This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development. The described protocol is optimized for high degrees of viability and purity, which are essential for next-generation sequencing applications. Demonstrating the procedure will be Shelby Cain, a graduate student from the Hershey Laboratory.
Start removing eyes from the euthanized neonatal mouse by cutting away the skin and membrane over the eye with a perpendicular incision to the eyelid using dissection scissors. Then use the forceps to press down above and below the eye so that the eye moves out of the socket. Carefully pinch underneath the eye with the forceps and cut the optic nerve that keeps the eye attached.
Then place both the eyes from the mouse in a well of the 48-well plate in the freshly prepared ice-cold PBS until finished harvesting. To isolate mouse retinal tissue, suspend the eyes in the dissection pad in a Petri dish filled with 500 microliters of ice-cold PBS using a transfer pipe pad with a wide tip. Working under the dissection microscope, hold the optic nerve with one fine forceps and use the second forceps to pierce the hole through the anterior chamber of the eye where the cornea and sclera connect.
Then tear the hole in a circle about 75%of the way around the cornea. While holding the optic nerve with one forceps, use the second forceps to gently tear the sclera and the vitreous body off the retinal tissue. To remove lens and hyaloid plexus vessels, reach into the retina with open forceps until almost touching it and then close the forceps to grab and pull the lens and hyaloid plexus vessels out from the retina.
When all vessels are removed, place the retinas in the two milliliter micro-centrifuge tubes filled with 500 microliters of one x ice-cold PBS. Remove excess PBS from the two milliliter micro-centrifuge tube with a pipette leaving about 100 microliters of PBS in the tube to completely cover the retinal tissue. Then add 500 microliters of the freshly prepared digestion solution to the tube.
Use a P1000 pipetta and pipette tip to pipette up and down the retinal tissue in the digestion solution five times. When done, incubate the digestion mixture in a 37 degrees Celsius water bath for 20 minutes with pipetting the digestion mixture up and down every five minutes as explained before. After incubation, the retinal tissue will dissolve into a single cell suspension turning the digestion mixture cloudy.
Next, pellet the cells by centrifugation at 375g for five minutes at four degrees Celsius. Following centrifugation, remove the PBS from the washed cell pellet by pipetting and then immunostain the cells by resuspending the pellet in 100 microliters of antibody staining solution per 0.5 times 10 to the sixth cells. Incubate the single cell suspension with the antibody staining solution for 30 minutes on ice in the dark with tapping the tube every 10 minutes to gently mix the cells.
For fluorescence-activated cell sorting, pellet the cells by centrifugation at 375g for five minutes at four degrees Celsius. After removing the supernatant, wash and pellet the cells in 500 microliters of PBS as described before. And then resuspend the cells in 300 microliters of fluorescence-activated cell sorting or FACS buffer by gently mixing with the pipetta.
Next, add propidium iodide or PI as a viability marker to the sample tubes containing the FACS buffer to get a final concentration of 0.5 micrograms per milliliter. Use a pipette to transfer the cell suspensions through a cell strainer snap cap containing a 35 micron filter into a five milliliter test tube. After filtration, keep the FACS tube on ice in the dark and then transfer the tube to the cell sorter.
Turn on the FACS instrument and the computer and open the FACS software to run and operate the FACS instrument. Perform routine quality control tests. Load the control stain samples into the FACS instrument to adjust the axes.
Start with IgG antibody only cells to generate and adjust forward scatter and side scatter. Then CD31 antibody only to generate and adjust CD31. And then CD45 antibody only to generate and adjust CD45.
Record the data from the control samples. Next, load the stained sample cells into the FACS instrument and run the sample. Gait the cells based on forward scatter or FSC and side scatter or SSC parameters.
Gait cells by FSCA and FSCH to identify cell doublets and only collect single cells. Gait cells by PI and SSCA to identify viable cells. Make a CD31+CD45-gait with controls Make a CD31+CD45-gait with controls and gait the cells by CD31 and CD45.
Insert a collection tube with 250 microliters of FACS buffer in the instrument. Next, start sorting cells that are CD31+CD45-into the installed collection tube. Keep the collection tubes on ice for further analysis.
The samples can be processed for next-generation sequencing applications. In the study, varying the digestion time affected the cell yield and viability percentage. The digestion time of 20 minutes resulted in an optimal yield with a low percentage of non-viable endothelial cells.
Subsequent propidium iodide staining demonstrated that the isolated endothelial cells stayed viable for 60 minutes after isolation. Cell population purity was assessed by quantitative PCR or qPCRR analysis of the expression of endothelial cell genes showing strong enrichment for endothelial cell genes in the CD31+CD45-population. Purifying RNA from the isolated endothelial cells converting and amplifying cDNA through library preparation and sequencing The cDNA library resulted in more than 16, 000 genes identified in nine independent samples.
A drop-off in transcripts per million reads or TPM was observed in genes below this threshold. The single cell RNA sequencing of the isolated retinal endothelial cells through the 10x Genomics pipeline resulted in a median of 1, 171 genes per cell, 15, 247 total genes detected, and a median of 2, 255 unique molecular identifier or UMI counts per cell in 917 cells. The critical steps in this protocol are retinal tissue isolation and tissue digestion, which should be performed accurately as described to optimize cell yield and viability.
The isolated endothelial cells can be used in advanced sequencing techniques such as whole-transcriptome RNA sequencing, single cell RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin sequencing. The use of this optimized method in combination with next-generation sequencing methods and by computational approaches can further elucidate the mechanisms of interconnected signaling pathways that regulate vascular developments.
