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1.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-33806404

RESUMEN

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Medios de Cultivo Condicionados/química , Proteoma/análisis , Animales , Bovinos , Cromatografía Liquida , Células HeLa , Humanos , Proteómica/métodos , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/aislamiento & purificación , Espectrometría de Masas en Tándem , Factor de Necrosis Tumoral alfa/farmacología
2.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804138

RESUMEN

Mesenchymal stem (MS) cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells are known for their ability to differentiate into different lineages, including chondrocytes in culture. However, the existing protocol for chondrocyte differentiation is time consuming and labor intensive. To improve and simplify the differentiation strategy, we have explored the effects of interactions between growth factors (transforming growth factor ß1 (Tgfb1) and colony stimulating factor 3 (Csf3), and culture environments (2D monolayer and 3D nanofiber scaffold) on chondrogenic differentiation. For this, we have examined cell morphologies, proliferation rates, viability, and gene expression profiles, and characterized the cartilaginous matrix formed in the chondrogenic cultures under different treatment regimens. Our data show that 3D cultures support higher proliferation rate than the 2D cultures. Tgfb1 promotes cell proliferation and viability in both types of culture, whereas Csf3 shows positive effects only in 3D cultures. Interestingly, our results indicate that the combined treatments of Tgfb1 and Csf3 do not affect cell proliferation and viability. The expression of cartilaginous matrix in different treatment groups indicates the presence of chondrocytes. We found that, at the end of differentiation stage 1, pluripotent markers were downregulated, while the mesodermal marker was upregulated. However, the expression of chondrogenic markers (col2a1 and aggrecan) was upregulated only in the 3D cultures. Here, we report an efficient, scalable, and convenient protocol for chondrogenic differentiation of iPS cells, and our data suggest that a 3D culture environment, combined with tgfb1 and csf3 treatment, promotes the chondrogenic differentiation.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Condrogénesis/genética , Receptores del Factor Estimulante de Colonias/genética , Factor de Crecimiento Transformador beta1/genética , Animales , Cartílago/crecimiento & desarrollo , Diferenciación Celular/genética , Proliferación Celular/genética , Condrocitos/citología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Mesenquimatosas/citología , Ratones
3.
Anticancer Res ; 41(3): 1261-1269, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33788717

RESUMEN

BACKGROUND/AIM: Non-small cell lung cancer patients with epidermal growth factor receptor (EGFR) mutation have been shown to have a good response to erlotinib, a receptor tyrosine kinase inhibitor of EGFR. In this study, we found that the cell death pathways activated by erlotinib in 2D and 3D culture systems are different. MATERIALS AND METHODS: The cell death pathways induced by erlotinib were evaluated by flow cytometry and immunoblotting in both 2D and 3D culture systems of EGFR mutant lung cancer cells. RESULTS: Treatment with erlotinib induced caspase 8 activation and up-regulation of TNF-related apoptosis-inducing ligand (TRAIL) expression only in 3D cultures. Knockdown of TRAIL attenuated both erlotinib-induced activation of caspase-8 and apoptosis in 3D cultures. Erlotinib also increased LC3, an autophagy marker, expression and c-Jun N terminal kinase (JNK) activation. Both 3-MA as an autophagy inhibitor and SP600125 as a JNK inhibitor, significantly inhibited erlotinib-induced cell death. CONCLUSION: Erlotinib induces apoptotic cell death in 3D cultures through an autophagy-TRAIL-JNK pathway.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Clorhidrato de Erlotinib/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Apoptosis/efectos de los fármacos , Autofagia/fisiología , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Clorhidrato de Erlotinib/uso terapéutico , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Neoplasias Pulmonares/patología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología
4.
Methods Mol Biol ; 2265: 47-63, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704704

RESUMEN

In order to protrude within a dense tissue, tumor cells have to develop the ability to digest the extracellular matrix (ECM). Melanoma cells, similarly to other types of tumor cells, form invadopodia, membranous invaginations rich in filamentous actin and several other proteins including matrix metalloproteinases (MMPs). MMPs degrade ECM structural proteins such as collagens, fibronectin, or laminin. Here we describe an assay that allows the detection of gelatinase activity exhibited by tumor cells under 2D conditions and methods to present obtained data in both a quantitative and a qualitative manner.


Asunto(s)
Matriz Extracelular/enzimología , Gelatina/metabolismo , Melanoma/enzimología , Microscopía Confocal/métodos , Actinas/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Fluorescencia , Gelatinasas/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Melanoma/patología , Imagen Óptica , Podosomas/enzimología , Podosomas/metabolismo , Podosomas/patología
5.
Methods Mol Biol ; 2265: 65-71, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704705

RESUMEN

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points.


Asunto(s)
Movimiento Celular , Melanoma/patología , Cicatrización de Heridas , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Cicatrización de Heridas/efectos de los fármacos
6.
Methods Mol Biol ; 2265: 73-80, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704706

RESUMEN

Melanoma cells have high glycolytic capacity. Glucose uptake is a key rate-limiting step in glucose utilization. Here we describe a simple protocol for measuring direct glucose uptake in living melanoma cells by flow cytometry.


Asunto(s)
Citometría de Flujo/métodos , Glucosa/metabolismo , Melanoma/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/química , Transporte Biológico , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Desoxiglucosa/análogos & derivados , Desoxiglucosa/química , Fluorescencia , Colorantes Fluorescentes/química , Humanos
7.
Methods Mol Biol ; 2265: 81-89, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704707

RESUMEN

Cancer cells have deregulated metabolism that can contribute to the unique metabolic makeup of the tumor microenvironment. This can be variable between patients, and it is important to understand these differences since they potentially can affect therapy response. Here we discuss a method of processing and assaying metabolism from direct ex vivo murine and human tumor samples using seahorse extracellular flux analysis. This provides real-time profiling of oxidative versus glycolytic metabolism and can help infer the metabolic status of the tumor microenvironment.


Asunto(s)
Melanoma/metabolismo , Análisis de Flujos Metabólicos/métodos , Mitocondrias/metabolismo , Consumo de Oxígeno , Animales , Técnicas de Cultivo de Célula/métodos , Humanos , Análisis de Flujos Metabólicos/instrumentación , Ratones , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Fosforilación Oxidativa/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Microambiente Tumoral
8.
Methods Mol Biol ; 2265: 91-110, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704708

RESUMEN

Glutamine is a major substrate for biosynthesis. It contributes to multiple pathways required for cell proliferation, supports antioxidant defense via glutathione synthesis, and sustains the tricarboxylic acid (TCA) cycle through anaplerosis. Glutamine-fueled anaplerosis and related biosynthesis can be studied in detail in melanoma using stable isotope (13C) labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis of metabolite amounts and labeling. Detailed protocols for the assay of polar metabolites (including amino acids, TCA cycle, and glycolysis metabolites) and fatty acids by these methods following cell treatment with 13C-glutamine or 13C-glucose are presented.


Asunto(s)
Cromatografía de Gases y Espectrometría de Masas/métodos , Glucosa/metabolismo , Glutamina/metabolismo , Marcaje Isotópico/métodos , Melanoma/metabolismo , Aminoácidos/metabolismo , Isótopos de Carbono , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Ciclo del Ácido Cítrico/fisiología , Glucólisis/fisiología , Humanos
9.
Methods Mol Biol ; 2265: 111-118, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704709

RESUMEN

Within the adaptive and innate immune system, effector lymphocytes known as cytotoxic T cells (CTLs) or natural killer (NK) cells play an essential role in host defense against tumor cells and virally infected cells. Here we describe a flow cytometry-based method to quantify CTLs or NK cell cytotoxic activity against melanoma cells. In this assay, spleen cells, peripheral blood mononuclear cells (PBMCs), or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line. The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells. After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications.


Asunto(s)
Pruebas Inmunológicas de Citotoxicidad/métodos , Citometría de Flujo/métodos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Técnicas de Cultivo de Célula/métodos , Muerte Celular/inmunología , Línea Celular Tumoral , Técnicas de Cocultivo/métodos , Femenino , Colorantes Fluorescentes , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Bazo/citología , Bazo/inmunología
10.
Methods Mol Biol ; 2265: 119-128, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704710

RESUMEN

Tumor-associated macrophages (TAMs) are one of most important components of the tumor microenvironment. Although many assays have been developed to differentiate monocytes into macrophages (Mϕ) for studying the biology of TAMs in vitro, little is known whether the macrophages induced by these approaches can recapitulate the biology of TAMs present in the tumor microenvironment. We have developed a novel assay to differentiate human monocytes into TAMs using modified melanoma-conditioned medium, which is derived from the concentrated tumor cell culture medium. Characterization of these modified melanoma-conditioned medium-induced macrophages (MCMI-Mϕ) by multiple flow cytometry, Luminex, microarray, and immunohistochemistry analyses indicates that MCMI-Mϕ are phenotypically and functionally highly similar to the TAMs present in the tumor microenvironment.


Asunto(s)
Diferenciación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Macrófagos/citología , Melanoma/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Melanoma/patología , Monocitos/citología , Monocitos/efectos de los fármacos , Microambiente Tumoral
11.
Methods Mol Biol ; 2265: 129-138, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704711

RESUMEN

Lymph node invasion by tumor cells is an important process in the progression of melanoma and is a poor prognostic factor for patients with this cancer. Before they are able to spread to regional lymph nodes, though, melanoma cells must first adhere to lymphatic endothelium and transmigrate into the lymphatic vasculature. In order to study melanoma cell adhesion to lymphatic endothelial cells and the factors that regulate this process, we have developed an in vitro flow cytometry-based assay to measure melanoma cell attachment to lymphatic endothelial cells. This assay will be a useful tool for investigating the interactions that take place between melanoma cells and lymphatic endothelial cells during the adhesion process.


Asunto(s)
Adhesión Celular , Células Endoteliales/metabolismo , Endotelio Linfático/metabolismo , Citometría de Flujo/métodos , Melanoma/metabolismo , Melanoma/patología , Técnicas de Cultivo de Célula/métodos , Endotelio Linfático/citología , Humanos
12.
Methods Mol Biol ; 2265: 155-171, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704713

RESUMEN

Researchers often aim to incorporate microenvironmental variables such as the dimensionality and composition of the extracellular matrix into their cell-based assays. A technical challenge created by introduction of these variables is quantification of single-cell measurements and control of environmental reproducibility. Here, we detail a methodology to quantify viability and proliferation of melanoma cells in 3D collagen-based culture platforms by automated microscopy and 3D image analysis to yield robust, high-throughput results of single-cell responses to drug treatment.


Asunto(s)
Antineoplásicos/farmacología , Técnicas de Cultivo de Célula/métodos , Proliferación Celular/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador/métodos , Melanoma/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Análisis de la Célula Individual/métodos , Supervivencia Celular/efectos de los fármacos , Colágeno , Imidazoles/farmacología , Melanoma/patología , Oximas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Esferoides Celulares
13.
Methods Mol Biol ; 2265: 185-199, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704715

RESUMEN

Sphere assays are widely used in vitro techniques to enrich and evaluate the stem-like cell behavior of both normal and cancer cells. Utilizing three-dimensional in vitro sphere culture conditions provide a better representation of tumor growth in vivo than the more common monolayer cultures. We describe how to perform primary and secondary sphere assays, used for the enrichment and self-renewability studies of melanoma/melanocyte stem-like cells. Spheres are generated by growing melanoma cells at low density in nonadherent conditions with stem cell media. We provide protocols for preparing inexpensive and versatile polyHEMA-coated plates, setting up primary and secondary sphere assays in almost any tissue culture format and quantification methods using standard inverted microscopy. Our protocol is easily adaptable to laboratories with basic cell culture capabilities, without the need for expensive fluidic instruments.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Melanoma/patología , Células Madre Neoplásicas/citología , Esferoides Celulares/patología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Ratones , Células Madre Neoplásicas/patología , Ensayos Antitumor por Modelo de Xenoinjerto
14.
Methods Mol Biol ; 2265: 213-222, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704717

RESUMEN

Within the last decade, circulating tumor cells (CTCs) have emerged as a promising biomarker for prognostication, treatment monitoring, and detection of markers of treatment resistance, and their isolation can be used as a minimally invasive means of profiling tumors across multiple body sites. However, CTCs represent a minuscule fraction of the total circulating cells in a patient. Therefore, sensitive isolation methods are needed for the detection and downstream analysis of these cells. Herein we describe a sensitive method for melanoma CTC isolation using a multi-marker immunomagnetic bead method. This method has been purposely optimized to detect CTCs in melanoma patients.


Asunto(s)
Antígenos de Neoplasias/inmunología , Separación Inmunomagnética/métodos , Leucocitos Mononucleares/metabolismo , Melanoma/sangre , Antineoplásicos Inmunológicos/inmunología , Biomarcadores de Tumor/metabolismo , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Melanoma/metabolismo , Melanoma/patología , Células Neoplásicas Circulantes
15.
Methods Mol Biol ; 2265: 265-276, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33704721

RESUMEN

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.


Asunto(s)
Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Dispositivos Laboratorio en un Chip , Melanoma/metabolismo , Microfluídica/instrumentación , Microfluídica/métodos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Técnicas Biosensibles/instrumentación , Técnicas de Cultivo de Célula/métodos , Humanos , Inmunoensayo/instrumentación , Biopsia Líquida/métodos , Melanoma/genética , Melanoma/patología , Mutación , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Proteínas Proto-Oncogénicas B-raf/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología
16.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-33672998

RESUMEN

With increasing global health threats has come an urgent need to rapidly develop and deploy safe and effective therapies. A common practice to fast track clinical adoption of compounds for new indications is to repurpose already approved therapeutics; however, many compounds considered safe to a specific application or population may elicit undesirable side effects when the dosage, usage directives, and/or clinical context are changed. For example, progenitor and developing cells may have different susceptibilities than mature dormant cells, which may yet be different than mature active cells. Thus, in vitro test systems should reflect the cellular context of the native cell: developing, nascent, or functionally active. To that end, we have developed high-throughput, two- and three-dimensional human induced pluripotent stem cell (hiPSC)-derived neural screening platforms that reflect different neurodevelopmental stages. As a proof of concept, we implemented this in vitro human system to swiftly identify the potential neurotoxicity profiles of 29 therapeutic compounds that could be repurposed as anti-virals. Interestingly, many compounds displayed high toxicity on early-stage neural tissues but not on later stages. Compounds with the safest overall viability profiles were further evaluated for functional assessment in a high-throughput calcium flux assay. Of the 29 drugs tested, only four did not modulate or have other potentially toxic effects on the developing or mature neurospheroids across all the tested dosages. These results highlight the importance of employing human neural cultures at different stages of development to fully understand the neurotoxicity profile of potential therapeutics across normal ontogeny.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Reposicionamiento de Medicamentos/métodos , Células Madre Pluripotentes Inducidas/citología , Células-Madre Neurales/citología , Neuronas/química , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neuronas/efectos de los fármacos
17.
J Vis Exp ; (168)2021 02 12.
Artículo en Inglés | MEDLINE | ID: mdl-33645570

RESUMEN

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient's blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Leucocitos Mononucleares/citología , Miocitos Cardíacos/citología , Diferenciación Celular , Proliferación Celular , Reprogramación Celular , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Células Madre Pluripotentes Inducidas/citología , Técnicas de Placa-Clamp , Proteínas Wnt/metabolismo
18.
J Vis Exp ; (168)2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33645580

RESUMEN

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.


Asunto(s)
Células Madre Adultas/citología , Técnicas de Cultivo de Célula/métodos , Fibras Musculares Esqueléticas/citología , Células Madre/citología , Animales , Células Cultivadas , Colagenasas/metabolismo , Ratones Endogámicos C57BL , Desarrollo de Músculos , ARN Interferente Pequeño/metabolismo , Regeneración , Fijación del Tejido , Transfección
19.
Nat Protoc ; 16(4): 1802-1829, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33649566

RESUMEN

Lung and airway epithelial cells generated in vitro from human pluripotent stem cells (hPSCs) have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here, we describe a strategy for directed differentiation of hPSCs into mature lung and airway epithelial cells obtained through maturation of NKX2.1+ hPSC-derived lung progenitors in a 3D matrix of collagen I in the absence of glycogen synthase kinase 3 inhibition. This protocol is an extension of our previously published protocol on the directed differentiation of lung and airway epithelium from hPSCs that modifies the technique and offers additional applications. This protocol is conducted in defined media conditions, has a duration of 50-80 d, does not require reporter lines and results in cultures containing mature alveolar type II and I cells as well as airway basal, ciliated, club and neuroendocrine cells. We also present a flow cytometry strategy to assess maturation in the cultures. Several of these populations, including mature NGFR+ basal cells, can be prospectively isolated by cell sorting and expanded for further investigation.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Linaje de la Célula , Imagenología Tridimensional , Pulmón/citología , Células Madre Pluripotentes/citología , Animales , Biomarcadores/metabolismo , Células Cultivadas , Endodermo/citología , Células Madre Embrionarias Humanas/citología , Humanos , Ratones , Virus de la Parainfluenza 3 Humana/fisiología
20.
Int J Mol Sci ; 22(5)2021 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-33673496

RESUMEN

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Hidrogeles/química , Leucemia Linfocítica Crónica de Células B , Andamios del Tejido/química , Línea Celular Tumoral , Humanos , Células Madre Mesenquimatosas , Oligopéptidos , Porosidad , Succinimidas/química
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