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

Vereinfachung von Angiogenese-Studien mit einem modifizierten Matrix-Gel-Plug-Assay

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 im Rampenlicht: Untersuchung der Angiogenese und Gefäßpermeabilität durch einen modifizierten Matrix-Gel-Plug-Assay

Modifizierter In-vivo-Matrix-Gel-Plug-Assay für Angiogenese-Studien

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.

Vorbereitung von Matrix-Gel mit Gefäßzellen für den In-vivo-Matrix-Gel-Plug-Assay

Bereiten Sie zunächst das 10-fache M199-Kulturmedium vor, indem Sie M199-Pulver in 90 Millilitern deionisiertem Wasser auf die 10-fache Konzentration auflösen, gefolgt von der Zugabe von 10 Millilitern FBS. Filtern Sie die Lösung durch einen 0,22-Mikron-Filter. Das gefilterte Medium kann bis zu zwei Monate bei vier Grad Celsius gelagert werden.Verwenden Sie für die Herstellung des Matrixgels mit Gefäßzellen etwa 2 Millionen Gefäßzellen. Kühlen Sie das sterile 1,5-Milliliter-Röhrchen mit dem entsprechend vorbereiteten Zellpellet in einem Eisbad ab. Tauen Sie das Matrix-Gel in einem Eisbad vollständig auf, ohne es zu mischen oder zu wirbeln.Kühlen Sie in ähnlicher Weise das vorbereitete 10-fache M199-Nährmedium zusammen mit dem Testreagenz oder dem Arzneimittel von Interesse auf vier Grad Celsius ab. Kombinieren Sie als nächstes unter Beibehaltung eines Volumenverhältnisses von 8,8 zu 1 zu 0,2 das vorgekühlte Matrixgel mit dem vorgekühlten 10-fachen M199-Medium mit 10 % FBS und dem vorgekühlten Testreagenz, um die Matrixgelmischung herzustellen. Zum Schluss werden die vorgekühlten Zellen in 400 Mikrolitern der vorbereiteten Matrixgelmischung durch vorsichtiges Mischen wieder suspendiert, um Blasenbildung zu vermeiden.Halten Sie das Röhrchen mit den Zellen im Matrixgel auf Eis, bis die Mäuse für die Matrixgel-Injektion bereit sind.

Research

JoVE Journal

Biology

Preparing Matrix Gel with 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 ...

Forschung

Biologie

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Vorbereitung von Gefäßzellen für den In-vivo-Matrix-Gel-Plug-Assay

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Injektion einer Matrix-Gel-Mischung in die Maus für den Matrix-Gel-Plug-Assay

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

Dextran-FITC-Injektion und Matrix-Gel-Plug-Entnahme aus der Maus für den Matrix-Gel-Plug-Assay