Developmental Biology
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Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells
Chapters
Summary September 18th, 2019
Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.
Transcript
This 3D co-culture spheroid system is designed to mimic physiologic angiogenesis. It will be effective for evaluating potential angiogenic modulators and provides predictable information in advance of in vivo studies. This system utilizes co-culture spheroids formed by two vascular progenitor cells and can be used to investigate cellular interactions, sprouting, and the tubular maturation of physiologic angiogenesis.
If we can use patient cells to form co-culture spheroids, we can use this system to develop personalized treatments for abnormal angiogenesis-related diseases, such as cancer. This assay system can increase the sensitivity and efficiency of studies of angiogenesis and drug development. Begin by using 05%trypsin-EDTA to detach ECFCs and MSCs from 80 to 90%confluent cell cultures for three or five minutes in a cell culture incubator, respectively.
Inactivate trypsin with fresh complete DMEM medium, and pipette the cells a few times to generate a single cell suspension. Collect the cells by centrifugation, followed by two washes with serum-free medium. After the second wash, dilute the ECFCs to a three times 10 to the six cells per milliliter of medium concentration and the MSCs to a two times 10 to the six cells per millimeter of medium concentration in individual two-milliliter microcentrifuge tubes.
Pellet the cells by centrifugation, and carefully aspirate all but the last 15 to 25 microliters of supernatant from each cell sample. Next, resuspend each pellet in 250 microliters of diluent C from a fluorescent dye kit, and gently pipette the cell suspensions to ensure a complete dispersion. Add 250 microliters of freshly prepared 20-micromolar PKH67 to the tube of ECFCs.
Add 250 microliters of 12-micromolar PKH26 to the tube of MSCs. Immediately mix both cell suspensions. After five minutes at room temperature with shaking, protected from light, stop the staining process with a one-minute incubation in 0.5 milliliters of FBS per tube.
At the end of the incubation, collect the cells by centrifugation, and carefully remove the supernatant, followed by two washes in two milliliters of fresh complete medium per wash. Then suspend the cells in respective medium in 15-milliliter tubes. For spheroid generation, after the second wash, first dilute the cell suspensions in the appropriate concentration of endothelial cell growth medium MV2 supplemented with 20%methyl cellulose solution and 5%fetal bovine serum concentration.
Next, add each cell suspension into a sterilized polystyrene rectangular reservoir. Use a multichannel pipette to add approximately 125-microliter droplets of cells onto the cover of a 150-millimeter culture plate. When all of the cells have been added, invert the cover over the bottom of a dish containing 15 milliliters of PBS, and place the plates in the cell culture incubator for 24 hours.
The next day, rinse the dish covers with five milliliters of PBS into one 50-milliliter tube per dish. Rinse the covers with an additional five milliliters of PBS to collect any remaining spheroids, and pellet the spheroids by centrifugation. Carefully discard all but the last 100 to 200 microliters of supernatant, and gently tap on the wall of the tube so that the spheroids are freely suspended in the remaining supernatant.
Add the appropriate volume of endothelial cell growth medium MV2 supplemented with 5%fetal bovine serum and 40%methyl cellulose solution to each tube to resuspend the spheroids at a 100 spheroids per milliliter of medium concentration. Use a one-milliliter pipette tip cut to a three-to five-millimeter diameter to gently mix the spheroid suspensions. Use a new wide-tip, one-milliliter pipette to mix the spheroid suspension solution with a neutralized type I collagen gel at a one-to-one ratio on ice.
Add 900 microliters of each spheroid-suspending collagen gel solution into the wells of a pre-warmed 24-well plate. Allow the suspensions to polymerize for 30 minutes in the cell culture incubator, before covering the spheroids with 100 microliters of endothelial cell growth medium MV2 supplemented with 2.5%FBS and 50 nanograms per milliliter of VEGF to the ECFC-only spheroids and medium supplemented with FBS only to the MSC and ECFC-plus-MSC spheroid cultures. Then place each plate in a real-time cell recorder installed in a cell culture incubator, and randomly focus on five to 10 spheroids at a 10x magnification to monitor the sprouting formation of each fluorescence-labeled spheroid every hour for 24 hours.
To quantify the spheroid sprouting, import the image files to ImageJ, and measure the number and length of sprouts expressing the appropriate fluorescent signal from five randomly selected spheroids per experimental group. For ECFC-plus-MSC spheroids, the number of sprouts and cumulative sprout length are significantly higher compared to those of ECFC-only spheroids at all time points. MSCs-only spheroids do not form sprouts but demonstrate an individual migration of the MSC outside of spheroids.
The labeling of ECFC with green-fluorescent dye and MSC with red-fluorescent dye before combining to generate ECFC-plus-MSC spheroids demonstrates that ECFC-mediated sprout structures are covered with MSCs, suggesting that combined MSCs function as perivascular cells during sprout formation, enhancing sprout stability and durability by the tight association between two vascular cells. ECFC-plus-MSC spheroids pretreated with an angiogenesis inhibitor show a decreased sprout number and a cumulative sprout length in a dose-dependent manner compared to control ECFC-plus-MSC spheroids. In parallel experiments, ECFC-only spheroids pretreated with the inhibitor followed by stimulation with VEGF also demonstrate a decreased growth factor-induced sprout number and cumulative sprout length in a dose-dependent manner.
Of note, a higher concentration of angiogenesis inhibitor is needed to inhibit ECFC-plus-MSC spheroids compared to ECFC-only spheroids. Tumor growth is significantly inhibited in high dose angiogenesis inhibitor-treated animals compared to both control-treated animals and low dose angiogenesis inhibitor-treated animals in a human xenograft tumor mouse model. The plasma concentration of bevacizumab in high dose-treated mice was 568 plus or minus 40.62 micrograms per milliliter, which was closer to the IC50 value of bevacizumab for inhibiting cumulative sprout length in ECFC-plus-MSC spheroids rather than ECFC-only spheroids.
This suggests that the ECFC-plus-MSC co-culture spheroid system is suitable for predicting effective plasma concentrations in advance of an animal study. Using a wide-tip, one-mL pipette to gently mix the spheroids while they are in collagen gel on ice is important for protecting the integrity of the spheroids. We can modify this system to introduce other cell types such as immune cells and other extracellular matrices to investigate cell-matrix crosstalk during angiogenesis.
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