preparing vascular cells for in vivo matrix gel plug assay

Simplifying angiogenesis studies with a modified matrix gel plug assay(수정된 매트릭스 겔 플러그 어세이를 통한 혈관신생 연구 간소화)

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.

저자 스포트라이트: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay (수정된 매트릭스 겔 플러그 분석을 통한 혈관신생 및 혈관 투과성 조사)

혈관신생 연구를 위한 수정된 In Vivo Matrix Gel Plug Assay

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 Assay를 위한 혈관 세포 준비

먼저 460ml의 내피 세포 배지를 사용하여 완전한 내피 배양 배지를 준비하고 FBS 50ml, 페니실린 스트렙토마이신 5ml, 내피 세포 성장 보충제 5ml를 첨가합니다. 준비된 미디어는 섭씨 4도에서 한 달 동안 보관할 수 있습니다. 다음으로, 제조된 완전한 내피 배양 배지 8ml에서 0.1백만 개의 혈관 세포를 포함하는 100mm 조직 배양 접시에서 세포를 섭씨 37도, 5% 이산화탄소에서 70%의 공동 유창성이 달성될 때까지 배양합니다.그런 다음 접시에서 배양 배지를 제거하고 PBS로 세포를 두 번 헹구어 부착되지 않은 세포와 파편을 제거합니다. PBS를 제거한 후 2.21 밀리몰 EDTA를 함유 한 0.25 % 트립신 3 밀리리터를 세포에 넣고 섭씨 37도에서 1 분 동안 접시를 배양합니다. 광학 현미경으로 40배 배율로 세포 분리를 확인합니다.7mL의 완전한 내피 배양 배지로 트립신을 중화하고 배양 접시에서 세포를 부드럽게 헹굽니다. 셀 현탁액을 15G에서 400밀리리터 튜브에 수집한 후 10분 동안 원심분리합니다. 상층액을 제거한 후 5mL의 완전한 내피 배양 배지에 세포를 다시 현탁시킨 다음 혈구측정기를 사용하여 세포를 계수하고 200만 개의 세포를 포함하는 계산된 현탁액 부피를 멸균된 1.5mL 튜브로 옮깁니다.상층액을 제거하기 전에 400G에서 5분 동안 현탁액을 원심분리하여 세포를 펠렛화합니다. 혈관 세포는 이제 매트릭스 겔 플러그 분석을 위해 매트릭스 겔과 혼합할 준비가 되었습니다.

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 ...

연구

생물학

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

In Vivo Matrix Gel Plug Assay를 위한 혈관 세포가 있는 Matrix Gel 준비

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Matrix Gel Plug Assay를 위해 마우스에 Matrix Gel Mixture 주입

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

Dextran-FITC 주입 및 매트릭스 겔 플러그 수집(Matrix Gel Plug Assay)을 위한 마우스의 Matrix Gel Plug Collection