Diese Arbeit wurde durch ein Forschungsstipendium von den National Institutes of Health (HL068915 zu DBC), einem New Researcher Award von der Thrasher Research Fund (CAP), ein Grant-in-Aid von der American Heart Association (12GRNT11910008 zu DBC) unterstützt ein Forschungsstipendium von der Kinderherzstiftung (DBC) und Spenden an die Kinderklinik Boston Erregungsleitungsfonds, der Ryan Familiengröße, und von David Pullman.

Hier werden wir die Isolierung von COL1 aus Lammfell zu demonstrieren. Wie geschrieben, kann dieses Protokoll auch verwendet, um erfolgreich von Kaninchen und die menschliche Haut zu isolieren COL1 werden. 1. Bereiten Dermal Beispiel <ol><li> Ins Gleichgewicht alle Reagenzien auf 4 ° C vor der Verwendung. </li><li> Spülen Hautprobe (25-50 g) in eiskaltem dH 2 O und entfernen Sie alle Wolle, Fell oder Haare mit Enthaarungscreme. </li><li> Verwenden Sie einen einzelnen Rasierklinge, um die Probe frei von Bindegewebe und Fett zu kratzen. </li><li> Die Probe in eiskaltem dH 2 O spülen </li><li> Schneiden Sie die Hautprobe in 1 cm x 1 cm große Stücke mit einer einzigen Rasierklinge. </li></ol> 2. Entfernen kollagenen Solubilized Werkstoff <ol><li> Wiegen Sie 5 g der Probe pro 50 ml konischen Röhrchen und 30 ml eiskaltem 0,5 M Natriumacetat. </li><li> Mix Röhrchen für 1 min bei 6 m / sec Einstellung mit einem 50-ml-Tube-Adapter in der Tisch-Homogenisator. </li><li> Entsorgen supernatant und wiederholen Sie für insgesamt 7 Natriumacetat Waschzyklen. </li><li> Spülen Sie die Probe in eiskaltem dH 2 O und mischen Sie einmal, um Rest Natriumacetat zu entfernen. </li><li> Verwenden Sie einen Spachtel, um die Probe gegen die Seite des Rohres zu komprimieren, um überschüssige Flüssigkeit zu entfernen und dann übertragen auf ein frisches 50 ml konischen Röhrchen. </li></ol> 3. Typ-I-Kollagen-Extraktion <ol><li> Die Probe zweimal Waschen in 2 ml / g 0,075 M Natriumcitrat-Puffer für 1 min bei 6 m / sec in der Tisch-Homogenisator Komprimieren der Probe und Verwerfen des Überstandes nach jeder Wäsche. </li><li> Hinzufügen eines neues 2 ml / g Aliquot von 0,075 M Natriumcitrat-Puffer zu der Probe. </li><li> Führen sequentielle 6 1 min, 6 m / sec Tisch Homogenisator Mix Zyklen der Agitation, ohne den Puffer zwischen jedem Zyklus. </li><li> Übertragen Sie die dicke, klare Überstand in ein Sammelrohr. </li><li> Fügen Sie eine weitere 1 ml / g 0,075 M Natriumcitrat-Puffer auf die Probe und führen einen letzten Tisch-Homogenisator agitation Zyklus. </li><li> In diese letzte Überstand in ein Sammelrohr. </li><li> Das Sammelröhrchen zentrifugiert bei 3200 g für 10 min bei 4 ° C. </li><li> Den Überstand in der oberen Kammer eines Molekulargewicht von 100.000 abgeschnitten Kreiselfiltereinrichtung. </li><li> Zentrifugieren bei 3200 × g für 30 min bei 4 ° C </li><li> Übertragen Sie die gereinigten COL1 aus dem oberen Fach zu einer sauberen konischen Rohr und bei 4 ° C </li></ol>

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Die Autoren haben keine finanziellen Interessen im Wettbewerb offen zu legen.

Ein wesentlicher Bestandteil dieses Verfahrens ist die wiederholte effiziente Kompression der Hautprobe, um überschüssige Flüssigkeit zu entfernen. Es ist wichtig, so viel wie möglich Natriumacetat in Schritt 2.5 durch Komprimieren der Probe zu entfernen. Wir verwenden einen Spatel, um die Haut gegen die Seite der Röhre zu verdichten, jedoch könnte Gaze verwendet werden, obwohl dies die Entfernung der Probe aus dem Rohr erforderlich und erhöht die erforderliche Zeit, um diese Schritte durchzuführen. Ebenso ist e...

Seit Jahrzehnten haben Wissenschaftler und kommerzielle Anbieter solubilisierten COL1 aus einem Sortiment von Gewebequellen, einschließlich Haut und Sehnen mit irgendeiner Variation eines einfachen Säure-Extraktionsprotokolls, gefolgt von Neutralisation, die in einer Resuspendierung aus einer Matrix organisiert COL1 Fibrillen führt, die verwendet werden können, isoliert für eine Vielzahl von biomedizinischen Anwendungen 1-4. Zwar gibt es viele Beispiele für klinische Anwendungen für COL1, einige von diesen beschäftigen autolog stamm COL1 weil Herstellung dieses Proteins erfordert langwierige Extraktionen unter Tage oder Wochen auf 5-7 durchzuführen. Als Ergebnis der Forschung Forscher und Ärzte verwenden in der Regel teuer, Handelspräparate, die unter Verwendung von COL1 nicht-menschlichen Geweben wie Ratten, Rinder-, Schweine-oder Lederhaut oder über die Haut von Leichen entfernt oder nach der Beschneidung von Männern zubereitet werden. Für regenerative Anwendungen sind viele dieser Produkte zeigen Variabilität in Bezug auf ihre Fähigkeit, feste bildenMatrices oder Gewebe-Konstrukte in der Lage ist die Zellhaftung und Wachstum. Folglich gibt es einen deutlichen Bedarf für eine neue standardisierte Methode entwickelt, um schnell zu extrahieren COL1 aus zugänglich und reichlich autologen Gewebequellen. Ein derartiges Verfahren würde Zeit und Geld in die Forschung, in dem COL1 konnte aus dem Tiermodell von Interesse extrahiert und für die präklinische Prüfung von technischen Geweben auf Kollagenbasis Gerüste montiert werden zu speichern. Gleichzeitig würde mit der Möglichkeit, eine schnell isoliert, autolog stamm COL1 in der Klinik die allgemeine Sicherheit für die Patienten, die sonst erhalten würde allogenen oder xenogenen Vorbereitungen zu erhöhen. Bei Patienten, die eine autogenen Vorbereitung, würde dies die vereinfachte Verfahren stark das Intervall zwischen Biopsie-Sammlung und Kollagen-Anwendung zu reduzieren. Aus diesen Gründen haben wir versucht, auf eine lange etablierte COL1 Isolierung und Reinigung Verfahren unter Verwendung von High-Speed-ag verbessernitation und Größenausschluss-Zentrifugation. Dieses Verfahren ist einfach durchzuführen unter Verwendung von Standardpuffern und in vielen Labors gefunden. Durch den Einsatz von High-Speed-Agitation, reduziert unsere Protokoll die notwendige Zeit, um lösliche, Haut COL1 von etwa 10 Tagen auf weniger als 3 Stunden 8 zu isolieren. Wichtig ist, dass diese Methode kann leicht in der Klinik, um autolog stamm COL1 zur Anwendung bei Patienten während einer einzigen Reihe von Verfahren durchgeführt werden, vorzubereiten.

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			<td height="43" style="height:43px;width:123px;">FastPrep-24 System</td>
			<td style="width:136px;">MP Biomedicals</td>
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			<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

Lösliche Typ-1-Kollagen (COL1) wird weitgehend als Klebstoff Substrat für Zellkulturen sowie als Zellgerüst für regenerative Anwendungen verwendet. Klinisch wird dieses Protein häufig für kosmetische Chirurgie, Hautinjektionen, Knochentransplantation, und rekonstruktive Chirurgie. Die Quellen der COL1 für diese Verfahren sind allgemein nicht-menschlichen, welche das Potential für eine Entzündung und Ablehnung sowie xenobiotischen Krankheitsübertragung erhöht. Im Hinblick darauf wäre ein Verfahren, um schnell und effizient zu reinigen COL1 von begrenzten Mengen von autolog abgeleiteten Gewebe umgehen viele dieser Probleme, jedoch sind Standardisolierungsprotokolle langwierig und erfordern große Mengen an kollagenem Gewebe oft. Hier zeigen wir ein effizientes COL1 Extraktionsmethode, die die benötigte Zeit, um von ungefähr 10 Tage zu isolieren und zu reinigen, dieses Protein auf weniger als 3 h reduziert. Wir wählten die Dermis als unsere Gewebequelle wegen seiner Verfügbarkeit in vielen chirurgischen Verfahren. Tseine Methode verwendet traditionelle Extraktionspuffer kombiniert mit kraftvollen Bewegung und Zentrifugalfiltration zu hochreinem, lösliche COL1 von kleinen Mengen von Lederhaut erhalten. Kurz gesagt, Hautbiopsien werden gründlich in eiskaltem dH 2 O nach Entfernen von Fett, Bindegewebe und Haare gewaschen. Die Hautproben werden von nicht-kollagenen Proteinen und Polysacchariden unter Verwendung von 0,5 M Natriumacetat und eine Hochgeschwindigkeitslabortisch-Homogenisators abgestreift. Kollagen von restlichen Feststoffen wird anschließend mit einer 0,075 M Natriumcitrat-Puffer unter Verwendung eines Homogenisators extrahiert. Diese Extrakte werden mit 100.000 MW Cut-off-Filter, die Kreisel COL1 Zubereitungen der vergleichbare oder überlegene Qualität, kommerzielle Produkte oder solche mit herkömmlichen Verfahren gereinigt, erhalten. Wir erwarten, dieses Verfahren die Verwendung von autologen abgeleiteten COL1 für eine Vielzahl von Forschungs-und klinischen Anwendungen zu erleichtern.

Diese COL1 Isolation Protokoll benötigt etwa 2 Stunden und 15 Minuten in Anspruch. Fig. 1 zeigt ein schematisches Diagramm, das die Hauptschritte in diesem Verfahren. Herstellung der Probe und Durchführen der 7 Zyklen von Hochgeschwindigkeits-Agitation und spült mit Natriumacetat dauert etwa 35 min (Fig. 1A-C). Durchführen einer Agitation und spülen Zyklus mit dH 2 O dauert ca. 5 min (1D und 1E). Hinzufügen von Natriumcitrat und Unterwerfen der Probe ei...

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

Traditionelle Verfahren zur Isolierung von löslichen Typ-1-Kollagen (COL1) benötigen etwa 10 Tage von Anfang an wegen der langwierigen und mühsamen Puffer Inkubationen Resuspensionen von Fibrillen zu beenden. Hier beschreiben wir ein Mittel, um COL1 von kleinen Hautbiopsien in weniger als 3 Stunden zu reinigen.

Ein verbessertes Verfahren zur Herstellung von Typ I-Kollagen aus der Haut

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 Aufrufe.  Boston Children's Hospital.  Traditionelle Verfahren zur Isolierung von löslichen Typ-1-Kollagen (COL1) benötigen etwa 10 Tage von Anfang an wegen der langwierigen und mühsamen Puffer Inkubationen Resuspensionen von Fibrillen zu beenden. Hier beschreiben wir ein Mittel, um COL1 von kleinen Hautbiopsien in weniger als 3 Stunden zu reinigen. 

Serie: 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.

Organotypische Collagen I Assay: A Temperguss Platform to Cell Verhalten in 3-Dimensional Kontext beurteilen

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

Forschung

Biotechnik

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.

Verbesserte Methode zur Präparation einer menschlichen Zelle-basierte, Kontakt Modell der Blut-Hirn-Schranke

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.

Eine verbesserte mechanische Testverfahren zu Knochen-Implantat-Anchorage bewerten

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.

Engineering 3D cellularized Kollagengele für Gefäßgeweberegeneration

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.

Zerkleinerte Gewebe in komprimierter Collagen: Ein zellhaltigen Biotransplant für einstufige rekonstruktive Reparatur

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.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<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.

Bewertung der Keratinozyten Verbreitung auf zwei- und dreidimensionale Typ I Kollagen Substrate

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.

Gleichzeitige Quantifizierung ausgewählter Kynurenine, analysiert durch Flüssigchromatographie-Massenspektrometrie 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 ...

Eine neuartige minimal-invasive langsame Injektionsmethode zur Reduzierung von Gewebeverletzungen

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.

Autor im Rampenlicht: Eine neuartige Zellinjektionsmethode mit minimaler Invasion  

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

Eine neuartige Zellinjektionsmethode mit minimaler Invasion

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