Nous remercions nos collègues de RIKEN de BSI pour leurs soutiens et conseils gentiment fructueux au cours de ce travail. Je tiens à exprimer mes sincères remerciements au Professeur Shigemi Mori, professeur G Bor Juh SZ, les Drs. Kiyoji Matsuyama, Katalin AK kesi et rp d Dobolyi qui m&#39;a conseillé d&#39;étudier la méthode de microdialyse au cours de troisième cycle à l&#39;University Graduate for Advanced Studies, au Japon. Enfin, nous remercions M. Chris Sarnecky et le Dr Akitsugu Takasu pour leur soutient dans l&#39;enregistrement vidéo.

Etape 1. Préparation de l&#39;implantation de sonde <ol><li> Anesthésie (non montré dans la vidéo) Anesthetize chaque oiseau avec une injection intraperitoneale d&#39;une kétamine (12,5 mg / kg) et de xylazine (25 mg / kg) de mélange pour produire un niveau stable de l&#39;anesthésie chirurgicale. </li><li> Correction d&#39;un tête à un cadre stéréotaxique Utilisez Xylocaine (TM) de gelée pour réduire les douleurs locales des bars de pression oreille ». Le chef de l&#39;oiseau est fixée dans un appareil stéréotaxique composée de barres d&#39;oreilles et un porte-bec qui tient la tête à un angle de 45 °. Pendant la chirurgie stéréotaxique pour l&#39;implantation, réchauffer le corps de l&#39;oiseau par le pad chirurgicale avec contrôle thermique (température = 34 ~ 36 ° C). </li><li> Désinfecter zone chirurgicale Désinfecter le domaine chirurgical avec isodine (TM) avant la chirurgie. </li></ol> Etape 2. Positionnement stéréotaxique <ol><li> L&#39;alignement du point de référence Aligner la sonde fictive (verre micropipette) de la Lambda (Y-point) et la marque Y-point avec la pipette remplies de teinture. Restant Dextran-TMR de l&#39;étude de suivi dernières a été utilisé dans la vidéo. </li><li> Incision de la peau Enlever la peau et exposer la surface du crâne. Ensuite, nettoyer la surface du crâne avec des cotons-tiges. </li><li> Positionnement de la canule factice (dans le sens horizontal) Déplacer la canule factice pour le site cible (AP axe et la coordination latérale) </li><li> Craniotomie petites et incision de la dure-mère. Utilisez une pince fine pour une craniotomie et une aiguille d&#39;injection fines (30 G) pour les incisions de la dure-mère. Ensuite, nettoyer la surface du crâne. </li><li> Positionnement final (implantation) de la canule factices Localisez la pointe de la canule guide à 0,30 mm au-dessus de la sonde de microdialyse destiné région cible. Enfin, nettoyer la surface du crâne à nouveau, car une surface propre et sec est nécessaire pour le collage difficile. </li></ol> Étape 3. Prétraitement de la surface crânienne (La procédure similaire pour la «restauration remplissage direct» du produit dentaire ne doit être employée.) <ol><li> Traitement de Primer <ol><li> Mélanger PRIMAR liquide A à B sur un plat. </li><li> Appliquez le mélange sur la surface du crâne avec une petite brosse. </li><li> Laisser reposer pendant 30 secondes. </li><li> Sécher avec flux d&#39;air doux. (Ne pas laver.) </li></ol></li><li> Application de liant <ol><li> Appliquer l&#39;agent de liaison à la surface du crâne avec une petite brosse. </li><li> Appliquer un courant d&#39;air doux. (Ne pas laver.) </li><li> Photopolymériser pendant 20 secondes. </li></ol></li></ol> Étape 4. Montage en résine composite <ol><li> Monter avec une résine plugger plaine Si un fouloir ordinaire n&#39;est pas disponible, une spatule micro-peut être utilisé à la place. </li><li> Photopolymérisation </li><li> Incrémental de montage Incrémental de montage est recommandé pour assurer une réaction complète de la résine. Pour le montage différentiel, appliquer l&#39;agent de collage avant chaque montage. </li><li> Mouler la résine composite et photopolymérisable pour la finale de durcissement. </li><li> Fin de la chirurgie </li></ol> Nettoyez la zone chirurgicale, appliquer aux antibiotiques (pommade gentamicine), et enlever l&#39;animal de la flamme stéréotaxique. Ensuite, garder l&#39;animal dans une chambre de thermo-contrôlée (température = 33-35 ° C) pour la récupération post-anesthésiques. Comme le montre la vidéo, qu&#39;aucun comportement anormal n&#39;a été observée dans aucun des animaux le jour suivant après la chirurgie.

<ol>
	<li>Adell, A., Artigas, F., Boulton, A., Baker, G., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Brain+microdialysis:+Principles+and+applications.">In vivo Brain microdialysis: Principles and applications.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Kendrick, K., Robinson, T., Justice, J., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+in+large+unrestrained+animals:+neuroendocrine+and+behavioral+studies+of+acetylcholine,+amino+acid,+monoamine+and+neuropeptide+release+in+the+sheep.">Microdialysis in large unrestrained animals: neuroendocrine and behavioral studies of acetylcholine, amino acid, monoamine and neuropeptide release in the sheep.</a> Microdialysis in the Neuroscience. 7, 327-348 (1991).</li><li>Oakley, B., Schafer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Experimental Neurobiology: A laboratory Manual. , (1978).</li><li>Okumura, T., Yamashita, Y., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+Finch+I.+A+Biological+study.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese Finch I. A Biological study.</a> Soc. Neurosci Abst. 33, (2007).</li><li>Boulton, A., Baker, G., Robinson, T., Bateson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relation+of+brain+electrical+activity+to+behavior.">The relation of brain electrical activity to behavior.</a> In Vivo Neuromethods: Neuromethods. 32, 325-357 (1998).</li><li>Yamashita, Y., Okumura, T., Okanoya, K., Tani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+the+sensori-motor+nucleus+NIf+in+generation+of+complex+syntactical+song+in+the+Bengalese+finch.">Function of the sensori-motor nucleus NIf in generation of complex syntactical song in the Bengalese finch.</a> , 101-104 (2007).</li></ol>

Des expériences ont été menées en conformité avec les directives pour l&#39;expérimentation animale de l&#39;Institut des sciences du cerveau, RIKEN (RIKEN-BSI) et l&#39;institut des animaux comité d&#39;éthique de leur approbation.

Comparé à l&#39;application de résines acryliques auto conventionnelle durcissement, la technique a présenté plusieurs avantages importants: 1. Collage direct: Acryliques guérir soi peut être attaché à l&#39;os pendant un moment mais elle n&#39;adhère pas à l&#39;os. Par conséquent, les vis d&#39;ancrage sont nécessaires pour fixer une base de montage (voir figure 2a). En revanche, la résine photopolymérisable peut se lier à l&#39;os directement sorte que les vis ne sont pas nécessaires (figure 2B). Pa...

1A La photographie montre la sonde de microdialyse (a), un écrou borgne (b) et une canule guide (c). Sur cette photo, la canule guide est entourée par de la résine dentaire qui adhère au crâne directement. Photo B montre un oiseau durant la perfusion de médicaments. Sur le dessus de la tête, il ya une sonde de microdialyse qui est relié à un tube Tefron fine. Barres d&#39;échelle = 1 cm pour A et 2 cm pour les B, respectivement. <img src="http://www.jove.com/files/249_2008_8_26_3/249_Figure-01.png" /> Figure 1 - Photographies d&#39;une sonde de microdialyse (A) et un oiseau durant la perfusion de médicaments (B)

application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

Dans les expériences de l&#39;enregistrement chronique, auto-guérison dentaires résines acryliques ont été utilisés comme une base de montage d&#39;électrodes ou de microdialyse-sondes. Puisque ces acryliques ne liaison à l&#39;os, les vis ont été utilisés comme points d&#39;ancrage. Cependant, dans de petits animaux de laboratoire, comme des pinsons ou de la souris, leurs crânes sont très fragiles et ne parvient pas à maintenir les ancres. Dans ce rapport, nous proposons une nouvelle application de résines photopolymérisables dentaire pour la base de montage des électrodes ou des sondes de microdialyse dans des expériences chroniques. Ce matériel permet un collage direct sur le crâne. Par conséquent, les vis d&#39;ancrage ne sont pas nécessaires et le champ chirurgical peut être considérablement réduit. Les expériences passées montrent que l&#39;effet de collage maintient plus de 2 mois. Fenêtre de résine classique de temps où les matériaux sont souples et réalisable est de quelques minutes. Toutefois, la fenêtre du temps de travail pour ces adhésifs dentaires est nettement plus large et réglable.

Watch this Scientific Journal Video about Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments at JoVE.com

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Dans ce rapport, nous proposons une nouvelle application de résines photopolymérisables dentaire pour la base de montage des électrodes ou des sondes de microdialyse dans des expériences chroniques. Ce matériel permet un collage direct sur le crâne.

Application de résine photopolymérisable adhésif dentaire pour les électrodes de montage ou des sondes de microdialyse dans les expériences chroniques

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

application-light-cured-dental-adhesive-resin-for-mounting-electrodes

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Biology

15.3K vues.  RIKEN. Dans ce rapport, nous proposons une nouvelle application de résines photopolymérisables dentaire pour la base de montage des électrodes ou des sondes de microdialyse dans des expériences chroniques. Ce matériel permet un collage direct sur le crâne.

Vidéo: Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Préparation de bicouches artificielle pour des expériences d&#39;électrophysiologie

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Recherche

Biologie

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Fabrication d&#39;électrodes ampérométriques

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Robust 3D DNA FISH Using Directly Labeled Probes

3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.

We describe a robust and versatile protocol for analyzing nuclear architecture by 3D DNA FISH using directly labeled fluorescent probes.

Robuste FISH 3D DNA Utilisation des sondes directement marquées

3D DNA FISH has become a major tool for  analyzing three-dimensional organization of the nucleus, and several variations  of the technique have been ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicouche de montage pour à long terme Fiche microscopie optique de poisson zèbre

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolement et culture des cellules souches épithéliales dentaire de l&#39;adulte de souris Incisive

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

L&#39;Assemblée et l&#39;application des «Shear Anneaux»: A Novel Endothelial Modèle pour Orbital, unidirectionnel et périodique Fluid débit et contrainte de cisaillement

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&amp;#8226;) Probes, the geNOps, for Real-time Imaging of NO&amp;#8226; Signals in Single Cells

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

Application de Codé génétiquement fluorescent oxyde nitrique (NO •) Sondes, les geNOps, pour l&#39;imagerie en temps réel de NO • Les signaux dans les cellules simples

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Inducing Apical Periodontitis in Mice

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical periodontitis (AP). Apical periodontitis is a common pathology of an inflammatory nature in the periodontal tissues surrounding the tooth root. In order to better understand the nature and mechanism of this pathology it is advantageous to perform the procedure in mice. The induction of this odontogenic inflammation is achieved by drilling into the mouse tooth until the dental pulp is exposed. Next, the tooth pulp remains exposed to be contaminated by the natural oral flora over time, causing apical periodontitis. After this time period, the animal is sacrificed, and the tooth and the jaw bone can be analyzed in various ways. Typical analyses include micro-CT imaging (to evaluate bone resorption), histological staining, immunohistochemistry, and RNA expression. This protocol is useful for research in the field of oral biology to better understand this inflammatory process in an in vivo experimental setting with uniform conditions. The procedure requires a careful handling of the mice and the isolated jaw, and a visual demonstration of the technique is useful. All technical aspects of the procedures leading to induced apical periodontitis and its characterization in a mouse model are demonstrated.

Here, we present a protocol to locally induce apical periodontitis in mice. We show how to drill a hole in the mouse&#39;s tooth and expose its pulp, in order to cause local inflammation. Analysis methods to investigate the nature of this inflammation, such as micro-CT and histology, are also demonstrated.

Induire la parodontite apicale chez les souris

The mechanisms involved in local induced inflammation can be studied using several available animal models. One of these is the induction of apical ...

Systematic Assessment of Mammalian Skull Specimens for Dental and Temporomandibular Joint Pathology

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental pathology, and anatomic variations in many mammalian species. Studying the teeth and jaws of an array of species can present a challenge requiring attention to detail and understanding of a species&#39; normal anatomy. In the present article, a systematic and precise protocol for examining skull specimens is discussed that has been applied to a variety of mammals to define characteristic diseases in the oromaxillofacial region. The procedure outlined is simultaneously precise, repeatable, and adaptable to the highly differing skull and tooth shapes and anatomy across species. Specifically, specimens are examined for missing teeth, periodontal disease, endodontal disease, TMJ pathology, and anatomical variations. Results gleaned from research on museum specimens may reflect the natural history, health, and disease status of individuals and species. Furthermore, these data can inform ecological and conservation research efforts, as well as the care of captive individuals.

The present protocol describes the techniques for the systematic assessment of skull specimens to characterize anatomical and developmental variations and abnormalities of the teeth, periodontal disease, endodontal disease, and temporomandibular joint pathology.

Évaluation systématique de spécimens de crânes de mammifères pour la pathologie dentaire et de l’articulation temporo-mandibulaire

Museum skull specimens represent a non-invasive, informative, and readily available means to study temporomandibular joint (TMJ) lesions, dental ...

Établissement de la culture primaire de cellules souches de la pulpe dentaire

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

Pleins feux sur l’auteur : Faire progresser la régénération tissulaire et la modélisation des maladies avec les cellules souches de la pulpe dentaire

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

Culture primaire de cellules souches de la pulpe dentaire

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...