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

Semplificazione degli studi di angiogenesi con un test Matrix Gel Plug

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.

Autore in primo piano: Studio dell'angiogenesi e della permeabilità dei vasi attraverso un saggio di tappo in gel a matrice modificata

Saggio modificato in vivo del gel plug della matrice per gli studi sull'angiogenesi

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.

Preparazione del gel matrice con cellule vascolari per il saggio in vivo del gel in gel

Per iniziare, preparare 10 volte il terreno di coltura M199 sciogliendo la polvere M199 a 10 volte la concentrazione in 90 millilitri di acqua deionizzata, seguita dall'aggiunta di 10 millilitri di FBS. Filtrare la soluzione attraverso un filtro da 0,22 micron. Il mezzo filtrato può essere conservato a quattro gradi Celsius per un massimo di due mesi.Per preparare il gel della matrice con cellule vascolari, utilizzare circa 2 milioni di cellule vascolari. Raffreddare la provetta sterile da 1,5 millilitri contenente il pellet cellulare opportunamente preparato in un bagno di ghiaccio. Scongelare completamente il gel della matrice in un bagno di ghiaccio senza mescolarlo o vorticarlo.Allo stesso modo, raffreddare il terreno di coltura preparato 10 volte M199 insieme al reagente di prova o al farmaco di interesse a quattro gradi Celsius. Successivamente, mantenendo un rapporto di volume di 8,8 a 1 a 0,2, combinare il gel matrice preraffreddato con il terreno M199 preraffreddato 10 volte contenente il 10% FBS e il reagente di prova preraffreddato per produrre la miscela di gel a matrice. Infine, risospendere le celle pre-raffreddate in 400 microlitri della miscela di gel matrice preparata mescolando delicatamente per evitare la formazione di bolle.Tenere la provetta contenente le cellule nel gel della matrice sul ghiaccio fino a quando i topi non sono pronti per l'iniezione del gel della matrice.

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 ...

Ricerca

Biologia

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Preparazione delle cellule vascolari per il saggio in vivo Matrix Gel Plug

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Iniezione di miscela di gel Matrix nel topo per il saggio Matrix Gel Plug

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

Iniezione di destrano-FITC e raccolta di spine in gel a matrice dal topo per il saggio a spina di gel a matrice