Ce travail a été soutenu par une subvention de recherche des Instituts nationaux de la santé (HL068915 DBC), une nouvelle bourse de nouveau chercheur du Fonds de recherche Thrasher (pour PAC), un Grant-in-Aid de l&#39;American Heart Association (12GRNT11910008 à DBC) , une subvention de la Fondation des enfants de coeur (DBC), et les dons à l&#39;hôpital conduction cardiaque Fonds pour enfants de Boston, la Fondation de famille de Ryan, et par David Pullman recherche.

Ici, nous allons démontrer l&#39;isolement de COL1 de peau d&#39;agneau. Comme écrit, ce protocole peut également être utilisé pour isoler avec succès COL1 de lapin et de la peau humaine. Une. Préparer cutanée échantillon <ol><li> Tous les réactifs s&#39;équilibrer à 4 ° C avant utilisation. </li><li> Rincer échantillon cutanée (25-50 g) dans de la glace-froid dH 2 O et retirez tout laine, fourrure, ou les cheveux avec de la crème dépilatoire. </li><li> Utilisez une lame de rasoir unique de pointe pour gratter l&#39;échantillon de tissu propre et la graisse. </li><li> Rincer l&#39;échantillon dans de la glace-froid dH 2 O. </li><li> Couper l&#39;échantillon de peau de 1 cm x 1 cm morceaux avec une lame de rasoir unique de bord. </li></ol> 2. Retirer collagènes solubilisés Matériel <ol><li> Peser 5 g d&#39;échantillon de 50 ml par tube conique et ajouter 30 ml de 0,5 M d&#39;acétate de sodium par de la glace. </li><li> Mélanger les tubes pendant 1 min à la mise en m / s ou 6 à l&#39;aide d&#39;un adaptateur de tube de 50 ml dans l&#39;homogénéisateur de paillasse. </li><li> Jeter supernatant et répéter pour un total de 7 acétate de sodium cycles de lavage. </li><li> Rincer l&#39;échantillon dans de la glace-froid dH 2 O et mélanger une fois pour éliminer l&#39;acétate de sodium résiduel. </li><li> Utilisez une spatule pour comprimer l&#39;échantillon contre le côté du tube pour enlever l&#39;excès de liquide, puis les transférer à un 50 ml nouveau tube conique. </li></ol> 3. Collagène de type I Extraction <ol><li> Laver deux fois dans l&#39;échantillon de 2 ml / g 0,075 M de tampon citrate de sodium pendant 1 min à 6 m / sec dans la paillasse homogénéisateur comprimant l&#39;échantillon et élimination du surnageant après chaque lavage. </li><li> Ajouter une 2 ml / g de portion aliquote fraîche du tampon citrate 0,075 M de sodium à l&#39;échantillon. </li><li> Effectuez 6 séquentielle 1 min, 6 m / s paillasse homogénéisateur mélange cycles d&#39;agitation sans enlever le tampon entre chaque cycle. </li><li> Transférer l&#39;épaisse surnageant clair à un tube de collecte. </li><li> Ajouter un tampon de citrate de sodium 0,075 M supplémentaire de 1 ml / g de l&#39;échantillon et effectuer une dernière paillasse homogénéisateur agitationcycle tion. </li><li> Ajouter cette surnageant final à un tube de collecte. </li><li> Centrifuger le tube de prélèvement à 3200 xg pendant 10 min à 4 ° C. </li><li> Transférer le surnageant dans le compartiment supérieur d&#39;un poids moléculaire de 100.000 couper dispositif de filtre centrifuge. </li><li> Centrifuger à 3200 g pendant 30 min à 4 ° C. </li><li> Transférer la COL1 purifié à partir du compartiment supérieur à un tube conique et conserver à 4 ° C. </li></ol>

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Les auteurs n&#39;ont aucun intérêt financier concurrents à divulguer.

Un élément essentiel de cette méthode est la compression efficace répétée de l&#39;échantillon cutané pour éliminer l&#39;excès de liquide. Il est essentiel d&#39;éliminer l&#39;acétate de sodium, autant que possible à l&#39;étape 2.5 en comprimant l&#39;échantillon. On utilise une spatule pour compacter la peau contre le côté du tube, mais étamine pourrait également être utilisé, bien que cela nécessiterait le retrait de l&#39;échantillon à partir du tube et augmenter le temps nécessaire pour e...

Pendant des décennies, les chercheurs et les fournisseurs commerciaux ont isolé solubilisé COL1 à partir d&#39;un assortiment de sources tissulaires, y compris la peau et des tendons à l&#39;aide des variations d&#39;un protocole d&#39;extraction de l&#39;acide simple, suivie d&#39;une neutralisation, ce qui conduit à une remise en suspension d&#39;une matrice de fibrilles COL1 organisés qui peut être utilisé pour une multitude d&#39;applications biomédicales 4.1. Bien qu&#39;il existe de nombreux exemples d&#39;applications cliniques pour COL1, quelques-uns de ces emploient COL1 autologue dérivée parce que la préparation de cette protéine nécessite de longues extractions de prendre des jours ou des semaines pour effectuer 5-7. En conséquence, les enquêteurs de la recherche et les médecins utilisent généralement coûteux, les préparations commerciales de COL1 qui sont préparés à partir de tissus non humains tels que le rat, le boeuf, ou corium porcine ou en utilisant la peau retirée de cadavres ou après la circoncision. Pour les applications de régénération, beaucoup de ces produits présentent la variabilité par rapport à leur capacité à former solidedes matrices ou des constructions de tissu capables de supporter la fixation et la croissance cellulaires. Par conséquent, il ya un net besoin d&#39;une nouvelle méthode normalisée conçu pour extraire rapidement COL1 à partir de sources de tissus autologues accessibles et abondantes. Une telle méthode permettrait d&#39;économiser à la fois temps et argent dans le cadre de la recherche où COL1 pourrait être extrait du modèle animal d&#39;intérêt et utilisé pour les essais précliniques de tissus modifiés assemblés sur des échafaudages à base de collagène. Dans le même temps, ayant la possibilité d&#39;utiliser, d&#39;une manière autologue COL1 dérivé rapidement isolées en clinique augmenterait la sécurité globale des patients qui seraient autrement recevoir allogénique ou xénogénique préparations. Pour les patients recevant une préparation autogène, cette méthode simplifiée permettrait de réduire considérablement l&#39;intervalle entre la collecte de biopsie et de l&#39;application de collagène. Pour ces raisons, nous avons cherché à améliorer une méthode d&#39;isolement et de purification COL1 établie de longue date en utilisant ag haut débititation et d&#39;exclusion de taille centrifugation. Cette méthode est simple à réaliser en utilisant des tampons et les équipements standards trouvés dans de nombreux laboratoires. En employant une agitation à grande vitesse, notre protocole réduit le temps nécessaire pour isoler soluble, cutanée COL1 d&#39;environ 10 jours à moins de 3 h 8. Fait important, cette méthode peut être facilement réalisée dans le cadre clinique pour préparer COL1 dérivé autologue pour une utilisation chez les patients au cours d&#39;une seule série de procédures.

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
			<td style="width:147px;">116004500</td>
			<td style="width:139px;">-</td>
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			<td height="190" style="height:190px;width:123px;">Centrifuge</td>
			<td style="width:136px;">Beckman</td>
			<td style="width:147px;">J6-MI</td>
			<td style="width:139px;">Swing bucket rotor to accommodate 50 mL conical tubes at 3200 g.</td>
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an improved method for the preparation of type i collagen from skin

Type de collagène soluble 1 (COL1) est largement utilisé comme un substrat adhésif pour des cultures de cellules et comme échafaudage pour des applications de régénération cellulaire. Cliniquement, cette protéine est largement utilisé pour la chirurgie esthétique, les injections par voie cutanée, greffe osseuse, et la chirurgie reconstructrice. Les sources de COL1 pour ces procédures sont souvent non-humain, ce qui augmente le potentiel de l&#39;inflammation et de rejet ainsi que la transmission de la maladie des xénobiotiques. Compte tenu de cela, une méthode pour purifier efficacement et rapidement COL1 de quantités limitées de tissus autologue dérivés serait de contourner la plupart de ces questions, mais les protocoles d&#39;isolement classiques sont longues et nécessitent souvent de grandes quantités de tissus de collagène. Ici, nous démontrons une méthode d&#39;extraction COL1 efficace qui réduit le temps nécessaire pour isoler et purifier cette protéine d&#39;environ 10 jours à moins de 3 heures. Nous avons choisi le derme que notre source de tissus en raison de sa disponibilité au cours de nombreuses interventions chirurgicales. Tsa méthode utilise des tampons d&#39;extraction traditionnelles combinées avec une agitation énergique et filtration centrifuge pour obtenir très pur, COL1 soluble de petites quantités de corium. Brièvement, des biopsies dermiques sont lavées à fond dans glacée dH 2 O après élimination de la graisse, le tissu conjonctif, et les cheveux. Les échantillons de peau sont dépouillés de protéines non collagéniques et des polysaccharides à l&#39;aide de 0,5 M d&#39;acétate de sodium et d&#39;un homogénéisateur de paillasse à grande vitesse. Le collagène de solides résiduels est ensuite extrait avec un tampon de citrate de sodium 0,075 M à l&#39;aide de l&#39;homogénéisateur. Ces extraits sont purifiés en utilisant des filtres de 100 000 MW de coupure centrifuges qui produisent des préparations COL1 de qualité comparable ou supérieure à celle des produits du commerce ou de ceux obtenus en utilisant des procédures traditionnelles. Nous prévoyons que cette méthode facilitera l&#39;utilisation de COL1 autologue dérivée pour une multitude de recherche et les applications cliniques.

Ce protocole d&#39;isolement COL1 nécessite environ 2 heures et 15 minutes à compléter. La figure 1 montre un schéma décrivant les principales étapes de cette procédure. Préparation de l&#39;échantillon et d&#39;effectuer les 7 cycles de agitations à grande vitesse et se rince avec de l&#39;acétate de sodium prend environ 35 min (figures 1A-C). Effectuer une agitation et le cycle de rinçage avec dH 2 O prend environ 5 min (figures 1D et 1E). Ajout...

Watch this Scientific Journal Video about An Improved Method for the Preparation of Type I Collagen From Skin at JoVE.com

An Improved Method for the Preparation of Type I Collagen From Skin

Les procédures traditionnelles pour l&#39;isolement de soluble de type 1 collagène (COL1) nécessitent environ 10 jours à partir de début à la fin en raison d&#39;incubations de tampons de longues et laborieuses remises en suspension de fibrilles. Ici, nous décrivons un moyen de purifier COL1 de petites biopsies cutanées en moins de 3 heures.

Un procédé amélioré pour la préparation de collagène de type I de la peau

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

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Research

JoVE Journal

Bioengineering

22.7K vues.  Boston Children's Hospital.  Les procédures traditionnelles pour l'isolement de soluble de type 1 collagène (COL1) nécessitent environ 10 jours à partir de début à la fin en raison d'incubations de tampons de longues et laborieuses remises en suspension de fibrilles. Ici, nous décrivons un moyen de purifier COL1 de petites biopsies cutanées en moins de 3 heures. 

Vidéo: An Improved Method for the Preparation of Type I Collagen From Skin

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypiques de collagène I Dosage: Une plate-forme malléable pour évaluer le comportement des cellules dans un contexte 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Recherche

Bio-ingénierie

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Amélioration de la méthode pour la préparation d&#39;une base de cellules humaines, Contactez modèle de la barrière hémato-encéphalique

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

An Improved Mechanical Testing Method to Assess Bone-implant Anchorage

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a nano-scale. As such, traditional methods of describing implant surfaces -&#160;namely numerical determinants of surface roughness -&#160;are inadequate for predicting in vivo performance. Biomechanical testing provides an accurate and comparative platform to analyze the performance of biomaterial surfaces. An improved mechanical testing method to test the anchorage of bone to candidate implant surfaces is presented. The method is applicable to both early and later stages of healing and can be employed for any range of chemically or mechanically modified surfaces -&#160;but not smooth surfaces. Custom rectangular implants are placed bilaterally in the distal femora of male Wistar rats and collected with the surrounding bone. Test specimens are prepared and potted using a novel breakaway mold and the disruption test is conducted using a mechanical testing machine. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate and reproducible means for isolating an exact peri-implant region for testing.

An improved method to mechanically test bone anchorage to candidate implant surfaces is presented. This method allows for alignment of the disruption force exactly perpendicular, or parallel, to the plane of the implant surface, and provides an accurate means to direct the disruption forces to an exact peri-implant region.

Une méthode d&#39;essai mécanique améliorée pour évaluer Anchorage os-implant

Recent advances in material science have led to a substantial increase in the topographical complexity of implant surfaces, both on a micro- and a ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingénierie 3D cellularisé collagène Gels pour la régénération tissulaire vasculaire

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

Tissu haché en comprimé Collagen: Un Biotransplant contenant des cellules de réparation Reconstructive simple mise en scène

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Préparation de la 3D collagène Gels et microcanaux pour l&#39;étude des interactions 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Évaluation de la prolifération des kératinocytes sur deux et trois dimensions des substrats de collagène de Type I

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

Quantification simultanée de kynurenines sélectionnées analysées par chromatographie liquide-spectrométrie de masse dans le milieu recueilli à partir des cultures de cellules cancéreuses

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Une nouvelle méthode d’injection lente mini-invasive pour réduire les lésions tissulaires

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Pleins feux sur l’auteur : Une nouvelle méthode d’injection cellulaire avec une invasion minimale  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Une nouvelle méthode d’injection cellulaire avec un minimum d’invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...