Characterizing gene expression at the transcriptomic level is key to understanding cell function in health and disease. Our method focuses on capturing endothelial cells, the crucial layer of the vascular wall, from human donors'menesteric arteries for transcriptomic profiling with a single-cell and spatial resolution. There is an enriched population of endothelial cells from a vessel without a sorting step, which can cause cell loss.
Furthermore, other cell types exist which allow the investigation of cell-cell interactions in the tissue context. We have used this technique to investigate the arterial changes in diabetes and obesity. Depending on the availability of the vessel, these techniques can also be repurposed to explore biology in other vasculature and other diseases.
If it is the first time, two key steps require the most practice. One, scraping, though adequate force should be exerted to remove integral cells without detaching deeper layers. And two, cryostat sectioning.
Ensure sections are cold, flat, and are within the confines of the specialized slide. Demonstrating the procedure will be Yingjun Luo and Xiaofang Tang, two post-doctoral researchers from my laboratory. To begin wash the tissue with DPBS.
With sterile forceps and dissection scissors, remove the fat and outer connective tissues until the vessel is clean. Measure the vessel's length with a ruler placed outside the dish and take photographic records. Cut the mesenteric artery lengthwise with scissors to open the vessel lumen vertically.
Using needles, attach the vessel onto black wax on all four corners, exposing the intima. Add one milliliter of pre-warmed digestion buffer to intima. Using a sterile scalpel, gently scrape the lumen of the vessel twice.
Transfer the digestion buffer into a tube. Add one milliliter of digestion buffer to the intima and pipette up and down carefully to collect the remaining cells and transfer the cell suspension to a tube. Incubate cells at 37 degrees Celsius for five minutes in an incubator or on a rocker.
Add M199 medium to the cell suspension to quench the enzymatic reaction and mix gently. Centrifuge the suspension for five minutes at four degrees Celsius and 600 times G.At the end of the centrifugation, remove and store the supernatant separately as a control culture to observe if all cells are captured in the pellet. Then, re-suspend the cell pellet in one milliliter of M199 medium.
Assess the cell viability by mixing 10 microliters of trypan blue with 10 microliters of cell stock. Observe the cell morphology and count the cells using a hemocytometer. Coat two wells of a six-well plate by pipetting 500 microliters of attachment reagent into wells for 30 minutes at room temperature.
After washing the wells with sterile DPBS, dispense the full cell stock into one well, and the supernatant is kept as a control into the second well. Centrifuge previously prepared cell stock at four degrees Celsius and 600 times G for five minutes. Remove the supernatant and re-suspend the pellet gently with a P-1000 pipette in 0.04%BSA in DPBS.
Mix well to ensure a single-cell suspension. Pass the solution through a 40-micrometer strainer to remove cell debris. Then, centrifuge the suspension and remove the supernatant as demonstrated.
Re-suspend the pellet in 500 microliters of 0.04%BSA in DPBS using a P-1000 pipette and mix well to ensure a single-cell suspension. As demonstrated previously, assess the cell viability using trypan blue. Using a hemocytometer or automated cell counter, observe the single-cell suspension morphology, check for tissue debris, and calculate the number of living and dead cells.
After adding isopentane to a metal canister, chill in liquid nitrogen or dry ice. Pour OCT compound in wells of labeled plastic cryo mold, preventing bubble formation. Leave cryo mold on dry ice to chill.
Cut at least two coronal sections of one centimeter in the vessel's length. Using 12-inch forceps, submerge the tissue in isopentane until frozen. Quickly submerge the tissue in OCT at orientation with the lumen visible in the center.
Grasp and place the cryo molds on dry ice and observe the freezing. The OCT will become white progressively from the outside in one to two minutes. Store these sections in a secured container at minus 80 degrees Celsius for up to six months.
Set up the desired cryostat temperature for the chamber and specimen head. Equilibrate OCT-embedded vessel sections, knives, brushes, and slides to minus 20 degrees Celsius in the cryostat for approximately 30 minutes. While equilibrating, clean the machine and all equipment that may touch the sections, including the blade, with 70%ethanol followed by RNase decontamination solution.
Attach the sample by dispensing a small amount of OCT onto the circular cryostat block and placing the sample on top before it freezes. After placing the block into the center of the specimen head, screw the block in place using the tall black handle on the left. Cut away any excess OCT surrounding the vessel.
Setting the cutting thickness to 10 micrometers on the cryostat, cut approximately 60 sections and place them in a pre-cooled 1.5-milliliter tube containing RNA extraction reagent. Vortex until the sections are completely dissolved. Extract the RNA as described in the text manuscript and dissolve the purified RNA pellet in five microliters of RNase-free water.
Cut 10 micrometer thick sections with anti-roll plate in place. Flip and carefully flatten by gently touching the section through the surrounding OCT. Wipe down the slide with ethanol and pre-warm the backside of the capture area with your finger.
Using RNase-free cryostat brushes or the slide directly, place the tissue section within the square using only the surrounding OCT. Immediately place one finger on the backside of the capture area to melt the section to the slide. Once the section adheres, place the slide onto the cryo bar to allow the section to freeze.
Cut 10-micrometer-thick tissue sections as demonstrated previously onto tissue optimization or gene expression slides. Transfer the slide to a slide mailer placed on dry ice. The figure depicts isolated ECs cultured using the described protocol displaying a distinct cobblestone-like morphology with minimal contaminating cells.
Expression of EC marker vascular endothelial cadherin was confirmed using immunofluorescence visualizing cell-to-cell junctions. Approximately 80%of cells were CD31-positive, indicating human mesenteric arterial ECs made up most of the freshly isolated cell population. Unsuccessful isolations result in cells with disturbed morphology, potentially expressing mesenchymal markers or elongating resembling a fibroblastic state.
The figure shows a representative uniform manifold approximation and projection isolated for mesenteric artery using the present protocol for single-cell RNA sequencing on isolated human mesenteric arterial ECS, with 34%cells clustered as ECs using PECAM1 and VWF as markers. RNA of high quality should show an RNA integrity number no less than six, and the ratio of 28S to 18S ribosomal RNA bands is as close to two as possible. A weak or absence of these ribosomal RNA signals may be observed when RNAs are degraded.
Hematoxylin and eosin staining visualized the morphology of the vessel, allowing regions-of-interest to be identified. Gene expression was visualized with spatial anchoring on the hematoxylin-and eosin-stained vessel. Handle tissue and cell stock carefully to maintain RNA quality, especially for spatial transcriptome profiling.
Ensure the tissue is placed on dry ice to reduce RNA degradation. Also, handle the sections and tissue with pre-cooled instruments. We have been able to explore EC gene expression changes, including LncRNAs in context to diabetes and their interaction with other cell types.
We can deduce the location of these transcriptomic changes using spatial transcriptome profiling techniques.
