Reagents. Fetal bovine serum (FBS, Cat. #10099-141), DMEM (Cat. #11995-065), and Cellection pan mouse IgG kit (Cat. #11531D) were purchased from ThermoFisher (Waltham, MA, USA). Rabbit anti-Factor VIII antibody and (Cat. #11778-1-AP) and FITC-conjugated goat anti-rabbit IgG (Cat. #SA00003-2) were obtained from Proteintech (Wuhan, China). Mouse anti-CD31 antibody (Cat. #MCA1334G) was from Bio-Rad (Hercules, CA, USA). Rabbit anti-CD34 antibody (Cat. #ab185732) was from Abcam (Cambridge, MA, US). Collagenase type II (Cat. #4176) was from Worthington (Lakewood, NJ, USA). Hank’s solution (Cat. #H1045), L-Glutamine solution (Cat. #G0200), Trypsin-EDTA solution (Cat. #T1300), 4',6-diamidino-2-phenylindole (DAPI, Cat. #C0065), and 4% Paraformaldehyde (Cat. #P1110) and MTT cell proliferation and cytotoxicity assay kit (Cat. #M1020) were from Solarbio (Beijing, China).
Cell isolation and culture. About 10-day-old Sprague-Dawley rats were purchased from Beijing Haidian Xinglong Experimental Animal Breeding Factory (animal license No.: SCXK [Jing] 2019-0010) and used for cell culture. The experimental protocol for cell isolation from rat intestinal mucosas was approved by the Animal Care and Protection Committee of Beijing University of Agriculture (No. BUA_ZT202001). All procedures were performed in accordance with the relevant guidelines and regulations by Beijing Association on Laboratory Animal Care and the ARRIVE guidelines. Isolation and culture of rat intestinal mucosal MVECs were performed as previously described with minor modification5.
In brief, rats were euthanized by cervical dislocation, and the abdominal cavities were opened. The jejuna were sampled and then cut into small segments, which were longitudinally cut and rinsed away the contents with Hank’s solution. The intestinal mucosas were scraped and minced in 0.2% collagenase type II solution. After about 60-min digestion at 37°C with pipetting every 15-20 min, the digested solution was filtered through a 100-µm cell strainer and centrifuged. The cell pellet was resuspended in DMEM containing 15% FBS, 2 mmol/L glutamine, 100U/mL penicillin, and 100 mg/mL streptomycin, and the cells were seeded at a density of 5×104 cells/mL and incubated in a 5% CO2 37°C incubator. After 2 hr of incubation, the nonadherent cells were washed away and the complete medium was replaced. When it grew to confluence, the cell culture was detached using 0.05% trypsin-0.005% EDTA and purified by magnetic separation as follows.
Magnetic cell separation. Magnetic cell separation was carried out using a Cellection pan mouse IgG kit via the direct technique with the KingFisher 96 automatic magnetic separation system. Briefly, the beads were washed and incubated with mouse anti-CD31 antibodies on a rotary shaker at 4°C overnight. Removing excess antibodies, the beads were resuspended in Buffer 1 (0.01 M PBS containing 0.1% BSA, pH 7.4) of the same original volume. The detached cells were filtered through a 40-µm cell strainer to prepare the single-cell suspension at a density of 1×107 cells/mL in Buffer 2 (0.01 M PBS containing 0.1% BSA and 2 mM EDTA, pH 7.4). Twenty-five microliters of CD31 antibody-coated beads were added into 1 mL of the cell sample. Over 30-min incubation on the rotary shaker at room temperature, CD31+ cells were separated using the magnetic separation system. The bead-bound cells were placed into Buffer 3 (DMEM with 1% FBS, 1mM CaCl2, and 5 mM MgCl2, Ph 7.2), and the DNase I solution was added according to the manufacture’s recommendation. The beads were released over 15-min incubation and removed using the magnetic separation system. The released cells were resuspended in the complete medium and seeded with the medium replacement every 3 days.
Parameter setting of the separation system. The KingFisher 96 automatic magnetic separation system (Kingfisher 96, ThermoFisher Scientific, Vantaa, Finland) was used to wash beads, separate cells, and remove the beads, respectively, which included the KingFisher 96 Magnetic Particle Processor and the BindIt 3.2 operating software. All procedures were processed in KingFisher 96 Deep-Well plates (Cat. #97002534), and five plates were used for beads washing and cells separating and two plates for beads removing. When washing, Beads were released quickly and mixed fast for 5 sec. When separating cells, beads were released and mixed slowly for 5 sec. When removing beads, the sample was shaken strongly for 5 sec and then mixed fast for 5 sec. In every procedure, the beads were collected 3 times for 3 sec each time.
Cell proliferation assay. The cell proliferation ability was assayed at passage 5, 10, and 20. To determine the proliferation parameters, cells were seeded at a density of 5, 000 cells per well in a 96-well plate, and the medium was replaced every 3 days. The cell proliferation was determined each day in quintuplicate by the MTT method according to the kit instruction. To determine the number parameters, cells were plated at a density of 50, 000 cells in a corning 12 well plate, and the medium of 1 mL per well was replaced every 3 days. Cells were dissociated in triplicate by trypsin digestion each day and counted using a cell counter (TC20, Bio-Rad). The cell growth curves were drawn from the absorbance values or cell numbers for 7 days.
Immunofluorescent staining. Cells were grown on cover glasses in 24-well plates for 48 h and fixed in 4% paraformaldehyde for 20 min at room temperature. The expression of CD31, CD34, and FVIII were detected using indirect immunofluorescent staining as described previously14. Briefly, after washing with PBS three times, the fixed pieces were blocked with 1% bovine serum albumin for 30 min and incubated with primary antibodies for CD31, CD34, or FVIII in blocking solution (10 µg/mL) overnight at 4°C. The cell pieces were washed with PBS three times and incubated with FITC-labeled secondary antibody in blocking solution (1:100 dilution) for 2 h at room temperature in the dark. After washing, cell nuclei were counterstained with 2 µg/m DAPI for 3 min. The stained cells were mounted in glycerol and photographed using a fluorescence microscope (IX71, Olympus, Tokyo, Japan) with 490 and 350-nm excitation, and then the single-channel images were merged. Their fluorescence intensities were measured using Image J software.
Data analysis. Data were presented as the mean ± standard deviation with three or more replicates. Statistical comparisons were performed by one-way ANOVA combined with Tukey’s post hoc multiple comparison test using GraphPad Prism 8.0 software, and P<0.05 was considered a significant difference.