Introduction

Regenerative therapy using chondrocyte sheets is effective for osteoarthritis. The clinical application of chondrocyte sheet therapy is expected to be further advanced by the use of a feasible cryopreservation technique. Previously, we developed a chondrocyte sheet vitrification method; however, it was too complex to be used for routine clinical application. Here, we aimed to develop a prototype method for vitrifying chondrocyte sheets for clinical practice.

Methods

We developed a “circulating vitrification bag” as a container to process cell sheets for vitrification in an efficient and sanitary fashion. Moreover, we invented the “vitrification storage box”, which is useful for the vitrification of cell sheets, long-term preservation, and transportation. These devices were used to vitrify rabbit chondrocyte sheets, which were then assessed for their structural characteristics and the viability of the component cells after rewarming.

Results

In all cell sheet samples (n = 7) vitrified by the circulating vitrification bag method, the integrity of the sheet structure was maintained, and the cell survival rate was similar to that of non-vitrified samples (91.0 ± 2.9% vs. 90.0 ± 3.0%). Proteoglycan and type II collagen, which are major components of cartilage, were densely and evenly distributed throughout the chondrocyte sheet subjected to vitrification similarly to that observed in the non-vitrified sheet. After long-term storage using the vitrification storage box, the cell sheets maintained normal structure and cell viability (survival rate: 81.2 ± 1.0% vs. 84.3 ± 1.8%) compared to the non-vitrified sheet.

Conclusion

Our results indicate that the circulating vitrification bag method is an effective approach for realizing the clinical application of vitrified chondrocyte sheets. The vitrification storage box is also useful for the long-term preservation of vitrified cell sheets, further enhancing the feasibility of the clinical application of cryopreserved chondrocyte sheets.

中文翻译：

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开发用于临床应用的软骨细胞片的有效玻璃化方法。

介绍

使用软骨细胞片的再生疗法对骨关节炎有效。软骨细胞片层疗法的临床应用有望通过使用可行的冷冻保存技术进一步推进。此前，我们开发了一种软骨细胞片玻璃化方法；然而，它过于复杂，无法用于常规临床应用。在这里，我们旨在开发一种用于临床实践的玻璃化软骨细胞片的原型方法。

方法

我们开发了一种“循环玻璃化袋”作为容器，以高效和卫生的方式处理细胞片进行玻璃化。此外，我们还发明了“玻璃化储存盒”，可用于细胞片的玻璃化、长期保存和运输。这些装置用于玻璃化兔软骨细胞片，然后评估其结构特征和复温后成分细胞的活力。

结果

在通过循环玻璃化袋法玻璃化的所有细胞片样品（n = 7）中，片状结构的完整性得以保持，细胞存活率与非玻璃化样品相似（91.0 ± 2.9% vs. 90.0 ± 3.0%）。作为软骨的主要成分的蛋白多糖和II型胶原蛋白密集且均匀地分布在经历玻璃化的整个软骨细胞片中，类似于在非玻璃化片中观察到的情况。使用玻璃化储存盒长期储存后，细胞片与非玻璃化片相比，保持正常的结构和细胞活力（存活率：81.2 ± 1.0% vs. 84.3 ± 1.8%）。

结论

我们的研究结果表明，循环玻璃化袋法是实现玻璃化软骨细胞片临床应用的有效途径。本发明的玻璃化储存盒还可用于玻璃化细胞片的长期保存，进一步增强了冷冻保存的软骨细胞片临床应用的可行性。