Fig 1.
Overview of tissue-engineered blood vessel (TEBV) fabrication workflow and experimental design.
(A) Fabrication and sterilization of clamps and other necessary materials for TEBV fabrication should be completed prior to the start of the experiment. TEBV fabrication workflow begins with preparation of collagen type I mixture with human neonatal dermal fibroblasts (hNDFs). In situ plastic compression and dehydration is performed to remove excessive water content from the TEBVs. Subsequently, endothelial cells (ECs) are injected into the vessel lumen. To facilitate uniform cell adhesion, the chamber undergoes a 12-hour rotation in the incubator. Gradual ramping of perfusion starts the following day. (B) Both human umbilical endothelial cells (HUVEC) and red fluorescent protein (RFP)-tagged HUVEC are used in this study. Optimization parameters including seeding density, rotation duration and flow application were explored. Figure created in Biorender (https://BioRender.com).
Fig 2.
Design and schematic of the TEBV Chambers and microscope holders.
(A) i) Side view of the TEBV viewing chamber top. The outer recessed area is optimized to accommodate the front lens assembly housing, while the inner recessed area is designed to facilitate the observation of 4 TEBVs in the chamber; ii) Design and position of TEBV viewing chamber holders. The TEBV chamber is designed to be placed on top of 3D-printed viewing chamber holders. The outer dimensions of the holder are compatible with the slide holder on the microscopy stage, facilitating movement of the chamber during imaging. A central cutout in the holder allows for imaging of all four TEBVs within the chamber. (B) Schematic for the TEBV viewing chamber with associated components. The viewing chamber top features a square void designed to accommodate a thin glass slide (part 2, dimensions: 17.5 mm 19 mm) for direct visualization. The core chamber component (part 5) accommodates four TEBVs. Positioned at the chamber corners are four pedicle screws and nuts (part 1 and 8), while O-rings (part 3) are inserted into the grooves to ensure a secure seal. The two side ports (part 4) are tailored for side loop perfusion. In experiments where imaging capabilities are unnecessary, the viewing chamber top can be substituted with the same piece used for the bottom of the chamber (part 7). Figure created in Biorender (https://BioRender.com).
Fig 3.
Design and schematic of the TEBV PDMS clamps.
(A) Mold for polydimethylsiloxane (PDMS) clamps. (B) Schematic of the PDMS clamps positioned within the TEBV chamber, showing how four clamps are required to securely hold the TEBVs in place.
Fig 4.
Increased endothelial cell seeding density and extended rotation duration lead to more uniform endothelial cell coverage within TEBV lumens under static conditions.
(A) Representative images of TEBV cross-sections injected with RFP-HUVECs at low (1 105 ECs/cm2), medium (1.5
105 ECs/cm2), and high (2
105 ECs/cm2) densities, with rotation durations ranging from 3 to 12 hours. Asymmetric adhesion of RFP-HUVECs is observed in the 3-hour rotation group. Scale bar: 200 µm. (B) Assessment of circumferential coverage revealed increased luminal coverage in the 12-hour rotation group. TEBVs fabricated with cell density of 1.5
105 ECs/cm2 with a 12-hour rotation period resulted in the highest endothelial cell coverage. Significance was determined by two-way ANOVA. Mean ± S.D., n = 3 TEBVs (3-hour rotation), n = 4, 3, 4 TEBVs (12-hour rotation). *P < 0.05 (C) Representative images of TEBV cross-sections injected with 1.5
105 ECs/cm2 and rotated for 3, 6, 12 hours before fixation. Scale bar: 200 µm. (D) Evaluation of circumferential coverage of TEBVs as a function of rotation duration. Prolonged 12-hour perfusion did not lead to a significant increase in EC coverage under static culture. Mean ± S.D., n = 9, 4, 11 TEBVs. Since the two-way ANOVA did not detect an effect due to seeding density between 1-2
105 cells/cm2, the coverage at different seeding densities in Fig 4B were pooled for the 3-hour and 12-hour rotation groups and compared with the 6 h rotation time at 1.5
105 cells/cm2 using a one-way ANOVA. **P < 0.01. The raw data used to generate this figure are provided in Supporting Information S4 Data.
Fig 5.
Gradual ramping perfusion promotes endothelium stability and rapid start perfusion causes endothelial cell loss.
(A) Shear stress applied to each TEBV over a 3-hour period of flow application. The graph on the left shows a rapid increase to a steady shear stress, while the graph on the right illustrates a stepwise increase in shear stress. (B) Evaluation of circumferential EC coverage between TEBVs with different rotation periods and flow application methods. TEBVs in the 24h perfusion groups were subjected to the two different perfusion schemes after EC seeding. n = 3 TEBVs. Data were analyzed using a two-way ANOVA, and no significant differences were observed. (C) Representative images of TEBV cross-sections injected with RFP-HUVECs at the density of 1.5 105 ECs/cm2 and rotated for 12 hours. After 7 days perfusion at a flow rate of 0.5 ml/min per TEBV (0.4 Pa shear stress), no EC signal was observed in the rapid start group. Scale bar: 200 µm. (D) TEBVs subjected to a gradual increase in perfusion exhibit greater circumferential EC coverage when compared to the group with rapid initiation of perfusion after 7 days. Significance was determined by Student’s t-test. mean ± S.D., n = 3, 4 TEBVs. **P < 0.01. The raw data used to generate this figure are provided in Supporting Information S4 Data.
Fig 6.
The trajectory of a microcarrier bead under specific rotational conditions.
Parameters included a rotational speed of 35 rpm, fluid viscosity g/(cm·s), microcarrier radius
µm, and density of the microcarrier,
g/cm3. The rotating wall vessel radius was 5 cm. The bead started at an initial position of r
cm and 90°.
Fig 7.
Effect of initial radial position and particle density on movement of a particle in a TEBV.
(A) The trajectories of cells initiated from radial positions of 0.001 cm, 0.005 cm, 0.01 cm, and 0.02 cm at a rotation speed of 15 rph. The viscosity of cell culture medium at 37°C was 0.0085 g/(cm·s). The inner TEBV wall, located at 0.04 cm, is indicated by a dashed red line. (B) The trajectories of cells starting from the same radial positions of 0.001 cm, 0.005 cm, 0.01 cm, and 0.02 cm at an increased rotation speed of 20 rph. The viscosity of cell culture medium at 37°C was 0.0085 g/(cm·s). The inner TEBV wall is highlighted by a dashed red line. (C) The trajectories of cells initiated from radial positions of 0.001 cm, 0.005 cm, 0.01 cm, and 0.02 cm at a rotation speed of 24 rph. The inner TEBV wall, located at 0.04 cm, is indicated by a dashed red line. (D) At 30 rph, the trajectories of cells starting from the same radial positions of 0.001 cm, 0.005 cm, 0.01 cm, and 0.02 cm. The dashed red line marks the inner TEBV wall at a radius of 0.04 cm. (E) At 15 rph, the trajectory represented in red corresponds to cells with a higher density, which forms a larger circular pattern compared to the trajectory of cells with a density of 1.05 g/cm3. (F) At a high rotation speed of 360 rph, the cell trajectories become more confined, indicating that the rotation speed has an impact on the trajectory formation, irrespective of the cell density.
Fig 8.
Evaluation of endothelialization and in situ imaging of TEBVs over time.
(A) Evaluation of endothelialization at selected timepoints. RFP-HUVECs remained adhered throughout the duration of perfusion. mean ± S.D., n = 3, 3, 3, 4 TEBVs. Data were analyzed using a one-way ANOVA, and no significant differences were observed. The 7-day gradual ramping results presented here are the same as those shown in Fig 5D, as these experiments were conducted simultaneously. (B) In situ imaging of TEBVs over a 7-day perfusion period using the 5X objective lens. Scale bar: 500 µm. The raw data used to generate this figure are provided in Supporting Information S4 Data.
Fig 9.
Maintenance of endothelium within TEBVs following perfusion.
(A) Cross-sectional view of TEBVs fabricated with 1.5 105 ECs/cm2 and perfused at a flow rate of 0.5 ml/min per TEBV (0.4 Pa shear stress) for 7 days. TEBVs show intact endothelium positive for vWF by immunostaining. Scale bar: 200 µm. (B) En face sections of TEBVs show HUVECs forming tight junctions with VE-Cadherin localization and aligning with the flow direction in the lumen after 7 days of perfusion under 0.4 Pa shear stress. Scale bar: 200 µm, 50 µm. (C) ECs show alignment in the direction of flow after perfusion.
Fig 10.
Endothelial cells in the TEBVs respond to inflammatory stimulus.
(A) THP-1 monocytes (white arrows) show increased adhesion to endothelium after TNF-α treatment. Scale bar: 250 µm. Significance was determined by Student’s t-test. mean ± S.D., n = 4 TEBVs. *P < 0.05 (B) Quantification of ICAM-1 expression in TEBVs after TNF-α treatment. Scale bar: 200 µm. mean ± S.D., n = 5, 9 TEBVs. ***P < 0.001. The raw data used to generate this figure are provided in Supporting Information S4 Data.