Nakajima Ryota, Kobayashi Toyoshige, Moriya Noboru, Mizutani Manabu, Kan Kazutoshi, Nozaki Takayuki, Saitoh Kazuo, Yamato Masayuki, Okano Teruo, Takeda Shizu
Central Research Laboratory, Hitachi Ltd, 2520 Hatoyama, Saitama, 350-0395, Japan.
CellSeed Inc, 33-8 Wakamatsu-cho, Shinjuku-ku, Tokyo, 162-0056, Japan.
J Tissue Eng Regen Med. 2015 Nov;9(11):1259-67. doi: 10.1002/term.1639. Epub 2012 Dec 14.
Automation technology for cell sheet-based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell-culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm-thick gas-permeable membrane, which was similar to the results with the cell-culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100-300 µm-thick gas-permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial-specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin-1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets.
基于细胞片的组织工程自动化技术需要优化细胞片制造工艺，稳定细胞片质量并降低生物污染风险。在临床环境中必须避免生物污染。封闭培养系统为此提供了解决方案。在本研究中，我们开发了一种名为细胞盒的封闭培养装置，用于在封闭细胞培养系统中制造角膜上皮细胞片。兔角膜缘上皮细胞与3T3饲养细胞一起在多孔膜表面培养，3T3饲养细胞与细胞盒以及作为对照的细胞培养插入物中的上皮细胞分开。为了制造分层细胞片，研究了焊接到细胞盒上的五种不同厚度的膜。在焊接到25μm厚透气膜的细胞盒中制造了多层角膜上皮细胞片，这与细胞培养插入物的结果相似。然而，焊接到100 - 300μm厚透气膜的细胞盒中未出现角膜上皮细胞片的分层。通过组织学分析评估制造的细胞片，以检查角膜上皮特异性标志物的表达。免疫组织化学分析表明，假定的干细胞标志物p63、角膜上皮分化标志物CK3和屏障功能标志物Claudin - 1在细胞片中的适当位置表达。这些结果表明细胞盒对于制造角膜上皮细胞片是有效的。
