Densely vascularized thick 3D tissue shows enhanced protein secretion constructed with intermittent positive pressure

Abstract

Constructing a dense vascular endothelial network within engineered tissue is crucial for successful engraftment. The present study investigated the effects of air-compressing intermittent positive pressure (IPP) on co-cultured mesenchymal stem cells and vascular endothelial cells and evaluated the potential of IPP-cultured cell sheets for transplantation therapy. The results demonstrated that the IPP (+) group exhibited a denser vascular endothelial network and significantly increased cell sheet thickness compared to the IPP (-) group. Furthermore, in vivo experiments showed that IPP-cultured cell sheets enhanced the secretion of Gaussian luciferase by genetically modified mesenchymal stem cells. These findings highlight the IPP method as a technique that simultaneously enables the thickening of planar tissues and the construction of vascular networks. This approach demonstrates promise for fabricating functional, transplantable, and thick tissues with dense vascularization and a high capacity for protein secretion, paving the way for novel applications in regenerative medicine.

Fig. 1. Comparison in planar co-cultured condition between applied intermittent positive pressure and non-pressurization.

a Diagram of the co-cultured planar condition experiment. b Representative image of GFP-HUVECs (green) under intermittent positive pressure (IPP) (−) on day 1 (left) and day 3 (middle, right). Scale bar: 500 µm. c Representative image of GFP-HUVECs (green) under IPP (+) on day 1 (left) and day 3 (middle, right). Scale bar: 500 µm. d The total number of endothelial network junctions on day 3 (n = 8, measured from 8 independent culture dishes, with the mean of 3 randomly selected fields of view per dish, p = 0.10, Cohen’s d = 0.66). e The length of the endothelial network on day 3 (n = 8, measured from 8 independent culture dishes, with the mean of 3 randomly selected fields of view per dish, p = 0.09, Cohen’s d = 0.90). f The ratio of lactate production to glucose consumption (L/G ratio) on day 3 (n = 9, independent samples, **p < 0.01, Cohen’s d = 0.90). g The total number of the cells on day 3 (n = 9, independent samples, **p < 0.01, Cohen’s d = 0.69). h The percentage of GFP-HUVECs of the total cell number in the co-cultured cells in planar conditions (n = 9, independent samples, p = 0.50, Cohen’s d = 0.10). d–h Plots in the graph show the value of each experiment and the bar shows the average. L/G: The ratio of lactate production to glucose consumption.

Fig. 2. Comparison of vascular endothelial cells network in co-cultured cell sheet condition applied intermittent positive pressure and non-pressurization.

a Diagram of the experiment on the state of the co-cultured cell sheets. According to the schedule shown in the figure, cell seeding, cell sheet production, and evaluation of each item were carried out. b Representative images of GFP-HUVECs under IPP (−) on day 1 (left) and day 5 (middle, right). Scale bar: 500 µm. c Representative images of GFP-HUVECs (green) under IPP (+) on day 1 (left) and day 5 (middle, right). Scale bar: 500 µm. d Total number of endothelial network junctions on day 5 (n = 8, measured from 8 independent culture dishes, with the mean of 3 randomly selected fields of view per dish, **p < 0.01, Cohen’s d = 0.91). e Total length of the endothelial network on day 5 (n = 8, measured from 8 independent culture dishes, with the mean of 3 randomly selected fields of view per dish, **p < 0.01, Cohen’s d = 1.29). d, e Plots in the graph show the value of each experiment and the bar shows the average.

Fig. 3. Comparison of morphological and compositional changes of co-cultured cell sheets applied intermittent positive pressure and non-pressurization.

a 3D-OCT images of the central section of a co-cultured cell sheet on day 5 days of IPP (−) (left) and IPP (+) (right). Scale bar: 50 µm. b The average thickness of cell sheets on day 5 measured using OCT images (n = 8, average thickness measured at 3 random locations on each cell sheet, **p < 0.01, Cohen’s d = 1.43). c Representative cross-sectional image of co-cultured cell sheets on day 5 cultured under IPP (−) (left) and IPP (+) (right) on day 5. Magnified views of the sections are shown alongside. The cell sheets were stained with hematoxylin and eosin (HE-stained). Scale bar: 100 µm. d Representative cross-sectional image of co-cultured cell sheets on day 5 cultured under IPP (−) (left) and IPP (+) (right). Magnified views of the sections are shown alongside. The cell sheet was stained with Sirius red. Scale bar: 100 µm. e The thickness of cell sheets on day 5 measured using HE-stained sections (n = 6, average thickness measured at 3 random locations on each cell sheet, *p < 0.05, Cohen’s d = 0.53). f The amount of collagen contained in the cell sheet (n = 4, independent samples, *p < 0.05, Cohen’s d = 0.91). g The total number of the cells in the cell sheet on day 5 (n = 8, independent samples, **p < 0.01, Cohen’s d = 0.95). h The percentage of GFP-HUVECs of the total cell number in the co-cultured cell sheet (n = 5, independent samples, p = 0.19, Cohen’s d = 1.47). i The percentage of cell viability in the co-cultured cell sheets on day 5 (n = 5, independent samples, **p < 0.01, Cohen’s d = 2.2). j The forward scatter of hASCs (left) (n = 3, independent samples, p = 0.91, Cohen’s d = 0.05) and GFP-HUVECs (right) (n = 3, independent samples, p = 0.43, Cohen’s d = 0.17) measured using a flow cytometry.

Fig. 4. Comparison of air saturation at the bottom of the dish applied intermittent positive pressure and non-pressurization.

a Schematic diagram for the measurement of air saturation at the bottom of the dish in the area surrounding the cell sheet. b Representative data showing relative changes in air saturation. c Relative air saturation ratio of each experiment measured under IPP (−) / IPP (+) after 12 h, 24 h, and 48 h (n = 3).

Fig. 5. Comparison of RNA expression in co-cultured cell sheets between intermittent positive pressure and non-pressurization.

a The relative expression of RNAs associated with endothelial mechanotransduction induced by shear stress (n = 3, independent samples). b The relative expression of RNAs associated with cell cycles (n = 3, independent samples). c The relative expression of RNAs associated with the extracellular matrix (ECM) and hemidesmosome (n = 3, independent samples). d The relative expression of RNAs associated with hypoxia-inducible factor (HIF) family (n = 3, independent samples). e The relative expression of RNAs associated with angiogenesis factors (n = 3, independent samples). a–e The comparison was made between co-cultured cell sheets cultured under IPP (−)/IPP (+). The cell sheets were cultured for 5 days.

Fig. 6. Effects of mild hypoxia on intermittent positive pressure-induced responses in co-cultured cell sheets and inhibition of shear stress pathways by L-NAME.

a The average thickness of cell sheets under mild hypoxia on day 5 measured using OCT images [IPP (−) vs IPP (+) (µm): n = 6, p = 0.94, Cohen’s d = 0.02]. b Total number of endothelial network junctions on day 5 [IPP (−) L-NAME (−) vs IPP (−) L-NAME (+) (count/mm²): n = 6, p = 0.56, Cohen’s d = 0.05], [IPP (−) L-NAME (+) vs IPP (+) L-NAME (+) (count/mm²): n = 6, p = 0.44, Cohen’s d = 0.50], [IPP ( + ) L-NAME (−) vs IPP (+) L-NAME (+) (count/mm²): n = 6, *p < 0.05, Cohen’s d = 1.14]. c The total length of the endothelial network on day 5 [IPP (−) L-NAME (−) vs IPP (−) L-NAME (+) (mm/mm²): n = 6, p = 0.84, Cohen’s d = 0.06], [IPP (−) L-NAME (+) vs IPP (+) L-NAME (+) (mm/mm²): n = 6, p = 0.31, Cohen’s d = 0.56], [IPP (+) L-NAME (−) vs IPP (+) L-NAME (+) (mm/mm²): n = 6, *p < 0.05, Cohen’s d = 1.31]. d Representative image of GFP-HUVECs (green) of each group on day 5. Scale bar: 500 μm. e The average thickness of cell sheets on day 5 measured using OCT images of each group [IPP (−) L-NAME (−) vs IPP −) L-NAME (+) (µm); 35.5 ± 4.9 vs 35.3 ± 4.9, n = 6, p = 0.91, Cohen’s d = 0.04], [IPP (−) L-NAME (+) vs IPP (+) L-NAME (+) (µm); 35.3 ± 4.9 vs 41.4 ± 6.3, n = 6, *p < 0.05, Cohen’s d = 1.09], [IPP (+) L-NAME (−) vs IPP (+) L-NAME (+) (µm); 40.4 ± 5.4 vs 41.4 ± 6.3, n = 6, p = 0.58, Cohen’s d = 0.17]. a, eN = 6, representing 6 cell sheets, with the average thickness measured at 3 random locations on each sheet. b, cN = 6, representing 6 cell sheets, with the evaluation of the endothelial vascular network performed in 3 randomly selected fields of view per sheet.

Fig. 7. Functional comparison of intermittently positive pressurized and non-pressurized cell sheets transplanted subcutaneously in rats.

a Diagram of the experiment on the transplantation of co-cultured cell sheets. b, e Representative stereomicroscope images of a co-cultured cell sheet. The cell sheets were detached from the dish on day 5. Scale bar: 1 mm. c, f Representative images of a co-cultured cell sheet cultured under just after transplantation (left) and 2 days after transplantation (right). The white arrowheads indicate the region of the engrafted cell sheet. Scale bar: 1 mm. d, g Representative fluorescence image of a cell sheet (c or f) on day 2. The cell sheets are indicated in red as a consequence of m-Scarlet-I. Scale bar: 1 mm. h Representative images of fluorescence immunostained sections of the engrafted cell sheet 2 days after transplantation, IPP (−) (left) and IPP (+) (right). The cell sheets are labeled in red with the anti-RFP antibody. Scale bar: 100 µm. i Thickness of the engrafted cell sheets. (n = 5, average thickness measured at 3 random locations on each cell sheet, *p < 0.05, Cohen’s d = 1.06). j The intensity of Gaussia luciferase (GLuc) luminescence in blood serum on day 2 following cell sheet transplantation. (n = 6, independent samples, p = 0.13, Cohen’s d = 0.96). k Representative image of a transplanted co-cultured cell sheet captured by a stereomicroscope (left) and fluorescence microscope (middle). The merged image is shown on the right. The engrafted GFP-HUVECs formed tubular structures, which are indicated by the white arrows. It appears that the tube was filled with blood from the recipient rat. Scale bar: 100 µm. l Perfusion ratio of transplanted cell sheets on day 2 following transplantation. (n = 7, independent samples, p = 0.54, Cohen’s d = 0.60). GLuc: Gaussia luciferase.

Fig. 8. Comparison of the ability of glucose-responsive insulin secretion of iGL cells tri-cultured in planar condition under intermittent positive pressure and non-pressurization.

a Diagram of tri-culture planar condition experiment including iGLs. b Representative image of tri-cultured cells under IPP (−; left) and IPP (+; right). The red color indicates iGLs, the green color indicates GFP-HUVECs and the blue color indicates the nucleus. Scale bar: 100 µm. c Glucose-stimulated insulin secretion index of iGLs cultured in planar condition under IPP (−) / IPP (+) on day 3. (n = 4, independent samples, *p < 0.05, Cohen’s d = 1.36).

Fig. 9. A pressurization bioreactor system used in the experiment.

a A pressurization bioreactor system, consists of a control unit, a pressurizing pump unit, and a custom-made pressure chamber. Air is introduced into the chamber through the pump’s rotation via the tube. The volume of the chamber is calculated to be 803,840 mm³, based on its dimensions of 80 mm × 80 mm × 3.14 × 40 mm. A pressure of +80 mmHg was applied to the chamber every 180 s. IPP: Intermittent positive pressure.