Translational Center Regenerative Therapies (TLC-RT), Fraunhofer Institute for Silicate Research (ISC), Würzburg, Germany.
Department of Ophthalmology, University Hospital Würzburg, Würzburg, Germany.
Sci Rep. 2023 Jul 28;13(1):12261. doi: 10.1038/s41598-023-38927-8.
In vitro culture and generation of highly specialized goblet cells is still a major challenge in conjunctival 3D in vitro equivalents. A model comprising all physiological factors, including mucus-secreting goblet cells has the potential to act as a new platform for studies on conjunctival diseases. We isolated primary conjunctival epithelial cells and fibroblasts from human biopsies. 3D models were generated from either epithelial layers or a combination of those with a connective tissue equivalent. Epithelial models were investigated for marker expression and barrier function. Full-thickness models were analyzed for goblet cell morphology and marker expression via immunofluorescence and quantitative real-time PCR. Simple epithelial models cultured at the air-liquid interface showed stratified multi-layer epithelia with pathologic keratinization and without goblet cell formation. The combination with a connective tissue equivalent to generate a full-thickness model led to the formation of a non-keratinized stratified multi-layer epithelium and induced goblet cell differentiation. In our model, a high resemblance to natural conjunctiva was achieved by the combination of conjunctival epithelial cells with fibroblasts embedded in a collagen-hydrogel as connective tissue equivalent. In the future, our conjunctival in vitro equivalent enables the investigation of goblet cell differentiation, conjunctival pathologies as well as drug testing.
在体外培养和生成高度特化的杯状细胞仍然是结膜 3D 体外等同物的主要挑战。包含所有生理因素的模型，包括分泌粘液的杯状细胞，有可能成为结膜疾病研究的新平台。我们从人类活检中分离出原代结膜上皮细胞和成纤维细胞。从上皮层或其与结缔组织等同物的组合生成 3D 模型。对上皮模型进行标记物表达和屏障功能的研究。通过免疫荧光和实时定量 PCR 对全厚度模型进行杯状细胞形态和标记物表达的分析。在气液界面培养的简单上皮模型显示出具有病理性角化且没有杯状细胞形成的分层多细胞上皮。与生成全厚度模型的结缔组织等同物的组合导致非角化的分层多细胞上皮的形成，并诱导杯状细胞分化。在我们的模型中，通过将结膜上皮细胞与成纤维细胞嵌入胶原水凝胶中作为结缔组织等同物的组合，实现了与天然结膜的高度相似性。在未来，我们的结膜体外等同物将能够用于研究杯状细胞分化、结膜病理以及药物测试。
