Angiogenesis is closely associated with wound healing, inflammation, rheumatoid arthritis, diabetic retinopathy, macular degeneration, tumor growth, and other conditions. During the growth process of organisms, angiogenesis is an important component of new tissue formation. In adulthood, apart from periodic events tightly regulated in specific organs, almost every normal tissue lacks significant physiological angiogenesis due to the balance between pro-angiogenic and anti-angiogenic endogenous factors. When the balance promoting angiogenesis is disrupted, endothelial cells (ECs) within microvessels transform into angiogenic phenotypes, initiating angiogenic reactions that can be either eliminated or accelerated.

Traditional in vivo angiogenesis experimental methods include corneal micropocket assay, mesentery assay, sponge/matrix implantation, disc analysis (DAS), zebrafish, etc. However, these experiments are not only technically demanding, expensive, and time-consuming, but also involve ethical issues. Compared to in vivo angiogenesis experiments, maintaining cell-generated vessels using specific matrices in vitro is a more economical and practical experimental approach. Among these, the most commonly used matrix for supporting cell growth is the matrix gel extracted from EHS mouse tumor, known as matrix gel.

The main components of matrix gel include laminin, type IV collagen, heparan sulfate proteoglycan (HSPG), fibronectin, and other growth factors such as TGF-beta, EGF, IGF, FGF, tissue plasminogen activator, and other growth factors inherent in EHS tumors. It provides support, tensile strength, and scaffold support for tissues and cells, serves as a three-dimensional substructure for cell adhesion and movement, and acts as a repository for growth factors, chemokines, and cytokines. Additionally, it serves as a signal for cell morphology and differentiation. Arcegel Matrix Gel developed and produced by Arcegen Biotechnology does not contain LDEV (lactate dehydrogenase elevating virus), has an ultra-low endotoxin content, and has been tested for mycoplasma to ensure no mycoplasma contamination, including different types of matrix gels such as basic concentration, high concentration, low growth factor, etc. 

 

Application directions

 

 

Product Features

High Safety: Free from LDEV (Lactate Dehydrogenase Elevating Virus)

Diverse Concentrations: Concentrations range from 8 to 20 mg/mL

Excellent Batch Stability: Strict production quality inspection process ensures stable performance between batches

Low Endotoxin: Endotoxin content ≤ 4 EU/mL

Contaminant Detection: Tested free of mycoplasma, bacteria, and fungal residues

High Batch Yield: Batch yield is above the L-grade level

Good Compatibility: Compatible with any type of cell culture medium

 

Angiogenesis Experiment

Preparation of Matrix Gel

1. The day before the experiment, remove Arcegel Matrix Gel from the freezer and thaw overnight in a 4°C refrigerator, while pre-cooling the consumables to be used.

2. Keep the Arcegel Matrix Gel constantly in the icebox before the experiment.

3. Open the packaging of the angiogenesis carrier slides, and remove the slides.

4. Add 10 μl of Arcegel Matrix Gel to each well, ensuring that the gel is added vertically above the inner hole to prevent residual gel from flowing through the upper hole.

Gelation

1. Cover the carrier slide with its lid, prepare a 10 cm culture dish, and place a wet paper towel inside to create a humid chamber.

2. Place the carrier slide in the culture dish and cover it with the lid of the dish.

3. Place the entire culture dish in a CO2 incubator, let it stand for about 30 minutes for gelation, while preparing the cell suspension.

Cell Seeding

1. Prepare the digested cells into a suspension with a density of 2*10⁵ cells/ml, and mix well.

2. Remove the carrier slides with the gel that has solidified.

3. Add 50 μl of the cell suspension to each well, ensuring that the pipette tip remains vertical above the upper hole without touching the gel in the lower hole.

4. Add cell culture medium, cover with a lid, let it stand, and after some time, all cells will settle and adhere to the surface of the matrix gel.

Image Acquisition

Observe and photograph at regular intervals according to the cell growth rate, and retain the images.

Immunofluorescence Staining (Optional)

1. Carefully remove the culture medium from the wells, without touching the gel or cell network. Dilute calcein AM in serum-free medium to a final concentration of 6~8 µg/ml.

2. Add the cell staining solution to completely immerse the cells, and incubate at room temperature in the dark for 30~40 minutes.

3. Wash three times with PBS, slowly adding PBS to the upper hole to avoid dislodging the cells.

4. Use wavelengths of Ex=485 nm and Em=529 nm for fluorescence observation.

 

Result Analysis

Measure and record the tube length, coverage area, number of rings, and number of nodes, and perform statistical analysis.

 

Figure: Angiogenesis Results

 

Figure: Immunofluorescence Staining of Blood Vessels

 

FAQ

1. What is the reason for the color change (from pale yellow to deep red) observed in the matrix received?

 For matrices containing phenol red, the main reason for the color change is due to the interaction between phenol red and bicarbonate with CO2, but the color difference will decrease upon equilibrium with 5% CO₂. After thawing, gently shake the reagent bottle to evenly disperse the matrix gel.

2. What precautions should be taken when handling matrix gel?

  All operations should be carried out under sterile conditions, and pre-cooled pipettors should be used to ensure that the matrix gel is uniformly dispersed.

3. How should matrix gel be aliquoted, frozen, and used?

  After thawing, Arcegel matrix gel can be aliquoted into multiple small tubes using pre-cooled cryovials. All aliquoting should be done using pre-cooled cryovials, rapidly frozen, and stored to avoid repeated freeze-thaw cycles. All items involved in use should be pre-cooled, including pre-cooled pipettes, tips, and tubes for handling the matrix gel. 

 

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