Our protocol enables unique insights into the molecular and bulk tissue level mechanisms of vasculogenesis that may aid in the engineering functional vasculature for cardiovascular disease therapy in modeling. This protocol allows the creation of robust, three-dimensional vascular networks for evaluating the vasculogenic potential of various platforms, and also provides a free, open source computational pipeline for analyzing final network topology. Begin by adding 250 microlitres of cell detachment solution per well of the D5D6 differentiated cell culture for 10 minutes at 37 degrees Celsius.
At the end of the incubation, use a P1000 pipette tip to dissociate the cells into single cell suspensions. Pool the cell solutions in a single 15 milliliter conical tube for centrifugation. We suspend the pellet in 200 microliters of ice cold sorting buffer and label the cells with five microliters of concentrated fluorescence conjugated CD34 antibody for 10 minutes at four degrees Celsius.
At the end of the incubation, wash the cells in five mililitres of ice cold sorting buffer. Filter the suspension through a 40 micrometer pour strainer into a five milliliter fluorescence activated cell sorting, or FACS, tube. Before running the sample, sort one times 10th of the fourth unlabeled cells on the fluocitometer, gating a region at a high fluorescence intensity that does not contain any of the unlabeled cells to serve as the negative control.
Then sort the labeled sample contain endothelial progenitors derived from induced pluripotent stem cells based on their high CD34 expression. At the end of the sort, transfer the endothelial progenitor cells to a microcentrifuge tube for centrifugation. For collagen hydrogel encapsulation of the progenitor cells, we suspend the sorted cell pellet in 200 microliters of Endothelial Growth Medium 2, supplemented with 10 micromiliter of the ROCK Inhibitor Y-27632.
Add the cells to 400 microliters of seeding medium in a 1.8 mililiter microcentrifuge tube on ice. Mix 350 microliters of collagen into the cell suspension. The solution will become pale yellow.
Next, mix ten microliters of 1-Muller sodium hydroxide into the collagen and cell solution. The solution will now become bright pink. Pipette 56 microliters of this neutralized collagen cell solution into individual wells of a 96 well ultra-low attachment U-bottom cell culture plate for a 30-minute incubation at 37 degrees Celsius.
At the end of the incubation, examine the wells under a bright field microscope to check that the cells have been evenly distributed. Then add 100 microliters of freshly prepared Endothelial Growth Medium 2, supplemented with ROCK Inhibitor and Vascular Endothelial Growth Factor to each well and return the plate to the 37 degree Celsius incubator. After one week of culture, add 250 microliters of 4%paraformaldehyde to one well of a 48 well plate per hydrogel and remove the medium from the hydrogels.
Then use fine-tipped tweezers to transfer the hydrogels to the paraformaldehyde containing wells. After 10 minutes at room temperature, rapidly wash the hydrogels with PBS and permeabilize the 3-D cultures with 250 microliters of a 0.5%non-ionic surfactant for five minutes at room temperature. Wash the hydrogels with two five minute washes at room temperature with 250 microliters of PBS, supplemented with 300 milliliter glycine per wash, followed by the immersion of each hydrogel in 250 microliters of blocking buffer for 30 minutes at room temperature.
Label the cells with the appropriate primary antibodies diluted in blocking buffer overnight at four degrees Celsius, followed by two washes in 0.5%emulsifying reagent and Dulbecco's PBS. After the second wash, label the cells with the appropriate species specific secondary antibody for two hours at room temperature protected from light, before washing the 3-D cultures two times in 0.5%emulsifying reagent and Dulbecco's PBS, as demonstrated. To visualize the cell nuclei, add DAPI, diluted at a one to 10000 concentration in PBS to the samples for a two minute incubation at room temperature, followed by two washes in 0.5%emulsifying reagent and Dulbecco's PBS.
Then use fine-tipped tweezers to transfer each sample to an appropriate viewing vessel. After differentiation, sorting, and encapsulation in collagen hydrogels, the cells will typically remain rounded for 24 hours, before beginning to migrate and form initial lumens. After about six days of culture, a primitive capillary plexus will be visible in the hydrogel when viewed with bright field microscopy.
After imaging the fixed, stained, cell laden hydrogels on a confocal microscope, the pre-processed images are converted to a skeleton which enables an analysis of the overall length and connectivity of the network. It is imperative to add antibiotics after FACS, as sterilizing the interior of this instrument is difficult, and to remove the antibiotics 24 hours after cell encapsulation. Using this technique, we were able to determine which physical and chemical characteristics extracellular matrix mimicking biomaterials govern the vasculogenic potential of iPSC-derived derived endothelial progenitors.
