preparing 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.

Vasculaire cellen voorbereiden voor in vivo matrix gel plug-assay

Begin met het bereiden van het volledige endotheelkweekmedium met behulp van 460 milliliter endotheelcelmedium en voeg er 50 milliliter FBS, vijf milliliter penicilline-streptomycine en vijf milliliter endotheelcelgroeisupplement aan toe. De voorbereide media kunnen een maand lang bij vier graden Celsius worden bewaard. Vervolgens, in een weefselkweekschaal van 100 millimeter met 0,1 miljoen vasculaire cellen bij acht milliliter van het bereide complete endotheelkweekmedium en kweek de cellen bij 37 graden Celsius en 5% koolstofdioxide totdat 70% co-fluency is bereikt.Verwijder vervolgens het kweekmedium uit de schaal en spoel de cellen twee keer met PBS om losse cellen en vuil te verwijderen. Voeg na het verwijderen van de PBS drie milliliter 0,25% trypsine, met 2,21 millimolaire EDTA toe aan de cellen en incubeer de schaal gedurende één minuut bij 37 graden Celsius. Controleer de celloslating onder een lichtmicroscoop met een vergroting van 40 keer.Neutraliseer de trypsine met zeven milliliter compleet endotheelkweekmedium en spoel de cellen voorzichtig van het kweekschaaltje. Centrifugeer de celsuspensie nadat deze gedurende 10 minuten in een buis van 15 milliliter bij 400 G is opgevangen. Breng de cellen opnieuw in vijf milliliter compleet endotheelkweekmedium na het verwijderen van het supernatant, tel vervolgens de cellen met behulp van een hemocytometer en breng een berekend volume suspensie met 2 miljoen cellen over naar een steriele buis van 1,5 milliliter.Pelleteer de cellen door de suspensie gedurende vijf minuten op 400G te centrifugeren voordat u het supernatant verwijdert. De vasculaire cellen zijn nu klaar om te mengen met de matrixgel voor matrixgelplug-assay.

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 ...

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

Matrixgel voorbereiden met vasculaire cellen voor in vivo matrixgelplugtest

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