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Masatoshi Hirayama, Tetsuya Kawakita, Takashi Tsuji, Kazuo Tsubota; Regeneration Of Lacrimal Gland By Bioengineered Organ Germ Method. Invest. Ophthalmol. Vis. Sci. 2012;53(14):635.
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© ARVO (1962-2015); The Authors (2016-present)
Regenerated lacrimal gland transplantation is thought to be an ultimate therapeutic model for dry eye disease. We developed a three-dimensional organ-germ culture method for bioengineering teeth. This research was conducted to confirm whether lacrimal gland could be bioengineered by such organ culture system in vitro.
Lacrimal gland germs were dissected from ED16 mice. Isolated lacrimal gland germs were incubated in 1.2 U/ml dispase II and 20 U/ml DNase I for 1.3 min at room temperature. The epithelial and mesenchymal tissues were separated using a fine needle. The epithelial tissues were treated twice at 37°C for 15 min in 100 U/ml collagenase I -PBS(-), then in Ca2+- and Mg2+-phosphate-buffered saline (PBS(-)) supplemented with 0.25% trypsin and 20 U/ml DNase I for 5 min at 37°C, and dissociated into single cells by gentle pipetting. Single cells of mesenchymal origin were also prepared by treatment with PBS(-) supplemented with 0.25% trypsin, 50 U/ml collagenase I, and 20 U/ml DNase I at 37°C for 10 min. The cell precipitate were mixed and injected (0.3-0.4 μl) into a collagen gel drop. The bioengineered lacrimal gland germs were incubated for 5 min at 37°C, placed on a cell culture insert and the explants were then incubated at 37°C in a humidified atmosphere of 5% CO2. The histological analysis of reconstituted explants was conducted.
After 2 or 3 days cultivation, we observed formation of the initial budding of bioengineered lacrimal gland and the germ was developed with branching. By the histological analysis, we observed acinar and duct like structure.
The bioengineered lacrimal gland germ using bioengineered organ germ method generated a structurally correct gland in vitro organ culture.
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