Massie Isobel, Spaniol Kristina, Barbian Andreas, Poschmann Gereon, Stühler Kai, Geerling Gerd, Metzger Marco, Mertsch Sonja, Schrader Stefan
Laboratory of Experimental Ophthalmology, University Clinic Düsseldorf, Düsseldorf, Germany.
Department of Ophthalmology, University Clinic Düsseldorf, Düsseldorf, Germany.
Invest Ophthalmol Vis Sci. 2017 Oct 1;58(12):5564-5574. doi: 10.1167/iovs.16-20759.
Dry eye syndrome (DES) can cause blindness in severe cases, but mainly palliative treatments exist. A tissue-engineered lacrimal gland (LG) could provide a curative treatment. We aimed to evaluate decellularized porcine jejunum (SIS-Muc) as a scaffold for porcine LG epithelial cells.
To evaluate SIS-Muc as a potential scaffold, basement membrane proteins in SIS-Muc and native LG were compared (immunohistochemistry [IHC]). Porcine LG epithelial cells cultured on plastic were characterized (immunocytochemistry), and their culture supernatant was compared with porcine tears (proteomics). Epithelial cells were then seeded onto SIS-Muc in either a static (cell crown) or dynamic culture (within a perfusion chamber) and metabolic (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and secretory capacities (β-hexosaminidase assay), protein expression (IHC), and ultrastructure transmission electron microscopy (TEM) compared in each.
Collagen IV and laminin were found in both native LG and SIS-Muc. When cultured on plastic, LG epithelial cells expressed pan-cytokeratin, Rab3D, HexA, and produced mucins, but lysozyme and lactoferrin expression was nearly absent. Some porcine tear proteins (lipocalin-2 and lactoferrin) were found in LG epithelial cell culture supernatants. When LG cells were cultured on SIS-Muc, metabolic and β-hexosaminidase activities were greater in dynamic cultures than static cultures (P < 0.05). In both static and dynamic cultures, cells expressed pan-cytokeratin, Rab3D, lysozyme, and lactoferrin and produced mucins, and TEM revealed cell polarization at the apical surface and cell-cell and cell-scaffold contacts.
SIS-Muc is a suitable scaffold for LG cell expansion and may be useful toward reconstruction of LG tissue to provide a curative treatment for DES. Dynamic culture enhances cell metabolic and functional activities.
干眼症（DES）在严重情况下可导致失明，但目前主要是姑息治疗。组织工程化泪腺（LG）有望提供一种治愈性治疗方法。我们旨在评估脱细胞猪空肠（SIS-Muc）作为猪LG上皮细胞支架的可行性。
为评估SIS-Muc作为潜在支架的性能，对SIS-Muc和天然LG中的基底膜蛋白进行比较（免疫组织化学[IHC]）。对在塑料上培养的猪LG上皮细胞进行特性分析（免疫细胞化学），并将其培养上清液与猪泪液进行比较（蛋白质组学）。然后将上皮细胞接种到SIS-Muc上，分别进行静态培养（细胞冠）或动态培养（在灌注室内），并比较每种培养方式下的代谢能力（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴盐）、分泌能力（β-己糖胺酶测定）、蛋白质表达（IHC）和超微结构（透射电子显微镜[TEM]）。
在天然LG和SIS-Muc中均发现了IV型胶原蛋白和层粘连蛋白。当在塑料上培养时，LG上皮细胞表达泛细胞角蛋白、Rab3D、己糖胺酶A，并产生粘蛋白，但几乎不表达溶菌酶和乳铁蛋白。在LG上皮细胞培养上清液中发现了一些猪泪液蛋白（lipocalin-2和乳铁蛋白）。当LG细胞在SIS-Muc上培养时，动态培养中的代谢和β-己糖胺酶活性高于静态培养（P < 0.05）。在静态和动态培养中，细胞均表达泛细胞角蛋白、Rab3D、溶菌酶和乳铁蛋白，并产生粘蛋白，TEM显示细胞在顶端表面极化，存在细胞间和细胞与支架的接触。
SIS-Muc是LG细胞扩增的合适支架，可能有助于LG组织重建，为DES提供治愈性治疗。动态培养可增强细胞代谢和功能活性。
