preparing vascular cells for in vivo matrix gel plug assay

Simplificando os estudos de angiogênese com um ensaio de tampão de gel de matriz modificada

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.

Autor em destaque: Investigando a angiogênese e a permeabilidade dos vasos por meio de um ensaio de tampão de gel de matriz modificada

Ensaio de plugue de gel de matriz in vivo modificado para estudos de angiogênese

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.

Preparando células vasculares para ensaio de plugue de gel de matriz in vivo

Comece preparando o meio de cultura endotelial completo usando 460 mililitros de meio de célula endotelial e adicionando a ele, 50 mililitros de FBS, cinco mililitros de penicilina estreptomicina e cinco mililitros de suplemento de crescimento de células endoteliais. O meio preparado pode ser armazenado a quatro graus Celsius por um mês. Em seguida, em uma placa de cultura de tecidos de 100 milímetros contendo 0,1 milhão de células vasculares a oito mililitros do meio de cultura endotelial completo preparado e cultura das células a 37 graus Celsius e 5% de dióxido de carbono até que 70% de cofluência seja alcançada.Em seguida, retire o meio de cultura da placa e lave as células duas vezes com PBS para eliminar células soltas e detritos. Após a remoção do PBS, adicionar três mililitros de tripsina a 0,25%, contendo 2,21 milimolares de EDTA às células e incubar a placa a 37 graus Celsius por um minuto. Verifique o descolamento celular em microscópio de luz com aumento de 40 vezes.Neutralizar a tripsina com sete mililitros de meio de cultura endotelial completo e enxaguar suavemente as células da placa de cultura. Centrifugar a suspensão celular após coletá-la em um tubo de 15 mililitros a 400G por 10 minutos. Ressuspender as células em cinco mililitros de meio de cultura endotelial completo após a remoção do sobrenadante, contar as células usando um hemocitômetro e transferir um volume calculado de suspensão contendo 2 milhões de células para um tubo estéril de 1,5 mililitro.Pastilhar as células centrifugando a suspensão a 400G por cinco minutos antes de remover o sobrenadante. As células vasculares estão agora prontas para a mistura com o gel de matriz para o ensaio do plugue de gel de matriz.

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 ...

Biologia

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

Preparando Matrix Gel com Células Vasculares para Ensaio In Vivo Matrix Gel Plug

Matrix Gel Mixture Injection into Mouse for Matrix Gel Plug Assay

Injeção de mistura de gel de matriz em mouse para ensaio de plugue de gel de matriz

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

Injeção Dextran-FITC e Coleção de plugue de gel de matriz do mouse para ensaio de plugue de gel de matriz