The overall goal of this experiment is to evaluate the performance of the putative hemangioblastoma neovascularization using the spheroid sprouting assay in vitro. The method can help answer the key questions in the angiogenic field, such as tumor angiogenesis. The main advantage of this technique is that the product of a comprehensive procedure to evaluate in vitro where the classic tumor angiogenesis exists in hemangioblastoma and it grows in hemangioblastoma.
The implication of this technique extends toward therapy of hemangioblastumor and vasculaturement because the result highlight the complexity of hemangioblastumor neovascularization, and that suggests that this common form of angiogenesis is only a complementary mechanism. So this method can provide insight into the study of hemangioblastumor neovascularization. It can also be applied to tumors, angiogenesis, and vasculogenesis related application in some solid tumor, such as tumor vasculogenic mimicry in glass tumor.
Real demonstration of this method is critical as the generation of these manipulated endothelial cell spheroid step are difficult to learn. Because the suitable condition are important. First, culture the HUVEC endothelial cells in DMEM culture medium supplemented with ten percent fetal bovine serum, penicillin, and streptomycin.
Then, maintain the culture in the incubator at 37 degrees Celsius with five percent carbon dioxide. Next, to synthesize the the shRNA fragment, digest the plasmid with ApaI and EcoRI restriction enzymes. Then to the digested plasmid, add both the forward and reverse oligos, 10x NEB buffer, and double distilled water to a final volume of 50 microliters.
After adding all the reagents, heat the mixture at 98 degrees Celsius for four minutes. Then, gradually cool down to room temperature in a span of few hours. Use T4 ligase to ligate the annealed oligos and the plasmid.
Incubate the tube at four degrees Celsius for overnight. The following day, add five microliters of ligation mix to 25 microliters of DH5alpha competent cells. In 500 microliters of DMEM medium, add the lentiviral vector or the scrambled vector, along with other packaging plasmids.
Then, incubate the lentiviral mixture for 25 minutes at 37 degrees Celsius. Post incubation, add 7 milliliters of the DMEM medium to the scrambled or lentiviral mixed solution. Culture the 293FT cells in DMEM medium without fetal bovine serum in a 10 centimeter culture dish.
Add one milliliter of the lentiviral or scrambled solution to the 293FT cells in the culture dish. After six hours, aspirate the old medium from the culture dish. Then, replace the old medium with fresh DMEM medium containing 10 percent fetal bovine serum.
After 48 hours, collect the medium. Transfer the HUVEC endothelial cells in the lentiviral medium and maintain for 72 hours. Next, add two micrograms per milliliter of puromycin to the HUVEC cell culture medium.
Finally, incubate the culture for another 24 hours. The next day, add one milliliter of trypsin EDTA to trypsinize HUVEC cells. Then resuspend the trypsinized cell suspension in DMEM medium with 10 percent fetal bovine serum.
Count the number of cells using a cell counter. After counting, seed the cells in a 3D round bottom 96-well plate. After seeding, incubate the cells at 37 degrees Celsius with five percent carbon dioxide for 72 hours continuously.
After 36 hours, replace half of the culture medium with fresh medium. First, thaw the gel solution at four degrees Celsius, and then dilute it at a ratio of one to five with the reduced serum medium. Suck the spheroids from the DMEM medium using a micropipette.
Next, wash the spheroids with five milliliters of reduced serum medium. Carefully transfer the suspended spheroids and the diluted gel. In a 15 well plate, embed 300 microlitters of the mixed liquid of spheroids and diluted gel.
Incubate the plate at 37 degrees Celsius and five percent carbon dioxide for one hour. Next, add 400 microliters of reduced serum medium to a single well. To prevent evaporation, fill the surrounding of the well with sterile water.
Then, culture the cells at 37 degrees Celsius, five percent carbon dioxide, and 100 percent humidity for an hour. After the incubation, aspirate the old medium and add 600 microliters of reduced serum medium with one percent endothelial cell growth supplement to the well and incubate for a day. Capture images using an inverted light microscope.
Here, spheroid sprouting of the cells is captured using inverted light microscope to study the effect of VHL gene silencing on the angiogenic potential of the endothelial cells. A spheroid is seen to sprout 12 hours after lentiviral treatment in both the control and VHL silenced endothelial cells. Next, statistical analysis is performed to quantify the length of the sprouts generated by the VHL gene silenced spheroids.
The mean sprout length seem to be about 125 micrometers in the VHL silenced groups while only being about 65 micrometers in the control group. The VHL silenced group's average cumulative sprout length is approximately 1250 micrometers while the control group's is 680 micrometers. These results indicate a two-fold sprout length increase in the VHL silenced groups.
Once mastered, this technique can be done in 48 hours if it's performed properly. Following this procedure and method like capillary formation assay can be performed in order to answer additional questions. Like exploring the angiogenic ability of vascular endothelial cells.
After this development, this technique paved the way for researchers in the field of angiogenesis to explore tumor inner vasculorization. After watching this video, you should have a good understanding of which genes loose a function in manipulated endothelial cells used for exploding assay.
