preparing matrix gel with vascular cells for in vivo matrix gel plug assay

Simplificación de los estudios de angiogénesis con un ensayo de tapón de gel de matriz modificada

Angiogenesis, the process of formation of neo-vessels from pre-existing vessels, plays a critical role in many physiological and pathological processes, such as embryonic development, wound healing, atherosclerosis, tumor development, etc.1,2,3,4,5. This dynamic process involves several steps, including the degradation of the matrix, vascular cell proliferation, migration and self-organization to form tubular structures and the stabilization of the neo-vessels6. Promoting angiogenesis has been demonstrated to be critical in the treatment of myocardial infarction, stroke and other kinds of ischemic diseases7 while inhibiting angiogenesis has been considered a promising strategy in the treatment of cancers8 and rheumatoid diseases9. Angiogenesis has been considered an organizing principle for drug discovery10. Thus, the construction of a reliable and convenient method to assess the extent of angiogenesis is critical for mechanical research or drug discovery in angiogenesis-dependent diseases.
Several in vitro and in vivo models have been developed to evaluate angiogenesis11. Among these, two-dimensional (2-D) models, like matrix gel tube formation assay12, cannot form functional tubular structures. The animal models, such as the hind limb ischemia model13,14, can reproduce the angiogenesis process but are complex and require a laser speckle blood flow imaging system. 3D models of vascular morphogenesis, like matrix gel plug assay, provide a simple platform that can mimic the process of angiogenesis in vivo15, but the detection of angiogenesis requires immunohistochemistry or immunofluorescence staining16,17,18, which are variable and poorly visualized.
Here, we describe a protocol for a modified matrix gel plug assay where vascular cells were mixed with matrix gel and subcutaneously injected into the back of mice to form a plug. In the plug, vascular cells need to degrade the matrix, proliferate, migrate, and self-organize to finally form functional vessels with blood flow in the internal environment. Thereafter, fluorescent-labeled dextran is injected via the tail vein, to flow through the plug, and the label is visualized to indicate neo-vessels. The content of angiogenesis can be quantitatively evaluated by the length of the vessels. This method can form functional vessels that cannot be produced in 2-D angiogenesis models12, and does not need complex stain process as in ordinary matrix gel plug assay11. It also does not require expensive specific instruments like laser speckle blood flow imaging system in hind limb ischemia model13,14,19. This method is versatile, low-cost, quantifiable, and easy to perform, and can be used to determine the pro- or anti-angiogenic capability of drugs or be used in mechanical research involved in angiogenesis.

Autor destacado: Investigación de la angiogénesis y la permeabilidad de los vasos a través de un ensayo de tapón de gel de matriz modificada

Ensayo de tapón de gel de matriz in vivo modificado para estudios de angiogénesis

Preparación de gel de matriz con células vasculares para el ensayo de tapón de gel de matriz in vivo

Para comenzar, prepare 10 veces el medio de cultivo M199 disolviendo el polvo de M199 a una concentración 10 veces mayor en 90 mililitros de agua desionizada, seguido de la adición de 10 mililitros de FBS. Filtre la solución a través de un filtro de 0,22 micras. El medio filtrado se puede almacenar a cuatro grados centígrados durante un máximo de dos meses.Para preparar el gel de matriz con células vasculares, utilice alrededor de 2 millones de células vasculares. Enfríe el tubo estéril de 1,5 mililitros que contiene el gránulo celular debidamente preparado en un baño de hielo. Descongele el gel de matriz completamente en un baño de hielo sin mezclarlo ni vórtice.Del mismo modo, enfríe el medio de cultivo preparado 10 veces M199 junto con el reactivo de prueba o el fármaco de interés a cuatro grados centígrados. A continuación, manteniendo una relación de volumen de 8,8 a 1 a 0,2, combine el gel de matriz preenfriado con el medio M199 preenfriado 10 veces que contiene 10% de FBS y el reactivo de prueba preenfriado para producir la mezcla de gel de matriz. Finalmente, vuelva a suspender las celdas preenfriadas en 400 microlitros de la mezcla de gel de matriz preparada mezclando suavemente para evitar la formación de burbujas.Mantenga el tubo que contiene las células en gel de matriz en hielo hasta que los ratones estén listos para la inyección de gel de matriz.

preparing-matrix-gel-with-vascular-cells-for-vivo-matrix-gel-plug

Research

JoVE Journal

Biology

Preparing Matrix Gel with Vascular Cells for In Vivo Matrix Gel Plug Assay

66 vistas. Para comenzar, prepare 10 veces el medio de cultivo M199 disolviendo el polvo de M199 a una concentración 10 veces mayor en 90 mililitros de agua desionizada, seguido de la adición de 10 mililitros de FBS. Filtre la solución a través de un filtro de 0,22 micras. El medio filtrado se puede almacenar a cuatro grados centígrados durante un máximo de dos meses.Para preparar el gel de matriz con células vasculares, utilice alrededor de 2 millones de células vasculares. Enfríe el tu...

Video: Preparación de gel de matriz con células vasculares para el ensayo de tapón de gel de matriz in vivo

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...