RESUMO
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that couples the binding of extracellular ligands, such as EGF and transforming growth factor-α (TGF-α), to the initiation of intracellular signaling pathways. EGFR binds to EGF and TGF-α with similar affinity, but generates different signals from these ligands. To address the mechanistic basis of this phenomenon, we have carried out cryo-EM analyses of human EGFR bound to EGF and TGF-α. We show that the extracellular module adopts an ensemble of dimeric conformations when bound to either EGF or TGF-α. The two extreme states of this ensemble represent distinct ligand-bound quaternary structures in which the membrane-proximal tips of the extracellular module are either juxtaposed or separated. EGF and TGF-α differ in their ability to maintain the conformation with the membrane-proximal tips of the extracellular module separated, and this conformation is stabilized preferentially by an oncogenic EGFR mutation. Close proximity of the transmembrane helices at the junction with the extracellular module has been associated previously with increased EGFR activity. Our results show how EGFR can couple the binding of different ligands to differential modulation of this proximity, thereby suggesting a molecular mechanism for the generation of ligand-sensitive differential outputs in this receptor family.
Assuntos
Células Cultivadas/fisiologia , Receptores ErbB/química , Ligantes , Transdução de Sinais/efeitos dos fármacos , Spodoptera/fisiologia , Fatores de Crescimento Transformadores/química , Animais , Humanos , Modelos MolecularesRESUMO
Cryopreserved human skin allografts (CHSAs) are used for the coverage of major burns when donor sites for autografts are insufficiently available and have clinically shown beneficial effects on chronic non-healing wounds. However, the biologic mechanisms behind the regenerative properties of CHSA remain elusive. Furthermore, the impact of cryopreservation on the immunogenicity of CHSA has not been thoroughly investigated and raised concerns with regard to their clinical application. To investigate the importance and fate of living cells, we compared cryopreserved CHSA with human acellular dermal matrix (ADM) grafts in which living cells had been removed by chemical processing. Both grafts were subcutaneously implanted into C57BL/6 mice and explanted after 1, 3, 7, and 28 days (n = 5 per group). A sham surgery where no graft was implanted served as a control. Transmission electron microscopy (TEM) and flow cytometry were used to characterise the ultrastructure and cells within CHSA before implantation. Immunofluorescent staining of tissue sections was used to determine the immune reaction against the implanted grafts, the rate of apoptotic cells, and vascularisation as well as collagen content of the overlaying murine dermis. Digital quantification of collagen fibre alignment on tissue sections was used to quantify the degree of fibrosis within the murine dermis. A substantial population of live human cells with intact organelles was identified in CHSA prior to implantation. Subcutaneous pockets with implanted xenografts or ADMs healed without clinically apparent rejection and with a similar cellular immune response. CHSA implantation largely preserved the cellularity of the overlying murine dermis, whereas ADM was associated with a significantly higher rate of cellular apoptosis, identified by cleaved caspase-3 staining, and a stronger dendritic cell infiltration of the murine dermis. CHSA was found to induce a local angiogenic response, leading to significantly more vascularisation of the murine dermis compared with ADM and sham surgery on day 7. By day 28, aggregate collagen-1 content within the murine dermis was greater following CHSA implantation compared with ADM. Collagen fibre alignment of the murine dermis, correlating with the degree of fibrosis, was significantly greater in the ADM group, whereas CHSA maintained the characteristic basket weave pattern of the native murine dermis. Our data indicate that CHSAs promote angiogenesis and collagen-1 production without eliciting a significant fibrotic response in a xenograft model. These findings may provide insight into the beneficial effects clinically observed after treatment of chronic wounds and burns with CHSA.
Assuntos
Aloenxertos/transplante , Queimaduras/cirurgia , Proliferação de Células/fisiologia , Criopreservação/métodos , Sobrevivência de Enxerto/fisiologia , Transplante de Pele/métodos , Cicatrização/fisiologia , Animais , Células Cultivadas/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Targeting activated fibroblasts, including myofibroblast differentiation, has emerged as a key therapeutic strategy in patients with idiopathic pulmonary fibrosis (IPF). However, there is no available therapy capable of selectively eradicating myofibroblasts or limiting their genesis. Through an integrative analysis of the regulator genes that are responsible for the activation of IPF fibroblasts, we noticed the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding protein, myristoylated alanine-rich C-kinase substrate (MARCKS), as a potential target molecule for IPF. Herein, we have employed a 25-mer novel peptide, MARCKS phosphorylation site domain sequence (MPS), to determine if MARCKS inhibition reduces pulmonary fibrosis through the inactivation of PI3K/protein kinase B (AKT) signaling in fibroblast cells. We first observed that higher levels of MARCKS phosphorylation and the myofibroblast marker α-smooth muscle actin (α-SMA) were notably overexpressed in all tested IPF lung tissues and fibroblast cells. Treatment with the MPS peptide suppressed levels of MARCKS phosphorylation in primary IPF fibroblasts. A kinetic assay confirmed that this peptide binds to phospholipids, particularly PIP2, with a dissociation constant of 17.64 nM. As expected, a decrease of phosphatidylinositol (3,4,5)-trisphosphate pools and AKT activity occurred in MPS-treated IPF fibroblast cells. MPS peptide was demonstrated to impair cell proliferation, invasion, and migration in multiple IPF fibroblast cells in vitro as well as to reduce pulmonary fibrosis in bleomycin-treated mice in vivo. Surprisingly, we found that MPS peptide decreases α-SMA expression and synergistically interacts with nintedanib treatment in IPF fibroblasts. Our data suggest MARCKS as a druggable target in pulmonary fibrosis and also provide a promising antifibrotic agent that may lead to effective IPF treatments.-Yang, D. C., Li, J.-M., Xu, J., Oldham, J., Phan, S. H., Last, J. A., Wu, R., Chen, C.-H. Tackling MARCKS-PIP3 circuit attenuates fibroblast activation and fibrosis progression.
Assuntos
Fibroblastos/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada/metabolismo , Fosfatidilinositóis/metabolismo , Fibrose Pulmonar/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Proliferação de Células , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Substrato Quinase C Rico em Alanina Miristoilada/genética , Fosfatidilinositóis/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fibrose Pulmonar/induzido quimicamenteRESUMO
In order to differentiate T cells in vitro, co-culture systems with Notch ligand-expressing feeder cells have been in use for a long time. Here we describe a feeder-free culture condition for differentiating T cells from hematopoietic cells that are cultured on Fc-DLL4-coated plate with T-lineage cytokines. This condition is capable of efficiently differentiating hematopoietic progenitor cells (HPCs) to immature T cells expressing both CD4 and CD8. To mature those cells into functional T cells, further stimulation and culture is necessary.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Meios de Cultura/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/instrumentação , Células Cultivadas/fisiologia , Citocinas/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/fisiologiaRESUMO
Regenerative therapeutic approaches for myocardial diseases often involve delivery of stem cells expanded ex vivo. Prior studies indicate that cell culture conditions affect functional and phenotypic characteristics, but relationship(s) of cultured cells derived from freshly isolated populations and the heterogeneity of the cultured population remain poorly defined. Functional and phenotypic characteristics of ex vivo expanded cells will determine outcomes of interventional treatment for disease, necessitating characterization of the impact that ex vivo expansion has upon isolated stem cell populations. Single-cell RNA-Seq profiling (scRNA-Seq) was performed to determine consequences of culture expansion upon adult cardiac progenitor cells (CPCs) as well as relationships with other cell populations. Bioinformatic analyses demonstrate that identity marker genes expressed in freshly isolated cells become undetectable in cultured CPCs while low level expression emerges for thousands of other genes. Transcriptional profile of CPCs exhibited greater degree of similarity throughout the cultured population relative to freshly isolated cells. Findings were validated by comparative analyses using scRNA-Seq datasets of various cell types generated by multiple scRNA-Seq technology. Increased transcriptome diversity and decreased population heterogeneity in the cultured cell population may help account for reported outcomes associated with experimental and clinical use of CPCs for treatment of myocardial injury.
Assuntos
Células-Tronco Adultas/fisiologia , Células Cultivadas/fisiologia , Miócitos Cardíacos/fisiologia , Transplante de Células-Tronco/métodos , Adulto , Células-Tronco Adultas/transplante , Animais , Diferenciação Celular/genética , Células Cultivadas/transplante , Biologia Computacional , Conjuntos de Dados como Assunto , Feminino , Perfilação da Expressão Gênica/métodos , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/terapia , Humanos , Camundongos , Miocárdio/citologia , Miocárdio/patologia , Miócitos Cardíacos/transplante , Cultura Primária de Células/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma/genética , Resultado do TratamentoRESUMO
Cattle are an economically important domestic animal species. In vitro 2D cultures of intestinal epithelial cells or epithelial cell lines have been widely used to study cell function and host-pathogen interactions in the bovine intestine. However, these cultures lack the cellular diversity encountered in the intestinal epithelium, and the physiological relevance of monocultures of transformed cell lines is uncertain. Little is also known of the factors that influence cell differentiation and homeostasis in the bovine intestinal epithelium, and few cell-specific markers that can distinguish the different intestinal epithelial cell lineages have been reported. Here we describe a simple and reliable procedure to establish in vitro 3D enteroid, or "mini gut", cultures from bovine small intestinal (ileal) crypts. These enteroids contained a continuous central lumen lined with a single layer of polarized enterocytes, bound by tight junctions with abundant microvilli on their apical surfaces. Histological and transcriptional analyses suggested that the enteroids comprised a mixed population of intestinal epithelial cell lineages including intestinal stem cells, enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We show that bovine enteroids can be successfully maintained long-term through multiple serial passages without observable changes to their growth characteristics, morphology or transcriptome. Furthermore, the bovine enteroids can be cryopreserved and viable cultures recovered from frozen stocks. Our data suggest that these 3D bovine enteroid cultures represent a novel, physiologically-relevant and tractable in vitro system in which epithelial cell differentiation and function, and host-pathogen interactions in the bovine small intestine can be studied.
Assuntos
Técnicas de Cultura de Células/veterinária , Diferenciação Celular , Células Epiteliais/fisiologia , Íleo/fisiologia , Animais , Bovinos , Técnicas de Cultura de Células/métodos , Células Cultivadas/fisiologia , Células Epiteliais/citologiaAssuntos
Diferenciação Celular , Células Endoteliais/citologia , Células-Tronco Mesenquimais/citologia , Neovascularização Patológica/sangue , Psoríase/patologia , Adulto , Estudos de Casos e Controles , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Células Endoteliais/fisiologia , Feminino , Humanos , Masculino , Células-Tronco Mesenquimais/fisiologia , Psoríase/sangue , Valores de ReferênciaRESUMO
Repair and regeneration of craniofacial tissues is particularly challenging because they comprise a complex structure of hard and soft tissues involved in intricate functions. This study combined collagen scaffolds and human adipose stem cells (hASCs) for oral mucosal and calvarial bone regeneration by using resveratrol (RSV), which affects the differentiation of mesenchymal stem cells. We have evaluated the effect of collagen scaffold-containing RSV (collagen/RSV) scaffolds both in vitro and in vivo for their wound healing and bone regeneration potential. Scanning electron microscopy and immunostaining results reveal that hASCs adhere well to and proliferate on both collagen scaffolds and collagen/RSV scaffolds. Oral mucosal lesion experiments demonstrated that the collagen/RSV scaffold is more effective in wound closure and contraction than the collagen scaffold. The micro-computed tomography (µCT) images of calvarial bone display regenerating bone in defects covered with hASCs on collagen/RSV scaffolds that are more visible than that in defects covered with hASCs on a collagen scaffolds. RSV was more effective at inducing hASC differentiation on the collagen scaffold, suggesting that collagen/RSV scaffolds can provide useful biological cues that stimulate craniofacial tissue formation.
Assuntos
Tecido Adiposo/transplante , Proliferação de Células/fisiologia , Colágeno/uso terapêutico , Anormalidades Craniofaciais/cirurgia , Resveratrol/uso terapêutico , Transplante de Células-Tronco/métodos , Engenharia Tecidual/métodos , Animais , Células Cultivadas/fisiologia , Humanos , Modelos Animais , Ratos , Alicerces TeciduaisRESUMO
Technological advances in microfabrication techniques in combination with organotypic cell and tissue models have enabled the realization of microphysiological systems capable of recapitulating aspects of human physiology in vitro with great fidelity. Concurrently, a number of analysis techniques has been developed to probe and characterize these model systems. However, many assays are still performed off-line, which severely compromises the possibility of obtaining real-time information from the samples under examination, and which also limits the use of these platforms in high-throughput analysis. In this review, we focus on sensing and actuation schemes that have already been established or offer great potential to provide in situ detection or manipulation of relevant cell or tissue samples in microphysiological platforms. We will first describe methods that can be integrated in a straightforward way and that offer potential multiplexing and/or parallelization of sensing and actuation functions. These methods include electrical impedance spectroscopy, electrochemical biosensors, and the use of surface acoustic waves for manipulation and analysis of cells, tissue, and multicellular organisms. In the second part, we will describe two sensor approaches based on surface-plasmon resonance and mechanical resonators that have recently provided new characterization features for biological samples, although technological limitations for use in high-throughput applications still exist.
Assuntos
Técnicas Biossensoriais/métodos , Células Cultivadas/fisiologia , Técnicas Eletroquímicas/métodos , Modelos Biológicos , Ressonância de Plasmônio de Superfície/métodos , Animais , Linhagem Celular Tumoral , Impedância Elétrica , Humanos , SomRESUMO
Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB-MSCs have a superior wound-healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB-MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB-MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB-MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB-MSCs. After a 3-day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB-MSC-treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast-treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB-MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing.
Assuntos
Células Cultivadas/fisiologia , Pé Diabético/terapia , Sangue Fetal , Fibroblastos/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/fisiologia , Cicatrização/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Feminino , Humanos , Masculino , Projetos Piloto , República da CoreiaRESUMO
OBJECTIVE: Endothelial cells (ECs) play an important role in neovascularisation, but are too limited in number for adequate therapeutic applications. Mesenchymal stem cells (MSCs) have the potential to differentiate into endothelial lineage cells, which makes them attractive candidates for therapeutic angiogenesis. The aim of this study was to investigate efficient differentiation of MSCs into ECs by inducing medium in vitro. METHODS: MSCs were isolated from bone marrow by density gradient centrifugation. The characterisation of the MSCs was determined by their cluster of differentiation (CD) marker profile. Inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied to differentiate the MSCs into ECs. Endothelial differentiation was quantitatively evaluated using flow cytometry. Real time quantitative PCR (qRT-PCR) was used to analyse mRNA expression of endothelial markers. Tube formation assay was further performed to examine the functional status of the differentiated MSCs. RESULTS: Flow cytometry analysis demonstrated that CD31+ and CD34+ cells increased steadily from 12% at 3 days, to 40% at 7 days, and to 60% at 14 days. Immunofluorescence staining further confirmed the expression of CD31 and CD34. qRT-PCR showed that expression of von Willebrand factor (vWF), vascular endothelial cadherin (VE-cadherin) and vascular endothelial growth factor receptor-2 (VEGFR-2) were significantly higher in the induced MSCs group compared with the uninduced MSCs group. The functional behavior of the differentiated cells was tested by tube formation assay in vitro on matrigel. Induced MSCs were capable of developing capillary networks, and progressive formation of vessel like structures was associated with increased EC population. CONCLUSIONS: These results provide a method to efficiently promote differentiation of MSCs into ECs in vitro for potential application in the treatment of peripheral arterial disease.
Assuntos
Diferenciação Celular/fisiologia , Citocinas/metabolismo , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Doença Arterial Periférica/terapia , Biomarcadores/metabolismo , Separação Celular/métodos , Células Cultivadas/fisiologia , Células Cultivadas/transplante , Centrifugação com Gradiente de Concentração/métodos , Meios de Cultura/metabolismo , Células Endoteliais/transplante , Citometria de Fluxo , Humanos , Neovascularização Fisiológica/fisiologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator A de Crescimento do Endotélio VascularRESUMO
Cell mechanics is a novel label-free biomarker for indicating cell states and pathological changes. The advent of atomic force microscopy (AFM) provides a powerful tool for quantifying the mechanical properties of single living cells in aqueous conditions. The wide use of AFM in characterizing cell mechanics in the past two decades has yielded remarkable novel insights in understanding the development and progression of certain diseases, such as cancer, showing the huge potential of cell mechanics for practical applications in the field of biomedicine. In this paper, we reviewed the utilization of AFM to characterize cell mechanics. First, the principle and method of AFM single-cell mechanical analysis was presented, along with the mechanical responses of cells to representative external stimuli measured by AFM. Next, the unique changes of cell mechanics in two types of physiological processes (stem cell differentiation, cancer metastasis) revealed by AFM were summarized. After that, the molecular mechanisms guiding cell mechanics were analyzed. Finally the challenges and future directions were discussed.
Assuntos
Células Cultivadas/fisiologia , Células Cultivadas/ultraestrutura , Interpretação de Imagem Assistida por Computador/métodos , Mecanotransdução Celular/fisiologia , Micromanipulação/métodos , Microscopia de Força Atômica/métodos , Animais , HumanosRESUMO
Targeted genome editing enables the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Therefore, broadly applicable and versatile methods for increasing its efficacy in cell populations are highly desirable. We designed a simple and robust coselection strategy for enrichment of cells with either nuclease-driven nonhomologous end joining (NHEJ) or homology-directed repair (HDR) events by harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems. Selection for dominant alleles of the ubiquitous sodium/potassium pump (Na+/K+ ATPase) that rendered cells resistant to ouabain was used to enrich for custom genetic modifications at another unlinked locus of interest, thereby effectively increasing the recovery of engineered cells. The process is readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells. The use of universal CRISPR reagents and a commercially available small-molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.
Assuntos
Sistemas CRISPR-Cas/genética , Células Cultivadas/fisiologia , Reparo do DNA/genética , Edição de Genes/métodos , Mutagênese Sítio-Dirigida , Marcadores Genéticos/genética , HumanosRESUMO
Mascarenhas et al. report that TRPV4 expression is upregulated in mast cells in response to the proteolytic cathelicidin fragment LL37 in a murine rosacea model and that TRPV4 loss of function attenuates mast cell degranulation. These findings render TRPV4 a translational-medical target in rosacea. However, signaling mechanisms causing increased expression of TRPV4 await elucidation. Moreover, we ask whether TRPV4-mediated Ca++-influx evokes mast cell degranulation.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Rosácea/genética , Rosácea/fisiopatologia , Canais de Cátion TRPV/genética , Animais , Sinalização do Cálcio/genética , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Modelos Animais de Doenças , Humanos , Mastócitos/citologia , Mastócitos/fisiologia , Camundongos , Pesquisa Translacional Biomédica , Regulação para Cima/genética , CatelicidinasAssuntos
Comunicação Celular/fisiologia , Inflamação/patologia , Telócitos/patologia , Telócitos/ultraestrutura , Animais , Biópsia por Agulha , Células Cultivadas/citologia , Células Cultivadas/fisiologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Fibrose/patologia , Haplorrinos , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica de Transmissão , Psoríase/patologia , Ratos , Valores de Referência , Escleroderma Sistêmico/patologia , Síndrome de Sjogren/patologiaRESUMO
By examining the use of tissue culture in post-war American biomedicine, this paper investigates how scientists experience and manage failure. I study how Leonard Hayflick forged his new definition of failure and ways of managing it by refuting Alexis Carrel's definition of failure alongside his theory of the immortality of cultured cells. Unlike Carrel, Hayflick claimed that every vertebrate somatic cell should eventually die, unless it transformed into a tumour cell. This claim defined cell death, which had been a problem leading to a laboratory failure, as a normal phenomenon. On the other hand, permanent life, which had been considered a normal cellular characteristic, became a major factor causing scientific failure, since it implied malignant transformation that scientists hoped to control. Hayflick then asserted that his cell strains and method would partly enable scientists to manage this factor-especially that occurred through viral infection-alongside other causes of failure in routine tasks, including bacterial contamination. I argue that the growing biomedical enterprise fostered this work of Hayflick's, which had repercussions in both his career and the uses of cells in diverse investigations. His redefinition of failure in the age of biomedicine resulted in the broad dissemination of his cells, medium, and method as well as his long struggle with the National Institutes of Health (NIH), which caused his temporarily failed career.
Assuntos
Células Cultivadas/fisiologia , Técnicas de Cultura de Tecidos/história , Pesquisa Biomédica/história , Morte Celular , História do Século XX , Estados UnidosRESUMO
Las ciencias básicas, la medicina oral y los nuevos avanzces en biotecnología y bioinformática constituyen un gran campo de investigación dentro de la odontología actual. En este sentido, dichos avances están proporcionandoun nuevo conjunto de estrategias terapéuticas para el manejo clínico de los pacientes con dolencias dentales y craneofaciales. Es importante destacar que las disciplinas relacionadas con las ciencias básicas, la medicina oral, la biotecnología y la bioinformática, han contribuido de manera trascendental al entendimiento de la fisiología y lasdiversas patologías que afectan las condiciones de normalidad del sistema bucal. La ingeniería tisular se considera como un enfoque prometedor para la odontología regenerativa, con el objetivofinal de reemplazar morfológica y funcionalmente los tejidos periodontales y/o los dientes perdidos a través dela síntesis in vitro de sustitutos análogos tisulares, considerando que el diente y las estructuras periodontales son importantes órganos del complejo craneofacial, los tratamientos utilizados para las enfermedades que los afectan no lo restauran completamente. La odontología clínica está incursionando en una nueva era en donde el enfoque terapéutico es el uso de terapia génica, terapia celular, ingeniería tisular y lamedicina regenerativa, ampliando el arsenal de posibilidades para nuestros pacientes. Una línea de investigaciónfundamental en ingeniería tisular y medicina regenerativa son las células madres. Como parte de los nuevos avances de la odontología a nivel mundial, científicos e investigadores del mundo aplican la bioingeniería para lograr reconstrucciones maxilofaciales,regeneraciones óseas y reconstrucciones de piezas dentales a partir de células madre como parte de tratamientos inovadores...
Assuntos
Humanos , Células-Tronco/fisiologia , Odontologia/tendências , Engenharia Tecidual , Técnicas de Cultura de Células/métodos , Bioengenharia/métodos , Células Cultivadas/fisiologia , Células Cultivadas/transplante , Doenças da Boca/terapia , Regeneração Óssea/fisiologia , Tecnologia OdontológicaRESUMO
The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles ductal cells morphologically. To study pancreatic ductal cell (PDC) and pancreatic intraepithelial neoplasia (PanIN)/PDAC biology, it is essential to have reliable in vitro culture conditions. Here we describe a methodology to isolate, culture, and passage PDCs and duct-like cells from the mouse pancreas. It can be used to isolate cells from genetically engineered mouse models (GEMMs), providing a valuable tool to study disease models in vitro to complement in vivo findings. The culture conditions allow epithelial cells to outgrow fibroblast and other "contaminating" cell types within a few passages. However, the resulting cultures, although mostly epithelial, are not completely devoid of fibroblasts. Regardless, this protocol provides guidelines for a robust in vitro culture system to isolate, maintain, and expand primary pancreatic ductal epithelial cells. It can be applied to virtually all GEMMs of pancreatic disease and other diseases and cancers that arise from ductal structures. Because most carcinomas resemble ductal structures, this protocol has utility in the study of other cancers in addition to PDAC, such as breast and prostate cancers.
Assuntos
Técnicas de Cultura de Células/métodos , Células Cultivadas/fisiologia , Ductos Pancreáticos/citologia , Animais , Técnicas de Cultura de Células/instrumentação , CamundongosRESUMO
PURPOSE: To assess the viability of cultured epithelium and preserved by freezing for periods varying from one month to one year. METHODS: Samples of cultured epithelium were incubated in cryoprotectant medium (Group A), packed in aluminum envelopes and packed in polystyrene boxes. The boxes were subjected to a temperature of-70ºC. After freezing for a period of time ranging from one to 12 months, cultured epithelial samples were assessed for their viability by vital staining (Trypan blue) and metabolic analysis based on glucose consumption and lactate production. Samples of not frozen cultured epithelium (Group B) were also tested for viability and the results obtained were used as comparison parameter for the variation of viability. RESULTS: Statistical analysis between the group A and B indicate that the mean age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical difference. In 30 days we obtained 37% of the original viability of cultured epithelium, 25% at six months and one year, less than 15%. This trend was confirmed statistically with a reduction of approximately 1.8% of the original viability epithelium cultured every 30 days of storage. In the analysis by lactate production, similar results were observed. In the analysis by the glucose consumption results were not significant. The viability indices show statistically significant difference between the group A and B (p<0.0001). CONCLUSIONS: Although cryopreserved cultured epithelium showed significant reduction of viability, all samples remained viable. It was also found that the viability of cryopreserved cultured epithelial decreased as a function of storage time.
Assuntos
Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Criopreservação/métodos , Pele , Sobrevivência de Tecidos/fisiologia , Aloenxertos/fisiologia , Meios de Cultura , Sobrevivência Celular/fisiologia , Células Cultivadas/fisiologia , Crioprotetores/farmacologia , Epitélio/fisiologia , Valores de Referência , Reprodutibilidade dos Testes , Estatísticas não Paramétricas , Fatores de TempoRESUMO
The regenerative potential of mesenchymal stromal cells (MSC) holds great promise in using them for treatment of a wide range of debilitating diseases. Several types of culture media and systems have been used for large-scale expansion of MSCs in vitro; however, the majority of them rely heavily on using foetal bovine serum (FBS)-supplement for optimal cell proliferation. FBS-based cultures pose the potential threat of spread of transmissible spongiform encephalopathy and bovine spongiform encephalopathy to MSCs and then to their recipients. A recent trend in cell culture is to change from serum-use to serum-free media (SFM). In this context, the current review focuses specifically on employment of various SFM for MSCs and discusses existences of various options with which to substitute FBS. In addition, we analyse MSC population growth kinetic patterns using various SFM for large-scale production of MSCs.