在纤维蛋白凝胶包埋成纤维细胞的三维共培养体系中呼吸道上皮的分化
Differentiation of respiratory epithelium in a 3-dimensional co-culture with fibroblasts embedded in fibrin gel.
作者信息
Albers Stefanie, Thiebes Anja Lena, Gessenich Kai L, Jockenhoevel Stefan, Cornelissen Christian G
机构信息
Department of Tissue Engineering & Textile Implants, Institute for Applied Medical Engineering, Helmholtz Institute of the RWTH University Hospital, Pauwelsstr. 20, 52074 Aachen, Germany.
Department for Internal Medicine - Section for Pneumology, University Hospital Aachen, Pauwelsstraße 30, Aachen, Germany ; Department of Tissue Engineering & Textile Implants, Institute for Applied Medical Engineering, Helmholtz Institute of the RWTH University Hospital, Pauwelsstr. 20, 52074 Aachen, Germany.
出版信息
Multidiscip Respir Med. 2016 Mar 1;11:6. doi: 10.1186/s40248-016-0046-3. eCollection 2015.
BACKGROUND
Tracheal tissue engineering is a promising option for the treatment of tracheal defects. In a previous study we proved the suitability of fibrin gel as a scaffold for tracheal tissue engineering. This study investigates whether the differentiation of respiratory epithelium can be increased by culturing epithelial cells in a three dimensional system containing fibroblasts embedded into fibrin gel.
METHODS
Respiratory epithelial cells were isolated from porcine trachea, seeded onto a fibrin gel and kept in air-liquid-interface culture for 33 days. Morphology as well as pan-cytokeratin, MUC5AC and claudin-1 expression of cells cultured on pure fibrin gel were compared to culture on gels containing fibroblasts.
RESULTS
After two weeks, cells seeded on pure fibrin gel were multilayered, showed hyperproliferation and dedifferentiation. Co-cultured cells built up a pseudostratified epithelium. The differentiation and organization of epithelial structure improved with respect to time. After four weeks, morphology of the co-cultured respiratory epithelium resembled native tracheal epithelium. Immunohistochemistry showed that respiratory epithelium co-cultured with fibroblasts had an increasing similarity of pan-cytokeratin expression compared to native trachea. Cells cultured without fibroblasts differed in pan-cytokeratin expression from native trachea and did not show any improvement of differentiation. Immunohistochemical staining of MUC5AC and claudin-1 proved seeded cells being respiratory epithelial cells.
CONCLUSIONS
This study indicates that adding fibroblasts to fibrin gel positively influences the differentiation of respiratory epithelium.
背景
气管组织工程是治疗气管缺损的一种有前景的选择。在先前的一项研究中，我们证明了纤维蛋白凝胶作为气管组织工程支架的适用性。本研究调查在含有包埋于纤维蛋白凝胶中的成纤维细胞的三维系统中培养上皮细胞是否能增强呼吸道上皮的分化。
方法
从猪气管中分离呼吸道上皮细胞，接种到纤维蛋白凝胶上，并在气液界面培养33天。将在纯纤维蛋白凝胶上培养的细胞的形态以及全细胞角蛋白、MUC5AC和闭合蛋白-1的表达与在含有成纤维细胞的凝胶上的培养情况进行比较。
结果
两周后，接种在纯纤维蛋白凝胶上的细胞形成多层，表现出过度增殖和去分化。共培养的细胞形成假复层上皮。上皮结构的分化和组织随着时间的推移而改善。四周后，共培养的呼吸道上皮形态类似于天然气管上皮。免疫组织化学显示，与天然气管相比，与成纤维细胞共培养的呼吸道上皮在全细胞角蛋白表达上的相似性增加。在没有成纤维细胞的情况下培养的细胞在全细胞角蛋白表达上与天然气管不同，并且没有显示出分化的任何改善。MUC5AC和闭合蛋白-1的免疫组织化学染色证明接种的细胞是呼吸道上皮细胞。
结论
本研究表明向纤维蛋白凝胶中添加成纤维细胞对呼吸道上皮的分化有积极影响。
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