Angiogenesis experiment
["Collaborating Experts | M.S. Jia Zhang", "Biology University of Three Gorges"], ["Reviewed by | Dr. Yifeng Nie", "Nano-biomedicine University of Chinese Academy of Sciences"]
Summary
In the process of tumor development, tumor tissues are often in a state of hypoxia and low nutrition. Under such conditions, due to the activation of angiogenic factors secreted by cancer cells, a large number of new blood vessels will be promoted to provide the oxygen and nutrients required for tumor growth.
Principle
Because tumor cells themselves can secrete a variety of growth factors to induce angiogenesis, so whether primary or secondary tumors are accompanied by angiogenesis, angiogenesis refers to the development of new blood vessels from existing capillaries or post-capillary venous development, vascular endothelial cells (HUVEC) in the Matrigel retained the endothelial cell proliferation and division of fast, fast migration rate HUVEC retain the characteristics of fast proliferation, division and migration of endothelial cells on Matrigel, therefore, the in vitro angiogenesis assay of endothelial cells can well simulate the process of tumor angiogenesis.
Appliance
In the study of tumor progression, abnormal blood vessel formation is considered a key factor in promoting tumor development. It can be used to study the effect of certain genes or drugs on this process, the revelation of the regulatory mechanism of angiogenesis, the development of vascular inhibitory drugs, and the screening of natural anti-angiogenic compounds.
Operation method
In vitro tube formation assay of human umbilical vein vascular endothelial cells (HUVEC)
Principle
Because tumor cells themselves can secrete a variety of growth factors to induce angiogenesis, so whether primary or secondary tumors are accompanied by angiogenesis, angiogenesis refers to the development of new blood vessels from existing capillaries or post-capillary venous development, vascular endothelial cells (HUVEC) in the Matrigel retained the endothelial cell proliferation and division of fast, fast migration rate HUVEC retain the characteristics of fast proliferation, division and migration of endothelial cells on Matrigel, therefore, the in vitro angiogenesis assay of endothelial cells can well simulate the process of tumor angiogenesis.
Materials and Instruments
Human umbilical vein endothelial cells, endothelial cell basal medium, hydrocortisone, fetal bovine serum, incubator
Plasmid containing target gene, 96-well plate, pipette, matrix gel, centrifuge, microscope, etc.
Move
I. Cell culture
1. Cultivate human umbilical vein endothelial cells (HUVECs) in endothelial cell growth medium: add 1 μg/mL hydrocortisone and 1 ng/mL epidermal growth factor to the endothelial cell basal medium, 10% fetal bovine serum, and penicillin/streptomycin, and cultivate the cells at 37 ℃, 5% CO2. 2.
2. When the fusion is 80%, divide the cells at a ratio of 1:3 per passaging. Using cells that have been passaged 2~5 times, there is no significant difference in the response of these cells to growth factors. After 6 passages, the cells begin to senesce and no longer form tubular cells on the gelatinized basal matrix.
II. Transfection
1. When the HUVECs have reached 70% fusion, transfect with the desired gene. 2.
2. For 100 mm plates, use 1 µg of empty pCDNA3 vector as control and 1 µg of pCDNA3 vector containing the gene of interest as experimental group. 3.
3. 1 µg of DNA with 3 µL of lipofectamine 2000 is diluted with diluted serum medium to a final volume of 200 µL, then the medium is replaced with fresh medium and the transfection solution is added to the cells. Incubate the cells at 37 °C in an incubator with 5% CO2. 4.
4. 3 hours after transfection, add 7 mL of completely fresh medium and further incubate the cells at 37 °C for 24-48 hours. The day after transfection, change to fresh complete medium.
Tube formation experiment
1. Cool the 96-well plate and pipette tips at 4 °C for 1 hour before preparing the gel substrate. 2.
2. Place the plate on ice at 0 °C and add 50 μL of cold substrate to each well while preparing the substrate.
3. Place the plate in an incubator at 37 °C, 5% CO2 for 30 minutes to allow the substrate to polymerize. 4.
4. Meanwhile, collect cells. Wash the HUVECs with 1 mL of 0.25% trypsin / 0.53 mM EDTA solution; then add 2 mL of trypsin EDTA solution and incubate at 37 ℃ for 5 minutes. The cell layer was observed to be completely dispersed under an inverted microscope. Then, the medium in which the cells were suspended was collected and centrifuged at 264 x g for 3 min to harvest the isolated cells. The cells were resuspended in serum-free culture medium. 5.
5. To count the cells, 0.2 mL of the cell suspension was added to 0.5 mL of PBS and 0.3 mL of 0.4% Taipan Blue solution. After 5 minutes at room temperature, cells are counted in a Bürker chamber. 6.
6. Resuspend the cells in complete medium at a concentration of 2×104 cells/50 μL. They are inoculated onto the gel substrate at a concentration of 2 x 104 cells per well. 7.
7. Incubate the cells in an incubator at 37 °C and 5% CO2 for 24 hours and then photograph tube formation.
8. In order to quantify the results of the tube formation analysis, the number of branch points with at least three tubes attached can be counted. Using image software (e.g. imageJ), count the number of branch points for each field in each micrograph and calculate the mean ± standard error (SE) for each experimental condition.
Caveat
1. When placing inoculated cells on the gel substrate, pay attention to the number of cells; too many or too few cells will not form tubes in the correct manner on the gel substrate. When observing tube formation: HUVECs are able to form tubes within 6 hours, but better results may be observed after 12-18 hours. 2.
2. It is advisable to starve the HUVEC cells for 3-6 hours by removing the serum double antibody before performing the tube formation assay. 3.
3. Spread the gel evenly over the entire well, taking care to avoid bubble formation.
Common Problems
Bubbles tend to form when performing Part III, Step 2. It is also important to note that the vials containing the substrate should be placed on ice during the experiment, as the substrate will turn solid at room temperature.
References:
[1] Hall H, Djonov V, Ehrbar M , et al. Heterophilic interactions between cell adhesion molecule L1 and alphavbeta3-integrin induce HUVEC process extension in vitro and angiogenesis in vivo.[J]. Angiogenesis, 2004, 7(3):213-223.
[2] Ma H, Jiang S,Du L,et al. Conditioned medium from primary cytotrophoblasts, primary placenta-derived mesenchymal stem cells, or sub-cultured placental tissue promoted HUVEC angiogenesis in vitro[J]. Stem cell research & therapy,2021, 12(1):141.
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