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

Simplification des études d’angiogenèse avec un dosage à matrice modifiée

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.

Pleins feux sur l’auteur : Étude de l’angiogenèse et de la perméabilité des vaisseaux à l’aide d’un dosage du bouchon de gel matriciel modifié

Essai de bouchon de gel matriciel in vivo modifié pour les études d’angiogenèse

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.

Préparation d’un gel matriciel avec des cellules vasculaires pour un test in vivo de bouchon de gel matriciel

Pour commencer, préparez 10 fois le milieu de culture M199 en dissolvant la poudre M199 à 10 fois la concentration dans 90 millilitres d’eau désionisée, suivie de l’ajout de 10 millilitres de FBS. Filtrez la solution à travers un filtre de 0,22 micron. Le milieu filtré peut être conservé à quatre degrés Celsius jusqu’à deux mois.Pour préparer le gel matriciel avec des cellules vasculaires, utilisez environ 2 millions de cellules vasculaires. Refroidissez le tube stérile de 1,5 millilitre contenant la pastille de cellule correctement préparée dans un bain de glace. Décongelez complètement le gel matriciel dans un bain de glace sans le mélanger ni le vortexer.De même, refroidissez le milieu de culture préparé 10 fois M199 avec le réactif de test ou le médicament d’intérêt à quatre degrés Celsius. Ensuite, en maintenant un rapport volumique de 8,8 à 1 à 0,2, combinez le gel matriciel pré-refroidi avec le milieu pré-refroidi 10 fois M199 contenant 10 % FBS, et le réactif de test pré-refroidi pour produire le mélange de gel matriciel. Enfin, remettre en suspension les cellules pré-refroidies dans 400 microlitres du mélange de gel matriciel préparé en mélangeant doucement pour éviter la formation de bulles.Conservez le tube contenant les cellules dans le gel matriciel sur de la glace jusqu’à ce que les souris soient prêtes pour l’injection de gel matriciel.

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 ...

Recherche

Biologie

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Préparation de cellules vasculaires pour un test in vivo de bouchon de gel matriciel

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Injection d’un mélange de gel matriciel chez la souris pour le dosage du bouchon de gel matriciel

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

Dextran-FITC Injection et collecte de bouchons de gel matriciels de souris pour le dosage des bouchons de gel matriciels