The integration of 3-D cell printing and mesoscopic fluorescence molecular tomography of vascular constructs within thick hydrogel scaffolds

Abstract

Developing methods that provide adequate vascular perfusion is an important step toward engineering large functional tissues. Meanwhile, an imaging modality to assess the three-dimensional (3-D) structures and functions of the vascular channels is lacking for thick matrices (>2 ≈ 3 mm). Herein, we report on an original approach to construct and image 3-D dynamically perfused vascular structures in thick hydrogel scaffolds. In this work, we integrated a robotic 3-D cell printing technology with a mesoscopic fluorescence molecular tomography imaging system, and demonstrated the capability of the platform to construct perfused collagen scaffolds with endothelial lining and to image both the fluid flow and fluorescent-labeled living endothelial cells at high-frame rates, with high sensitivity and accuracy. These results establish the potential of integrating both 3-D cell printing and fluorescence mesoscopic imaging for functional and molecular studies in complex tissue-engineered tissues.

Fig. 1

3-D vascular channel construct in perfusion system. (A) Schematics of channel construction procedure and cross-section view. (B) The actual perfusion pump system. Flow chamber is marked by a yellow rectangle. (C) Specially designed flow chamber. (D) Schematic diagram of cross –section view of the printed vascular channel construct in the flow chamber.

Fig. 2

Optimal regularization parameter chosen from L-curve analysis. (A) L-curve from experimental reconstructions is used to balance the size of residual (2-norm of Ax′ – b) and size of regularized solution (2-norm of x′). A is the whole Jacobian matrix computed based on a MC forward model; x′ is reconstructed fluorescence distribution; b is obtained from the fluorescence measurement. (B) Curvature of L-curve [22] with respect to regularization parameters is used to choose the best regularization parameter.

Fig. 3

Fluorescence inclusions at different depths can be resolved by MFMT system. (A) Reconstructed depths recovery at 488nm excitation and 658nm excitation in comparison to actual depths. (B) Comparison of reconstructed diameters at 488nm excitation and 658nm excitation with actual diameters in different depths.

Fig. 4

In-vitro validation of vascular structure mimicking in a collagen scaffold. (A) Schematic diagram of phantom design shows the locations of two capillaries filled with fluorescence dye in the collagen scaffold. (B) 3-D Surface based on threshold segmentation (T=25%) of reconstructed fluorescent capillaries by MFMT. (C) Localizations of glass walls of two capillaries in the collagen scaffold from micro CT scanner. (D) and (E) Merged 3-D image of (B) and (C). The red dashed cuboid shows the reconstructed volume (4mm×6mm×4mm). The green dashed cuboid shows the rendering volume by confocal microscope (0.89mm×0.89mm×3mm). (F-H) show top view, front view, and right side view of (E). (I) 3-D maximum intensity projection of capillaries obtained by confocal microscope (objective 10x, LSM 510Meta; Zeiss). The pink dashed lines shows the top capillary, and the pink dashed circle corresponds to the position of the deeper inclusion as located by micro-CT.

Fig. 5

Time-lapse phase contrast images of HUVECs under fluidic condition by a wide-field microscope (objective 10x). Perfusion started with gentle flow (2μl/min). Cells were round at this point. Flow rate was gradually increased until it reached 20ml/min. After 7 hours of perfusion, flow rate was increased to 200μl/min and HUVECs began to spread out. After 12 hours of perfusion, flow rate was increased to 2ml/min and maintained thereafter.

Fig. 6

(A) Schematic diagram of the printed vascular channel construct. (B) Fluorescence image of the printed vascular channel construct by a wide-field microscope (objective 4x). HUVECs were visualized in red color, beads flow in green color.

Fig. 7

Imaging of 3-D perfused vascular channel construct in thick collagen scaffold. (A) Localizations and dimensions of the open lumen from a micro CT scanner. (B) 3-D surface rendering based on threshold segmentation (T=35%) from MFMT image reconstruction of Open lumen (green) and luminal endothelial lining (red). (C) Fluorescence image of luminal endothelial lining in the printed vascular channel by a wide-field microscope (objective 4x). (D) 2-D maximum intensity Z projection of MFMT reconstruction of luminal endothelial lining. Scale: 1mm. (E) Merged 3-D image of (A) and (B). The reconstructed volume is marked by the blue dashed cuboid (2.5mm×7mm×3mm). (F) Merged 3-D image of reconstructed open lumen and (A). (G-J) are top view and right side view of (E) and (F), respectively.