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

Angiogenesestudies vereenvoudigen met een Modified 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.

Auteur in de schijnwerpers: onderzoek naar angiogenese en vatpermeabiliteit door middel van een gemodificeerde matrix-gelplugtest

Gemodificeerde In Vivo Matrix Gel Plug Assay voor Angiogenesestudies

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.

Matrixgel voorbereiden met vasculaire cellen voor in vivo matrixgelplugtest

Bereid om te beginnen 10 keer M199-kweekmedium voor door M199-poeder op te lossen tot 10 keer de concentratie in 90 milliliter gedeïoniseerd water, gevolgd door de toevoeging van 10 milliliter FBS. Filtreer de oplossing door een filter van 0,22 micron. Het gefilterde medium kan maximaal twee maanden bij vier graden Celsius worden bewaard.Gebruik voor het bereiden van de matrixgel met vasculaire cellen ongeveer 2 miljoen vasculaire cellen. Koel de steriele buis van 1,5 milliliter met de op de juiste manier bereide celpellet in een ijsbad. Ontdooi de matrixgel volledig in een ijsbad zonder het te mengen of te vortexen.Koel op dezelfde manier de bereide 10 keer M199-kweekmedia samen met het testreagens of het medicijn van belang tot vier graden Celsius. Combineer vervolgens, met een volumeverhouding van 8,8 tot 1 tot 0,2, de voorgekoelde matrixgel met de voorgekoelde 10-voudige M199-media die 10% FBS bevatten, en het voorgekoelde testreagens om het matrixgelmengsel te produceren. Breng ten slotte de voorgekoelde cellen opnieuw in 400 microliter van het bereide matrixgelmengsel door voorzichtig te mengen om belletjes te voorkomen.Houd het buisje met de cellen in matrixgel op ijs totdat de muizen klaar zijn voor de matrixgelinjectie.

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 ...

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Vasculaire cellen voorbereiden voor in vivo matrix gel plug-assay

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Matrix Gel Mengsel Injectie in Muis voor Matrix Gel Plug Assay

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

Dextran-FITC-injectie en Matrix Gel Plug Collectie van Mouse voor Matrix Gel Plug Assay