All methods were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki).
HUVEC maintenance
The HUVEC line was purchased from PromoCell (Heidelberg, Germany) (#C-12200). Frozen HUVECs were thawed and cultured according to the manufacturer’s protocol. Upon confluency, the HUVECs were detached by 15 min incubation with trypsin-EDTA (0.25%) (Invitrogen, Waltham, MA, USA) and seeded onto culture dishes at a density of 500,000 cells/cm2 in Endothelial Cell Growth Medium 2 (Ready-to-use) (PromoCell, #C-22011) supplemented with Endothelial Cell Growth Medium 2 SupplementMix (PromoCell, #C-39216) (hereafter referred as “culture medium”). Cells from passages five to eight were used in the recellularization experiments. Cell morphology was microscopically checked, and cultures with abnormal cell shapes were excluded from further experiments.
Preparation of decellularized porcine aorta slices
Decellularized porcine aorta slices were prepared as previously reported20. Briefly, fresh porcine aortas were purchased from Tokyo Shibaura Zouki, Tokyo, Japan and circularly cut with an inner diameter of 16 mm using a hollow punch after washing with saline. For decellularization, high hydrostatic pressure was applied at 1000 MPa and 30 °C for 10 min using a hydrostatic pressurization system (Dr. Chef, Kobelco, Tokyo, Japan); the samples were then washed with DNAse (0.2 mg/mL) and MgCl2 (50 mM) in saline at 4 °C for 7 days, followed by a change in the washing solution to 80% ethanol in saline at 4 °C for 3 days, and then to saline at 4 °C for 3 days to remove cell debris. The intima of the aorta was peeled off with tweezers and the resulting aortic slices were used for the experiment.
Pre-treatment of decellularized tissue with hL411 coating
To coat the aorta slices with hL411, we used iMatrix-411(#892041, Matrixome Inc., Suita, Japan), a recombinant E8 fragment of hL411. One microliter of the original titer was diluted in 200 µL of deionized water. The aorta slices were then coated with a 0.5 µg/cm2 of the diluted iMatrix-411 solution. To maintain a stable planar shape during the coating, the aorta slices were anchored using a stainless steel ring with 14 mm diameter (Fig. 1a). To check for fluid leakage, 200 µL of phosphate-buffered saline (PBS) was loaded into the ring. After completing the leakage check, PBS was discarded and the diluted iMatrix-411solution was added inside the stainless-steel ring. The outer region of the tissue was filled with PBS to maintain moist conditions. We incubated the tissue with the stainless ring in a 10 cm dish and placed the dish in a laminar flow cabinet at room temperature for approximately 2 h, in which the solution was expected to be almost vaporized. After the coating, the tissues were kept at 37 °C with 5% CO2 until the solution was fully vaporized but not completely dried.
Cell seeding onto the decellularized tissues
We placed the decellularized aorta slices in a 10-cm cell culture dish and anchored them using a stainless-steel ring (inner diameter 12 mm) to maintain the tissues in a planar shape. We then added 200 µL of PBS to the inner space of the stainless ring and kept it for 20 min at room temperature to check for fluid leakage. If PBS leakage was observed, the stainless-steel ring was repositioned. The PBS was discarded and cell suspension containing 2.0×106 of HUVECs and 500 µL of culture medium was added followed by incubation at 37 °C with 5% CO2 for 24 h. After incubation, we removed the stainless-steel ring, replaced the culture medium, and incubated the tissues at 37°C, with 5% CO2 for further 24 h.
Dynamic flow culture
A custom dynamic flow culture system was prepared (Tokai Hit Co. Ltd., Fujinomiya, Japan). Briefly, it was assembled into three parts: a flow-generating rotor, an incubation container, and a rotor speed controller (Fig.1a). The incubation container of the planar system was a stainless-steel platform with a thick glass cover having two input/output ports and was designed to fit a 35-mm cell culture dish. The vessel container for vascular-shaped tissues was made of polycarbonate; it was designed using SolidWorks 2019 software (Dassault Systems, Vélizy-Villacoublay, France) and fabricated using a milling machine (Fig. 4a). The container was designed in a separate format to facilitate tissue insertion. The inner diameter was 9 mm. The inner flow pattern (vector and streamline) was simulated using simulation software (SolidWorks Flow Simulation, Dassault Systems) (Fig. 4b).
To avoid bubble jamming in the incubation route, which may result in impaired cell culture, we rinsed the system with PBS prior to the culture medium. After washing with PBS, the fluid was replaced with the culture medium. After refilling the route with culture medium, we stopped the rotor, set the tissues installed in an incubation container, and restarted the dynamic flow culture. At the initial phase of the dynamic flow culture (1 h), we set the flow speed to 10% of the speed required for the maintenance phase (e.g., 10 rpm in 100 rpm of the maintenance rotor speed). After the initial phase, we increased the speed and maintained it at 200 rpm for planar tissue as well as for vascular-shaped tissue experiments. After confirming that the system was working without trouble, we placed the entire dynamic incubation system in a cell culture incubator (37 °C, 5% CO2) for further incubation. The incubation period was 2 weeks. The culture medium was changed every other day by refreshing approximately 10 mL of the culture medium in the culture medium reservoir. The rotor was stopped on day 14, and tissues were harvested for further examination.
Cell viability assay
To evaluate cell viability, we used Calcein AM (Invitrogen) as an indicator of live cells20. We moved the tissue during incubation from the incubation container to a 12-well plate on days 2, 4, 7, and 14 for planar tissue experiments, and on day 14 for vascular-shaped tissue experiments, and then treated the tissues with Calcein AM solution diluted 1:1000 in endothelial cell growth medium. We then incubated the tissues for at least 35 min at 37°C with 5% CO2. After incubation, the solution was discarded and the tissues 3 times with PBS. The fluorescence signal was measured using an all-in-one microscope (BZ-X800, Keyence, Osaka, Japan) and the attached software. Images were captured with a 5-layer overlay, where in each layer was 20 μm thick. The entire sample image was combined with 16 pieces of a single 2× field (n = 6). Area coverage was calculated as the percentage of fluorescence-positive areas across the entire culture area. Signal intensity was calculated as the average signal intensity. We carefully conducted procedures to avoid drying of the tissues during evaluation.
Histological evaluation
The recellularized tissues were collected and fixed overnight with 4% paraformaldehyde (PFA), followed by embedding in OCT compound (Sakura Finetek Japan, Tokyo, Japan) and freezing for further cryosectioning. Sections of 6 μm thickness were prepared and stained with hematoxylin and eosin. For immunofluorescence staining, sections were treated with Protein Block Serum Free (DAKO, Glostrup, Denmark) and incubated overnight at 4 °C with a primary antibody against human endothelial cells; rabbit polyclonal anti-CD31 antibody, ab28364 (Abcam, Cambridge, UK) (1:150). Then, anti-rabbit Alexa Fluor 488 (1:2000) was used as the secondary antibody, and the sections were incubated for 60 min at room temperature. Cell nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI) staining (0.1 µg/ml). All sections were photographed using an all-in-one microscope (BZ-X800; Keyence). The evaluations were repeated three times.
Clot formation assay
A clot formation assay was conducted as described in our previous report, with some modifications20. Frozen human single donor whole blood was purchased (Human Heparin sodium whole blood, single donor, CTSAG050, BioIVT, Westbury, NY, USA) and aliquoted into small volumes (2 mL/tube), stocked in -20°C freezer and pre-warmed using a 37 °C water bath before use. For activating the blood sample, 0.343% citric acid and 0.004 M CaCl2 was used. The samples obtained after 14 d of incubation (n = 6) were placed on a wet paper towel floating in a 37 °C water bath. We gently mixed activated whole blood and dropped 50 µL of blood for each sample. Samples were harvested at 4, 10, 20, and 40 min and then gently washed with PBS. The samples were then placed on white paper to observe the extent of clot formation (Fig. 4a). We then prepared 2 mL of deionized water in a 12-multiwell plate, immersed the samples with clots, and stored the samples overnight at 4 °C. We collected 1 mL of the supernatant, centrifuged it for 15 min at 1500 × g, 22 °C. The supernatant was added to a 96-multiwell plate (200 µL/well). The amount of dissolved hemoglobin was measured using a microplate reader at a wavelength of 540 nm (iMark, Bio-Rad, Hercules, CA, USA). %Blood coagulation rate was calculated as follows: (Sample read value - Blank read value)/(Positive control read value - Blank read value) × 100 (%). Glass coverslips were used as positive controls, mock samples (decellularized tissue without recellularization) were used as internal controls, and polytetrafluoroethylene (PTFE) sheets were used as negative controls. For the microplate reading, we used 50 µL of whole blood in 2 mL of deionized water as a positive control and deionized water as a negative control. This experiment was repeated six times.
Statistical analysis
Data were processed using GraphPad Prism 8 software version 8.3.0. (San Diego, CA, USA). Values are expressed as mean ± SD. Statistical analysis was performed using two-way repeated-measures analysis of variance with Tukey’s post-hoc test. Statistical significance was set at P < 0.05.