preparing 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

Our research deals with angiogenesis, the process of formation of new vessels from preexisting vessels. It plays a critical role in many physiological and pathological processes, such as embryonic development or curing atherosclerosis, tumor development, etc. The construction of a reliable model to evaluate the extent of angiogenesis is critical for myocardial research or the discovery of medicines for angiogenesis-depend diseases.However, most of the existing models are complex, expensive, and require specialized equipment. Here, we present a modified matrix gel plug assay that can be used to investigate angiogenesis and vessel permeability without staining in a simple, more intuitive, as well as efficient manner.

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

Comience preparando el medio de cultivo endotelial completo utilizando 460 mililitros de medio de células endoteliales y agréguele 50 mililitros de FBS, cinco mililitros de penicilina estreptomicina y cinco mililitros de suplemento de crecimiento de células endoteliales. Los medios preparados se pueden almacenar a cuatro grados centígrados durante un mes. A continuación, en una placa de cultivo de tejidos de 100 milímetros que contiene 0,1 millones de células vasculares a ocho mililitros del medio de cultivo endotelial completo preparado y se cultivan las células a 37 grados centígrados y 5% de dióxido de carbono hasta alcanzar una cofluidez del 70%.Luego, retire el medio de cultivo de la placa y enjuague las células dos veces con PBS para eliminar las células sueltas y los desechos. Después de retirar el PBS, agregue tres mililitros de tripsina al 0,25%, que contiene 2,21 milimolares de EDTA a las células e incube la placa a 37 grados centígrados durante un minuto. Verifique el desprendimiento de células bajo un microscopio óptico con un aumento de 40 veces.Neutralice la tripsina con siete mililitros de medio de cultivo endotelial completo y enjuague suavemente las células de la placa de cultivo. Centrifugar la suspensión celular después de recogerla en un tubo de 15 mililitros a 400 g durante 10 minutos. Vuelva a suspender las células en cinco mililitros de medio de cultivo endotelial completo después de eliminar el sobrenadante, luego cuente las células con un hemocitómetro y transfiera un volumen calculado de suspensión que contenga 2 millones de células a un tubo estéril de 1,5 mililitros.Granular las células centrifugando la suspensión a 400 G durante cinco minutos antes de eliminar el sobrenadante. Las células vasculares ahora están listas para mezclarse con el gel de matriz para el ensayo de tapón de gel de matriz.

Research

JoVE Journal

Biology

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

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 ...

Investigación

Biología

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

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

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Inyección de mezcla de gel de matriz en ratón para ensayo de tapón de gel de matriz

Dextran-FITC Injection and Matrix Gel Plug Collection from Mouse for Matrix Gel Plug Assay

Inyección de Dextran-FITC y colección de tapones de gel matriciales del ratón para el ensayo de tapones de gel matriciales