/estadística & datos numéricos , Análisis de la Célula Individual/estadística & datos numéricos , Aprendizaje Automático no Supervisado/estadística & datos numéricos , Animales , Teorema de Bayes , Benchmarking , Separación Celular/métodos , Cerebelo/química , Cerebelo/citología , Embrión de Mamíferos , Humanos , Hígado/química , Hígado/citología , Pulmón/química , Pulmón/citología , Ratones , Células Madre Embrionarias de Ratones/química , Células Madre Embrionarias de Ratones/citología , Páncreas/química , Páncreas/citología , Retina/química , Retina/citología , Análisis de la Célula Individual/métodos , Corteza Visual/química , Corteza Visual/citología , Cigoto/química , Cigoto/citología
11.
J Vis Exp ; (167)2021 01 23.
Artículo en Inglés | MEDLINE | ID: mdl-33554972

RESUMEN

Despite several advances in cardiac tissue engineering, one of the major challenges to overcome remains the generation of a fully functional vascular network comprising several levels of complexity to provide oxygen and nutrients within bioengineered heart tissues. Our laboratory has developed a three-dimensional in vitro model of the human heart, known as the "cardiac spheroid" or "CS". This presents biochemical, physiological, and pharmacological features typical of the human heart and is generated by co-culturing its three major cell types, such as cardiac myocytes, endothelial cells, and fibroblasts. Human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs or iCMs) are co-cultured at ratios approximating the ones found in vivo with human cardiac fibroblasts (HCFs) and human coronary artery endothelial cells (HCAECs) in hanging drop culture plates for three to four days. The confocal analysis of CSs stained with antibodies against cardiac Troponin T, CD31 and vimentin (markers for cardiac myocytes, endothelial cells and fibroblasts, respectively) shows that CSs present a complex endothelial cell network, resembling the native one found in the human heart. This is confirmed by the 3D rendering analysis of these confocal images. CSs also present extracellular matrix (ECM) proteins typical of the human heart, such as collagen type IV, laminin and fibronectin. Finally, CSs present a contractile activity measured as syncytial contractility closer to the one typical of the human heart compared to CSs that contain iCMs only. When treated with a cardiotoxic anti-cancer agent, such as doxorubicin (DOX, used to treat leukemia, lymphoma and breast cancer), the viability of DOX-treated CSs is significantly reduced at 10 µM genetic and chemical inhibition of endothelial nitric oxide synthase, a downstream target of DOX in HCFs and HCAECs, reduced its toxicity in CSs. Given these unique features, CSs are currently used as in vitro models to study heart biochemistry, pathophysiology, and pharmacology.


Asunto(s)
Bioingeniería/métodos , Corazón/fisiopatología , Esferoides Celulares/citología , Animales , Cardiotoxinas/farmacología , Recuento de Células , Separación Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Colágeno/farmacología , Doxorrubicina/farmacología , Doxorrubicina/toxicidad , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Fibroblastos/citología , Geles , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratas , Fijación del Tejido
12.
J Vis Exp ; (168)2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33616094

RESUMEN

Calcific aortic valve disease (CAVD), an active disease process ranging from mild thickening of the valve to severe calcification, is associated with high mortality, despite new therapeutic options such as transcatheter aortic valve replacement (TAVR). The complete pathways that start with valve calcification and lead to severe aortic stenosis remain only partly understood. By providing a close representation of the aortic valve cells in vivo, the assaying of T lymphocytes from stenotic valve tissue could be an efficient way to clarify their role in the development of calcification. After surgical excision, the fresh aortic valve sample is dissected in small pieces and the T lymphocytes are cultured, cloned then analyzed using fluorescence activated cell sorting (FACS). The staining procedure is simple and the stained tubes can also be fixed using 0.5% of paraformaldehyde and analyzed up to 15 days later. The results generated from the staining panel can be used to track changes in T cell concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific T cell subtypes of interest. In this study, we show the isolation of T cells, performed on fresh calcified aortic valve samples and the steps of analyzing T cell clones using flow cytometry to further understand the role of adaptive immunity in CAVD pathophysiology.


Asunto(s)
Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/citología , Válvula Aórtica/patología , Capa Leucocitaria de la Sangre/efectos de la radiación , Calcinosis/patología , Separación Celular/métodos , Células Nutrientes/citología , Citometría de Flujo/métodos , Linfocitos T/citología , Válvula Aórtica/metabolismo , Células Cultivadas , Células Nutrientes/metabolismo , Humanos , Linfocitos T/metabolismo
13.
J Vis Exp ; (167)2021 01 30.
Artículo en Inglés | MEDLINE | ID: mdl-33586700

RESUMEN

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrinα6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Plasticidad de la Célula , Células Epidérmicas/citología , Queratinocitos/citología , Esferoides Celulares/citología , Células Madre/citología , Biomarcadores/metabolismo , Proliferación Celular , Separación Celular , Rastreo Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Humanos , Masculino , Transcripción Genética
14.
J Vis Exp ; (168)2021 02 07.
Artículo en Inglés | MEDLINE | ID: mdl-33616101

RESUMEN

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-γ, IL-12 & IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host's defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers. This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases. In this paper, we polarized the naïve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Polaridad Celular , Separación Celular/métodos , Glucólisis , Macrófagos/citología , Animales , Bioensayo , Fraccionamiento Celular , Células Cultivadas , Metabolismo Energético , Eritrocitos/citología , Fémur/citología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Fenotipo
15.
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
16.
Methods Mol Biol ; 2273: 263-278, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33604860

RESUMEN

Tissue engineering is an elegant tool to create organs in vitro, that can help obviate the lack of organ donors in transplantation medicine and provide the opportunity of studying complex biological systems in vitro, thereby reducing the need for animal experiments. Artificial intestine models are at the core of Fish-AI, an EU FET-Open research project dedicated to the development of a 3D in vitro platform that is intended to enable the aquaculture feed industry to predict the nutritional and health value of alternative feed sources accurately and efficiently.At present, it is impossible to infer the health and nutrition value through the chemical characterization of any given feed. Therefore, each new feed must be tested through in vivo growth trials. The procedure is lengthy, expensive and requires the use of many animals. Furthermore, although this process allows for a precise evaluation of the final effect of each feed, it does not improve our basic knowledge of the cellular and molecular mechanisms determining such end-results. In turn, this lack of mechanistic knowledge severely limits the capacity to understand and predict the biological value of a single raw material and of their different combinations.The protocol described herein allows to develop the two main components essential to produce a functional platform for the efficient and reliable screening of feeds that the feed industry is currently developing for improving their health and nutritional value. It is here applied to the Rainbow Trout, but it can be fruitfully used to many other fish species.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Gelatina/química , Mucosa Intestinal/citología , Oncorhynchus mykiss , Andamios del Tejido/química , Acrilamidas/química , Alimentación Animal/análisis , Animales , Acuicultura/métodos , Materiales Biocompatibles/química , Separación Celular/métodos , Células Cultivadas , Norbornanos/química , Oncorhynchus mykiss/crecimiento & desarrollo
17.
Methods Mol Biol ; 2235: 27-35, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576968

RESUMEN

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.


Asunto(s)
Citometría de Flujo/métodos , Pericitos/citología , Pericitos/trasplante , Capilares/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Células Cultivadas , Células Endoteliales/citología , Humanos , Células Madre Mesenquimatosas/citología , Microvasos/citología , Pericitos/metabolismo , Fenotipo
18.
Methods Mol Biol ; 2235: 127-137, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576974

RESUMEN

Human pericytes are a perivascular cell population with mesenchymal stem cell properties, present in all vascularized tissues. Human pericytes have a distinct immunoprofile, which may be leveraged for purposes of cell purification. Adipose tissue is the most commonly used cell source for human pericyte derivation. Pericytes can be isolated by FACS (fluorescence-activated cell sorting), most commonly procured from liposuction aspirates. Pericytes have clonal multilineage differentiation potential, and their potential utility for bone regeneration has been described across multiple animal models. The following review will discuss in vivo methods for assessing the bone-forming potential of purified pericytes. Potential models include (1) mouse intramuscular implantation, (2) mouse calvarial defect implantation, and (3) rat spinal fusion models. In addition, the presented surgical protocols may be used for the in vivo analysis of other osteoprogenitor cell types.


Asunto(s)
Células de la Médula Ósea/metabolismo , Pericitos/metabolismo , Ingeniería de Tejidos/métodos , Tejido Adiposo/citología , Animales , Células de la Médula Ósea/citología , Regeneración Ósea/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular , Separación Celular/métodos , Humanos , Células Madre Mesenquimatosas/citología , Ratones , Osteogénesis/fisiología , Pericitos/citología , Ratas
19.
Methods Mol Biol ; 2235: 155-167, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33576976

RESUMEN

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte markers and are originally isolated from the embryonic dorsal aorta. From postnatal small vessels of skeletal muscle and heart, it is possible to isolate cells with similar characteristics to embryonic MABs. Adult MABs have the capacity to self-renew and to differentiate into cell types of mesodermal lineages upon proper culture conditions. To date, the origin of MABs and the relationship with other muscle stem cells are still debated. Recently, in a phase I-II clinical trial, intra-arterial HLA-matched MABs were proved to be relatively safe. Novel information on MAB pure populations is desirable, and implementation of their therapeutic potential is mandatory to approach efficacy in MAB-based treatments. This chapter provides an overview of the current techniques for isolation and characterization of rodent, canine, human, and equine adult MABs.


Asunto(s)
Diferenciación Celular/fisiología , Separación Celular/métodos , Pericitos/citología , Animales , Aorta/citología , Perros , Caballos , Humanos , Mesodermo/citología , Ratones , Desarrollo de Músculos , Músculo Esquelético/citología , Mioblastos/citología , Pericitos/fisiología , Ratas , Células Madre/citología
20.
J Vis Exp ; (167)2021 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-33522502

RESUMEN

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.


Asunto(s)
Permeabilidad de la Membrana Celular , Separación Celular/métodos , ADN/metabolismo , Espermatocitos/citología , Espermatogénesis , Animales , Bencimidazoles/metabolismo , Supervivencia Celular , Disección , Citometría de Flujo , Fluorescencia , Masculino , Meiosis , Ratones , Fase Paquiteno , Dispersión de Radiación , Espermátides/citología , Coloración y Etiquetado , Testículo/citología
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