preparing vascular cells for in vivo matrix gel plug assay

Simplifying Angiogenesis Studies with a 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.

Author Spotlight: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

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.

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

Begin by preparing the complete endothelial culture medium using 460 milliliters of endothelial cell medium, and adding into it, 50 milliliters of FBS, five milliliters of penicillin streptomycin, and five milliliters of endothelial cell growth supplement. The prepared media can be stored at four degrees celsius for a month. Next, in a 100 millimeter tissue culture dish containing 0.1 million vascular cells at eight milliliters of the prepared complete endothelial culture medium and culture the cells at 37 degrees Celsius and 5%carbon dioxide until 70%co-fluency is achieved.Then, remove the culture medium from the dish and rinse the cells twice with PBS to eliminate unattached cells and debris. After removing the PBS, add three milliliters of 0.25%trypsin, containing 2.21 millimolar EDTA to the cells and incubate the dish at 37 degrees Celsius for one minute. Verify cell detachment under a light microscope at 40 times magnification.Neutralize the trypsin with seven milliliters of complete endothelial culture medium and gently rinse the cells off the culture dish. Centrifuge the cell suspension after collecting it in a 15 milliliter tube at 400G for 10 minutes. Re-suspend the cells in five milliliters of complete endothelial culture medium after removing the supernatant, then count the cells using a hemo-cytometer, and transfer a calculated volume of suspension containing 2 million cells to a sterile 1.5 milliliter tube.Pellet the cells by centrifuging the suspension at 400G for five minutes before removing the supernatant. The vascular cells are now ready for mixing with the matrix gel for matrix gel plug assay.

Research

JoVE Journal

Biology

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

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

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