Este trabajo fue apoyado por una beca de investigación de los Institutos Nacionales de Salud (HL068915 de DBC), un nuevo Premio Investigador del Fondo de Investigación Thrasher (NAC), una subvención-en-Ayudas a la American Heart Association (12GRNT11910008 a DBC) , una beca de investigación de la Fundación del Niño del corazón (a DBC), y las donaciones al Hospital de Fondo de Niños de Boston cardíaco de conducción, la Fundación Ryan Familia, y por David Pullman.

Aquí vamos a demostrar el aislamiento de COL1 de la piel de cordero. Como está escrito, este protocolo también se puede utilizar para aislar con éxito COL1 de conejo y de la piel humana. 1. Preparar la muestra dérmica <ol><li> Que todos los reactivos a 4 ° C antes de su uso. </li><li> Enjuagar la muestra dérmica (25-50 g) en DH helada 2 O y eliminar cualquier lana, piel o cabello con crema depilatoria. </li><li> Utilice una hoja de afeitar de un solo filo para raspar la limpieza de la muestra de tejido conectivo y grasa. </li><li> Enjuagar la muestra en helada dH 2 O. </li><li> Cortar la muestra de piel en 1 cm x 1 cm pedazos con una cuchilla de afeitar de un solo filo. </li></ol> 2. Retire colagenosas solubilizado material <ol><li> Pesar 5 g de tubo cónico de la muestra por 50 ml y añadir 30 ml de acetato de sodio helado 0,5 M. </li><li> Mezclar los tubos durante 1 min a la configuración de 6 m / seg usando un adaptador de tubo de 50 ml en el homogeneizador de sobremesa. </li><li> Deseche supernatant y repita para un total de 7 ciclos de lavado de sodio acetato. </li><li> Enjuagar la muestra en dH helada 2 O y mezclar una vez para eliminar el acetato de sodio residual. </li><li> Utilice una espátula para comprimir la muestra contra el lado del tubo para eliminar el exceso de líquido y luego se transfieren a un tubo cónico de 50 ml fresco. </li></ol> 3. Colágeno tipo I Extracción <ol><li> Lavar la muestra dos veces en 2 ml / g 0,075 M de tampón de citrato de sodio durante 1 min a 6 m / seg en el homogeneizador de sobremesa compresión de la muestra y de desechar el sobrenadante después de cada lavado. </li><li> Añadir un nuevo 2 ml / g de parte alícuota de tampón de citrato de sodio 0,075 M a la muestra. </li><li> Realizar 6 secuencial 1 min, 6 m / seg ciclos de sobremesa mezcla homogeneizador de agitación sin quitar el tapón entre cada ciclo. </li><li> Transferir el grueso, claro sobrenadante a un tubo de colección. </li><li> Añadir un tampón citrato adicional de 1 ml / g de sodio 0,075 M a la muestra y realizar un homogeneizador última agitación de sobremesaciclo ción. </li><li> Añadir este último sobrenadante a un tubo de recogida. </li><li> Centrifugar el tubo de recogida a 3200 xg durante 10 min a 4 ° C. </li><li> Transferir el sobrenadante a el compartimiento de la parte superior de un peso molecular 100.000 cortar dispositivo de filtro centrífugo. </li><li> Se centrifuga a 3.200 g durante 30 min a 4 ° C. </li><li> Transferir el purificada COL1 del compartimiento superior a un tubo cónico limpia y se almacenan a 4 ° C. </li></ol>

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F., Liepmann, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vascular+smooth+muscle+cells+by+micropatterning.">Regulation of vascular smooth muscle cells by micropatterning.</a> Biochem. Biophys. Res. Comm. 307, 883-890 (2003).</li><li>Leighton, J., Justh, G., Esper, M., Kronenthal, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen-coated+cellulose+sponge:+three+dimensional+matrix+for+tissue+culture+of+Walker+tumor+256.">Collagen-coated cellulose sponge: three dimensional matrix for tissue culture of Walker tumor 256.</a> Science. 155, 1259-1261 (1967).</li></ol>

Los autores no tienen intereses financieros en competencia a revelar.

Un componente crucial de este método es la compresión eficiente repetida de la muestra dérmica para eliminar el exceso de líquido. Es crítico para eliminar el acetato de sodio tanto como sea posible en el paso 2.5 mediante la compresión de la muestra. Utilizamos una espátula para compactar la piel contra el lado del tubo, sin embargo, estopilla también podría ser utilizado, aunque esto requeriría la eliminación de la muestra desde el tubo y aumentar el tiempo necesario para llevar a cabo estos pasos. Asimismo...

Durante décadas, los investigadores y los proveedores comerciales han aislado solubilizado COL1 de una variedad de fuentes de tejidos incluyendo la piel y tendón utilizando alguna variación de un protocolo de extracción de ácido sencillo seguido de neutralización, lo que resulta en una resuspensión de una matriz de fibrillas COL1 organizadas que se puede utilizar para una multitud de aplicaciones biomédicas 1-4. Si bien hay muchos ejemplos de aplicaciones clínicas para COL1, algunas de ellas emplean COL1 derivada autóloga, porque la preparación de esta proteína requiere largas extracciones de tomar días o semanas para llevar a cabo 5-7. Como resultado, los investigadores de investigación y médicos generalmente usan preparaciones comerciales caros, de COL1 que se preparan utilizando tejidos no humanos, tales como la rata, bovino, porcino o corion o usando eliminado a partir de cadáveres de la piel o después de la circuncisión masculina. Para aplicaciones regenerativas, muchos de estos productos exhiben variabilidad con respecto a su capacidad para formar sólidamatrices o construcciones de tejido capaces de apoyar la unión de células y el crecimiento. En consecuencia, existe una clara necesidad de un nuevo método estandarizado diseñado para extraer rápidamente COL1 partir de fuentes de tejido autólogo accesibles y abundantes. Tal método podría ahorrar tiempo y dinero en el marco de la investigación donde COL1 podría extraerse del modelo animal de interés y se utiliza para los ensayos preclínicos de la ingeniería de tejidos montados en andamios a base de colágeno. Al mismo tiempo, tener la opción de utilizar un-aislado rápidamente, COL1 derivada autóloga-en la clínica aumentaría la seguridad general para los pacientes que de otro modo recibir alogénicas o xenogénicas preparaciones. Para los pacientes que reciben una preparación autógeno, este método simplificado reduciría enormemente el intervalo entre la toma de biopsias y la aplicación de colágeno. Por estas razones, hemos tratado de mejorar en un aislamiento y purificación método COL1 de larga data con ag de alta velocidadbilitación y la centrifugación de exclusión por tamaño. Este método es fácil de realizar utilizando tampones estándar y los equipos que se encuentran en muchos laboratorios. Mediante el empleo de la agitación de alta velocidad, nuestro protocolo reduce el tiempo necesario para aislar solubles, dérmica COL1 de aproximadamente 10 días a menos de 3 horas 8. Es importante destacar que este método se puede realizar fácilmente en el entorno clínico a fin de preparar COL1 derivada autóloga para su uso en pacientes durante un único conjunto de procedimientos.

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	<tbody>
		<tr height="43">
			<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>
		</tr>
		<tr height="190">
			<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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	</tbody>
</table>

an improved method for the preparation of type i collagen from skin

Tipo soluble 1 de colágeno (COL1) se utiliza ampliamente como un sustrato adhesivo para cultivos de células y como un andamiaje celular para aplicaciones regenerativas. Clínicamente, esta proteína es ampliamente utilizado para la cirugía estética, inyecciones dérmicas, injerto de hueso, y la cirugía reconstructiva. Las fuentes de COL1 para estos procedimientos son comúnmente no humano, lo que aumenta el potencial para la inflamación y el rechazo, así como la transmisión de enfermedades de xenobióticos. En vista de esto, un método para purificar de manera eficiente y rápidamente COL1 a partir de cantidades limitadas de tejidos derivados autóloga-sería eludir muchos de estos problemas, sin embargo, los protocolos estándar de aislamiento son largos y requieren a menudo grandes cantidades de tejidos de colágeno. Aquí, se demuestra un método de extracción COL1 eficiente que reduce el tiempo necesario para aislar y purificar esta proteína de aproximadamente 10 días a menos de 3 horas. Elegimos la dermis como nuestra fuente de tejido debido a su disponibilidad durante muchos procedimientos quirúrgicos. Tsu método utiliza buffers de extracción tradicionales combinados con agitación enérgica y filtración centrífuga para obtener alta pureza, COL1 soluble de pequeñas cantidades de corion. En pocas palabras, las biopsias dérmicas se lavan a fondo en DH helada 2 O después de la eliminación de grasa, tejido conectivo, y el cabello. Las muestras de piel se eliminan de las proteínas y los polisacáridos no colágenas utilizando 0,5 M de acetato de sodio y un homogenizador de alta velocidad de sobremesa. El colágeno de sólidos residuales se extrae posteriormente con un tampón de citrato de sodio 0,075 M usando el homogeneizador. Estos extractos se purificaron usando 100.000 MW de corte filtros centrífugos que producen preparaciones COL1 de calidad comparable o superior a los productos comerciales o los obtenidos utilizando procedimientos tradicionales. Anticipamos este método facilitará la utilización de derivados de forma autóloga-COL1 para una multitud de aplicaciones de investigación y clínicos.

Este protocolo de aislamiento COL1 requiere alrededor de 2 horas y 15 minutos en completarse. La figura 1 muestra un diagrama esquemático delineando los principales pasos de este procedimiento. Preparación de la muestra y la realización de los 7 ciclos de agitaciones de alta velocidad y se enjuaga con acetato de sodio tarda aproximadamente 35 minutos (las figuras 1A-C). Realización de una agitación y enjuague ciclo con dH 2 O toma aproximadamente 5 minutos (figuras 1D y ...

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

Los procedimientos tradicionales para el aislamiento de tipo soluble 1 colágeno (COL1) requieren unos 10 días a partir de principio a fin, a causa de largas incubaciones de amortiguación y resuspensiones laboriosas de fibrillas. A continuación, describimos un medio para purificar COL1 de pequeñas biopsias dérmicas en menos de 3 horas.

Un método mejorado para la preparación de colágeno tipo I De Piel

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

an-improved-method-for-the-preparation-of-type-i-collagen-from-skin

Research

JoVE Journal

Bioengineering

22.7K vistas.  Boston Children's Hospital.  Los procedimientos tradicionales para el aislamiento de tipo soluble 1 colágeno (COL1) requieren unos 10 días a partir de principio a fin, a causa de largas incubaciones de amortiguación y resuspensiones laboriosas de fibrillas. A continuación, describimos un medio para purificar COL1 de pequeñas biopsias dérmicas en menos de 3 horas. 

Video: 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.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

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

Investigación

Bioingeniería

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.

Método mejorado para la preparación de una base de células, Contacto Modelo Humano de la barrera hematoencefálica

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.

Una mejorada mecánica Método de ensayo para evaluar hueso implante 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 ...

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.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

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.

Tejido picado en colágeno comprimido: Un Bio Transplant que contiene células para la reparación reconstructiva-puesta en escena individual

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.

Preparación de geles de colágeno en 3D y microcanales para el estudio de las interacciones 3D<em&gt; En 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.

Evaluación de la proliferación del Keratinocyte en dos y tres dimensiones substratos de colágeno de tipo 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.

Cuantificación simultánea de kynureninas seleccionadas analizadas por cromatografía líquida-espectrometría de masas en medio recogido de cultivos celulares oncológicos

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

Un nuevo método de inyección lenta mínimamente invasivo para reducir la lesión tisular

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 destacado: Un nuevo método de inyección de células con una invasión mínima  

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

Un nuevo método de inyección celular con una invasión mínima

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