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Hui Lin, Guoying Sun, Hong he, Jennifer Elisseeff, Samuel Yiu; Functional rabbit acinar cells cultured on decellularized lacrimal gland scaffold. Invest. Ophthalmol. Vis. Sci. 2013;54(15):915.
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© ARVO (1962-2015); The Authors (2016-present)
Aqueous tear-deficient dry eye is a multifactorial chronic disorder in which the lacrimal glands fail to produce enough tears to maintain a healthy eye surface. The treatment primarily involves palliation using ocular surface lubricant. Construction of a bioengineered lacrimal gland having functional secretory epithelial cells is a potentially promising option for providing long-term relief to severe dye eye patients. Previously, we have successfully decellularized the rabbit lacrimal gland to create a novel scaffold for 3 dimensional culture of acinar cell. Current study investigates the secretory function of cells seeded and cultured in decellularized lacrimal gland tissue.
NZW rabbit lacrimal glands purchased from Pel Freeze were treated with 1% sodium dodecyl sulfate or 1% Triton X-100 for 36 to 40 hours for decellularization. The rabbit lacrimal gland acinar cells were isolated by enzyme mixtures of collagenase, hyaluronidase and DNase. Subsequently, the isolated cells were layered over 5% Ficoll and centrifuged at 50g for purification. Prior to seeding, the decellularized lacrimal gland scaffold was treated with Matrigel, fibronectin or collagen I in different groups, and with Peter’s complete medium (PCM) in control group. The lacrimal constructs were cultured in PCM for up to 2 weeks. The live/dead assay for cell viability was performed on day 2, 7 and 14. The beta-hexosaminidase secretion assay for assessing acinar cell function was performed on day 2 and 7. RNA was also extracted to evaluate the gene expression of beta-hexosaminidase.
Majority of the lacrimal acinar cells in the decellularized scaffold survived for at least 7 days in all groups, however, cell viability decreased at day 14. Use of extracellular matrix to treat the scaffold during the seeding process helped improve cell viability. Beta-hexosaminidase activity increased in the supernatant medium following stimulation with 100uM carbachol on day 2 and 7. Carbachol stimulation was also found to up-regulate mRNA level of beta-hexosaminidase in the reseeded cells.
Isolated acinar cells successfully cultured in decellularized lacrimal gland tissue and maintained the cell viability and secretory function for at least 1 week. Further investigation is required to optimize the decellularization and seeding process to improve the duration of cell viability and function.
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