preparing vascular cells for in vivo matrix gel plug assay

Modified Matrix Gel Plug Assayによる血管新生研究の簡素化

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.

著者スポットライト:Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

血管新生研究のための改変 In Vivo マトリックスゲルプラグアッセイ

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.

In Vivo Matrix Gel Plug Assay 用の血管細胞の調製

まず、460ミリリットルの内皮細胞培地を使用して完全な内皮培養培地を調製し、そこに50ミリリットルのFBS、5ミリリットルのペニシリンストレプトマイシン、および5ミリリットルの内皮細胞増殖サプリメントを加えます。調製した培地は、摂氏4度で1ヶ月間保存できます。次に、10万個の血管細胞を含む100ミリメートルの組織培養皿に、調製した完全内皮培養液8ミリリットルで、70%のコフルエントが得られるまで、細胞を摂氏37度、二酸化炭素5%で培養した。次に、培養液を皿から取り出し、PBSで細胞を2回すすぎ、付着していない細胞や破片を取り除きます。PBSを除去した後、2.21ミリモルのEDTAを含む0.25%トリプシン3ミリリットルを細胞に加え、37°Cで1分間インキュベートします。40倍倍の倍率の光学顕微鏡で細胞の剥離を確認します。7ミリリットルの完全内皮培養培地でトリプシンを中和し、培養皿から細胞を静かに洗い流します。細胞懸濁液を15ミリリットルのチューブに400gで10分間回収した後、遠心分離します。上清を除去した後、5ミリリットルの完全内皮培養液に細胞を再懸濁し、ヘモサイトメーターを使用して細胞をカウントし、200万個の細胞を含む計算された量の懸濁液を1.5ミリリットルの滅菌チューブに移します。懸濁液を400Gで5分間遠心分離して細胞をペレット化してから、上清を除去します。これで、血管細胞をマトリックスゲルと混合してマトリックスゲルプラグアッセイを行う準備が整いました。

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 ...

生物学

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

In Vivo Matrix Gel Plug Assay のための血管細胞を用いたマトリックスゲルの調製(英語)

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

マトリックスゲルプラグアッセイのためのマウスへのマトリックスゲル混合物注入

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

マウスからのデキストラン-FITC注入およびマトリックスゲルプラグ採取によるマトリックスゲルプラグアッセイ