preparing matrix gel with 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.

制备带有血管细胞的基质凝胶用于体内基质凝胶塞测定

首先，通过将 M199 粉末溶解至 10 倍浓度在 90 毫升去离子水中，然后加入 10 毫升 FBS，制备 10 倍 M199 培养基。通过0.22微米过滤器过滤溶液。过滤后的介质可在4摄氏度下储存长达两个月。为了制备含有血管细胞的基质凝胶，使用约200万个血管细胞。在冰浴中冷却含有适当制备的细胞沉淀的无菌1.5毫升管。在冰浴中完全解冻基质凝胶，不要混合或涡旋。同样，将制备的 10 倍 M199 培养基与感兴趣的测试试剂或药物一起冷却至 4 摄氏度。接下来，保持8.8比1比0.2的体积比，将预冷的基质凝胶与含有10%FBS的预冷10倍M199培养基和预冷的测试试剂混合，生产基质凝胶混合物。最后，通过轻轻混合将预冷的细胞重新悬浮在400微升制备的基质凝胶混合物中，以避免形成气泡。将含有基质凝胶中的细胞的试管放在冰上，直到小鼠准备好进行基质凝胶注射。

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 ...

科研

生物学

Preparing Vascular Cells for In Vivo Matrix Gel Plug Assay

制备用于体内基质凝胶塞测定的血管细胞

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