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

Commencez par préparer le milieu de culture endothélial complet en utilisant 460 millilitres de milieu de cellules endothéliales, et ajoutez-y 50 millilitres de FBS, cinq millilitres de pénicilline streptomycine et cinq millilitres de supplément de croissance des cellules endothéliales. Les milieux préparés peuvent être conservés à quatre degrés Celsius pendant un mois. Ensuite, dans une boîte de culture tissulaire de 100 millimètres contenant 0,1 million de cellules vasculaires à huit millilitres du milieu de culture endothélial complet préparé, cultivez les cellules à 37 degrés Celsius et 5 % de dioxyde de carbone jusqu’à ce que la co-fluidité de 70 % soit atteinte.Ensuite, retirez le milieu de culture de la boîte et rincez les cellules deux fois avec du PBS pour éliminer les cellules non attachées et les débris. Après avoir retiré le PBS, ajoutez trois millilitres de trypsine à 0,25 %, contenant 2,21 millimolaires d’EDTA dans les cellules et incubez la boîte à 37 degrés Celsius pendant une minute. Vérifiez le décollement des cellules au microscope optique à un grossissement de 40 fois.Neutralisez la trypsine avec sept millilitres de milieu de culture endothélial complet et rincez doucement les cellules de la boîte de culture. Centrifugez la suspension cellulaire après l’avoir recueillie dans un tube de 15 millilitres à 400G pendant 10 minutes. Remettre les cellules en suspension dans cinq millilitres de milieu de culture endothélial complet après avoir retiré le surnageant, puis compter les cellules à l’aide d’un hémocytomètre et transférer un volume calculé de suspension contenant 2 millions de cellules dans un tube stérile de 1,5 millilitre.Granulez les cellules en centrifugeant la suspension à 400G pendant cinq minutes avant de retirer le surnageant. Les cellules vasculaires sont maintenant prêtes à être mélangées avec le gel matriciel pour le dosage du bouchon de gel matriciel.

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 ...

Recherche

Biologie

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

Préparation d’un gel matriciel avec des 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