preparing vascular cells for in vivo matrix gel plug assay

使用改良基质凝胶塞测定简化血管生成研究

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.

作者聚焦：通过改良基质凝胶塞测定研究血管生成和血管通透性

用于血管生成研究 的改良体内 基质凝胶塞测定

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.

制备用于体内基质凝胶塞测定的血管细胞

首先使用460毫升内皮细胞培养基制备完整的内皮培养基，并在其中加入50毫升FBS，5毫升青霉素链霉素和5毫升内皮细胞生长补充剂。制备好的培养基可以在4摄氏度下储存一个月。接下来，在含有 010 万个血管细胞的 100 毫米组织培养皿中，用 8 毫升制备的完全内皮培养基，并在 37 摄氏度和 5% 二氧化碳下培养细胞，直到达到 70% 的共流畅度。然后，从培养皿中取出培养基并用PBS冲洗细胞两次，以消除未附着的细胞和碎屑。去除PBS后，向细胞中加入3毫升含有2.21毫摩尔EDTA的0.25%胰蛋白酶，并在37摄氏度下孵育培养皿一分钟。在40倍放大倍率的光学显微镜下验证细胞分离。用 7 毫升完全内皮培养基中和胰蛋白酶，然后轻轻冲洗培养皿中的细胞。将细胞悬液收集在15毫升管中以400G离心10分钟后。除去上清液后，将细胞重新悬浮在5毫升完全内皮培养基中，然后使用血细胞仪对细胞进行计数，并将含有200万个细胞的悬浮液的计算体积转移到无菌的1.5毫升管中。在除去上清液之前，通过将悬浮液以400G离心5分钟来沉淀细胞。血管细胞现在已准备好与基质凝胶混合，用于基质凝胶塞测定。

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 ...

科研

生物学

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

制备带有血管细胞的基质凝胶用于体内基质凝胶塞测定

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

将基质凝胶混合物注射到小鼠中进行基质凝胶塞测定

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

来自小鼠的葡聚糖-FITC 注射液和基质凝胶塞集合，用于基质凝胶塞测定