Orthotopic transplantation of the bioengineered lung using a mouse-scale perfusion-based bioreactor and human primary endothelial cells

Abstract

Whole lung engineering and the transplantation of its products is an ambitious goal and ultimately a viable solution for alleviating the donor-shortage crisis for lung transplants. There are several limitations currently impeding progress in the field with a major obstacle being efficient revascularization of decellularized scaffolds, which requires an extremely large number of cells when using larger pre-clinical animal models. Here, we developed a simple but effective experimental pulmonary bioengineering platform by utilizing the lung as a scaffold. Revascularization of pulmonary vasculature using human umbilical cord vein endothelial cells was feasible using a novel in-house developed perfusion-based bioreactor. The endothelial lumens formed in the peripheral alveolar area were confirmed using a transmission electron microscope. The quality of engineered lung vasculature was evaluated using box-counting analysis of histological images. The engineered mouse lungs were successfully transplanted into the orthotopic thoracic cavity. The engineered vasculature in the lung scaffold showed blood perfusion after transplantation without significant hemorrhage. The mouse-based lung bioengineering system can be utilized as an efficient ex-vivo screening platform for lung tissue engineering.

Keywords: Box-counting analysis; Decellularization; Lung bioengineering; Perfusion-based Bioreactor; Transplantation; Vascular engineering.

Figure 1

Decellularization of the mouse heart–lung block. Cannulation layout for the mouse heart–lung block (a). Representative images during the microscopic surgery (b and c). The pulmonary artery (PA) catheter was inserted via a window of the right ventricle (b). The tracheal catheter was inserted and secured just below the larynx (c). The cannulated heart–lung block was removed from the thoracic cavity (d). Sequential detergent administration was performed from the PA cannula and tracheal cannula.

Figure 2

Representative histological images of the native mouse lung and the decellularized mouse lung. Native lungs (a–f). Decellularized lungs (g–l). Hematoxylin–eosin staining (a and g). Masson-trichrome staining (b and h). Elastica-Masson staining (c and i). Periodic-acid-Schiff staining (d and j). Immunohistochemistry of laminin (e and k). Immunofluorescence of CD31 (f and l). Bars; 200 µm (a–e, g–k), bars; 100 µm (f and l).

Figure 3

Perfusion-based bioreactor culture of decellularized mouse lungs. Schematic illustration of in-house developed perfusion-based bioreactor (a). Decellularized mouse lungs were connected to the media circuit and the culture media was perfused using a pulsatile pump at a speed of 2 mL/min (b). Decellularized mouse lungs were incubated for 2–3 days in a glass chamber (c). After the perfusion organ culture, the recellularized mouse heart–lung blocks were harvested (d). A representative image of recellularized mouse lungs using 10 million HUVECs (e).

Figure 4

Comparison of revascularized lung scaffold using different numbers of HUVECs. 15 million (a–c), 30 million (d–f), or 60 million (g–i) of HUVECs were injected, and perfusion culture of the HLB was performed for 2 days. Representative images of hematoxylin–eosin staining (a, d, and g), immunofluorescence of CD31 (b, e, and h), and TUNEL staining (c, f, and i). Quantitative PCR was performed to quantify the gene expression difference of HUVECs before and after perfusion-bioreactor culture (j), from three biological replicate experiments. BR; Bioreactor. Arrowheads; TUNEL positive cells. Bars; 200 µm (a, d, and g), 50 µm (b, c, e, f, h, and i).

Figure 5

Fractal analysis of the recellularized lungs. A representative HE image of a recellularized lung (a). The scaffold area was selected, and the pixel numbers were counted (b). The HE images were converted into grayscale and binarized (c). The percentages of cell area in scaffold area are indicated in d. The schematic procedure of the box-counting methods (e). Log–log plotting of the box-counting obtained from the lung scaffold recellularized with 30 million HUVECs (f). Summary of fractal dimensions calculated from different samples (g).

Figure 6

Transmission electron microscope of engineered mouse lungs. Representative high-magnification images of native mouse lungs (a and b), decellularized mouse lungs (c), and recellularized mouse lungs using 30 million HUVECs and perfusion-bioreactor (d–h). Panel h explains how the caliber of the vascular tube was measured. The number of cells that were attached to the vascular tube with a specific diameter (bin width = 2) is shown as a histogram (i). *Red blood cell, Cap.: Capillary endothelial cells, ATII: Alveolar Type II cells, PA: Pulmonary artery.

Figure 7

Orthotopic transplantation of revascularized mouse lung. Representative intraoperative images of left lung transplantation of decellularized lungs (a–c) and recellularized mouse lungs using 30 million HUVECs and perfusion-bioreactor (d–f). Each panel is; before de-cramping the pulmonary artery (a and d), the ventral side of the transplanted left lung 1 min after reperfusion, and the dorsal side of the transplanted left lung 1 min after reperfusion. Representative histological images of transplanted lungs (g–l). Hematoxylin–eosin staining (g, h, i, and j). Immunohistochemistry of CD31 (k). Bars; 500 µm (g), 1000 µm (i), 100 µm (h, j and k). Arrowheads; Red blood cells.