preparing vascular cells for in vivo matrix gel plug assay

Forenkling af angiogeneseundersøgelser med et modificeret matrix gelstik-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.

Forfatter Spotlight: Undersøgelse af angiogenese og karpermeabilitet gennem et modificeret matrix gelstik assay

Modificeret In vivo Matrix Gel Plug Assay til angiogenese undersøgelser

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.

Forberedelse af vaskulære celler til in vivo matrix gel plug assay

Begynd med at forberede det komplette endotelkulturmedium ved hjælp af 460 ml endotelcellemedium og tilsæt 50 ml FBS, fem milliliter penicillin streptomycin og fem ml endotelcellevæksttilskud. Det forberedte medie kan opbevares ved fire grader celsius i en måned. Dernæst i en 100 millimeter vævskulturskål indeholdende 0,1 millioner vaskulære celler ved otte ml af det fremstillede komplette endotelkulturmedium og kultur cellerne ved 37 grader Celsius og 5% kuldioxid, indtil 70% co-fluency opnås.Fjern derefter dyrkningsmediet fra skålen og skyl cellerne to gange med PBS for at fjerne ikke-fastgjorte celler og snavs. Efter fjernelse af PBS tilsættes tre ml 0,25% trypsin, indeholdende 2,21 millimolær EDTA til cellerne og inkuberer skålen ved 37 grader Celsius i et minut. Kontroller cellefrigørelse under et lysmikroskop ved 40 gange forstørrelse.Neutraliser trypsinet med syv ml komplet endotelkulturmedium og skyl forsigtigt cellerne af kulturskålen. Centrifuger cellesuspensionen efter opsamling i et 15 ml rør ved 400 G i 10 minutter. Cellerne suspenderes igen i fem ml komplet endoteldyrkningsmedium efter fjernelse af supernatanten, derefter tælles cellerne ved hjælp af et hæmocytometer, og et beregnet suspensionsvolumen indeholdende 2 millioner celler overføres til et sterilt 1,5 ml rør.Pellets cellerne ved centrifugering af suspensionen ved 400 G i fem minutter, før supernatanten fjernes. De vaskulære celler er nu klar til blanding med matrixgelen til matrixgelstikassay.

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

Forberedelse af matrixgel med vaskulære celler til in vivo matrixgelpropanalyse

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Matrix gelblanding injektion i mus til matrix gel plug assay

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

Dextran-FITC injektion og matrix gel plug samling fra mus til matrix gel plug assay