Background

The utility of endoscopic transplantation of epithelial cell sheets to ulcer sites after endoscopic submucosal dissection (ESD) has been shown to prevent scar stenosis after ESD of early esophageal cancer. Previously, our group reported use of an endoscopic transplantation device fabricated with a 3-dimensional printer. Cell sheets are transplanted to the esophageal wound site with the following procedure: first, a cell sheet harvested from temperature-responsive culture dishes is placed on the device's deflated balloon surface and transported to the wound site with endoscopic forceps; second, by applying pressure from inflating the balloon locally at the wound site, the cell sheet is successfully transferred and adhered to the wound tissue; third, the balloon is deflated, and the device is removed. By repeating the procedure, several cell sheets can be safely transplanted to a wider ESD area. Nonetheless, possible damage to cell sheets using this procedure has not yet been assessed.

Objective

Effects of endoscopic transplantation balloon inflation on cell viability and damage of normal human epidermal keratinocyte sheets resident on the device's balloon surface were evaluated by histology after sheet placement onto lumenal surfaces in the ex vivo porcine submucosal dissection esophagus model. Endoscopic transplantation of these same cell sheets with conventional methods using a polyvinylidene fluoride (PVDF) cell sheet support membrane, balloon device transfer, and also using a novel modified balloon transfer procedure was also examined. Cell sheet transfer results obtained with these three procedures were compared.

Method

Normal human epidermal keratinocyte sheets were fabricated on temperature-responsive culture inserts. By temperature reduction to 20 °C, all cells were harvested as a single contiguous cell sheet. Freshly excised porcine esophagi purchased in a slaughter house were turned inside-out, and the exposed lumenal mucosa and submucosal layers were removed by Cooper scissors. This luminal surface was then utilized as a transplantation bed in ex vivo cell sheet experiments. Cell sheets were adhered to the endoscopic transfer device balloon, expanded by balloon inflation and resulting cell viability was evaluated by trypan blue exclusion test after cell sheet trypsinization and dispersion. Cell sheets were transferred onto the esophagus lumen ex vivo using forceps and the balloon device, and also using a modified balloon transfer method. The obtained results were compared with those without balloon expansion, and evaluated for sheet thickness and lumenal histology. Finally, TUNEL staining was performed to examine cell apoptosis.

Result

Cell sheets thinned after cell sheet balloon expansion, but no apoptosis was observed after these procedures.

Conclusion

Expanding keratinocyte cell sheets on a balloon endoscopic transfer device did not damage the cell sheets. This sheet transplantation method using the endoscopic balloon transfer device may be considered as a future standard cell sheet endoscopic transplantation procedure for repairing the esophagus.

中文翻译：

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在离体模型中评估黏膜下剥离后移植到猪切除食道上的人角质形成细胞片

背景

内镜黏膜下剥离术 (ESD) 后将上皮细胞层内镜移植到溃疡部位的效用已被证明可预防早期食管癌 ESD 后的瘢痕狭窄。此前，我们小组报告了使用由 3 维打印机制造的内窥镜移植装置。通过以下程序将细胞片移植到食管伤口部位：首先，将从温度敏感培养皿中采集的细胞片放置在装置的瘪了的球囊表面，并用内窥镜镊子运送到伤口部位；其次，通过在伤口部位局部对气囊充气施加压力，细胞片被成功转移并粘附在伤口组织上；第三，气球放气，取出装置。通过重复该过程，几个细胞片可以安全地移植到更广泛的 ESD 区域。尽管如此，尚未评估使用此程序可能对细胞表造成的损害。

客观的

在体外猪黏膜下剥离食管模型中，通过组织学评估内窥镜移植球囊膨胀对位于装置球囊表面的正常人表皮角质形成细胞片的细胞活力和损伤的影响。还检查了使用聚偏二氟乙烯 (PVDF) 细胞片支持膜、球囊装置转移以及还使用新型改良球囊转移程序的常规方法对这些相同细胞片的内窥镜移植。比较了用这三种程序获得的细胞片转移结果。

方法

正常人表皮角质形成细胞片是在温度响应性培养插入物上制造的。通过将温度降低到 20 °C，所有细胞都被收获为单个连续的细胞片。在屠宰场购买的新鲜切除的猪食道被翻转过来，并用库珀剪刀去除暴露的管腔粘膜和粘膜下层。然后将该腔表面用作离​​体细胞片实验中的移植床。将细胞片粘附到内窥镜转移装置球囊上，通过球囊充气膨胀，并在细胞片胰蛋白酶化和分散后通过台盼蓝排除试验评估所得细胞活力。使用镊子和球囊装置，以及使用改进的球囊转移方法将细胞片离体转移到食道内腔。将获得的结果与没有球囊扩张的结果进行比较，并评估薄片厚度和管腔组织学。最后，进行 TUNEL 染色以检查细胞凋亡。

结果

细胞片球囊扩张后细胞片变薄，但在这些程序后未观察到细胞凋亡。

结论

在球囊内窥镜转移装置上扩张角质形成细胞片不会损坏细胞片。这种使用内窥镜球囊转移装置的薄片移植方法可以被认为是未来用于修复食管的标准细胞薄片内窥镜移植程序。