Figure 2. Engraftment of the bioengineered gland germ.

(a) Schematic representation of the methods used to engraft a bioengineered germ into an adult lacrimal gland-defect mouse. (b) Phase-contrast images of bioengineered lacrimal gland germs with a PGA monofilament inserted after 2 days of culture. Scale bar, 100 μm. (c) Photographs of procedures for bioengineered gland germ engraftment. The extra-orbital lacrimal gland was completely removed (left, centre-left, white dotted line). The boxed area in the centre-left panel is the engraftment site shown at a higher magnification in the centre-right panel. The boxed area in the centre-right panel is shown at a higher magnification in the right panel. The PGA monofilament was inserted into the host lacrimal excretory duct (centre-right, right). The white dotted line in the right panel indicates the host lacrimal duct. The arrowhead in the centre-right and right panels indicates the engrafted bioengineered lacrimal gland with a PGA monofilament. Scale bar, 1 mm. (d) Photographs of bioengineered lacrimal glands at 30 days after engraftment (left). Scale bar, 500 μm. (e) Photographs of a bioengineered harderian gland at 30 days after engraftment (left). The boxed area in the left panel is shown at a higher magnification in the right panel. Scale bar, 500 μm. (f) Photographs of a bioengineered lacrimal gland reconstituted using epithelial cells and mesenchymal cells from GFP-transgenic mice. A merged image (left) and a fluorescent image (right) are shown. Scale bar, 500 μm. (g) Analysis of the duct connection using Evans blue dye injection. The arrowhead indicates the injection site. Scale bar, 500 μm. (h) Histological analysis of the duct connection. 3D images of the bioengineered lacrimal gland reconstituted between DsRed transgenic mice-derived epithelial cells (red) and normal mice-derived mesenchymal cells. Bioengineered lacrimal glands developed in vivo with the correct connection (arrowhead) to the recipient lacrimal excretory duct. FITC-gelatin conjugate (green), DAPI (blue) and excretory duct (dotted line) are shown. Scale bar, 100 μm. (i) HE-stained lacrimal gland (left) and harderian gland (right). A natural (upper) and bioengineered (lower) gland after 30 days of engraftment are shown. Scale bar, 50 μm.