Introduction: Tendons play a crucial role in connecting muscles and bones and are composed primarily of collagen, a dense connective tissue that is arranged in a regular pattern. Once a tendon is injured, most tendons require conservative rehabilitation. However, once a tendon is ruptured, it is challenging to heal due to the need for surgery. Therefore, artificial tendons are crucial in supporting these treatments. In this study, we aim to fabricate artificial tendons, or tenocyte sheets, in a form that can be easily utilized in surgical procedures. Moreover, maintain the quality of artificial tendons for an extended period by adding a "cryopreservation process" of artificial tendons or tenocyte sheets.

Methods: Tendon tissue collected from 3-week-old male Wistar rats was enzymatically treated and purified, and rat tenocytes were collected. The obtained tenocytes were cultured on type I collagen-coated culture dishes and used for the experiments (lower than passage 5). Collagen Vitrigel membrane, polyester (PET) membrane, and other extracellular matrix (ECM) membranes were used as carriers for the preparation of tenocyte sheets. The collagen Vitrigel membrane is a gel formed from high-density collagen fibers, comparable to connective tissue in vivo, and is both transparent and strong. The vitrification and slow-freezing methods were used to freeze the tenocyte sheets. The function of the tenocyte sheets was evaluated in vitro.

Results: First, the tenocytes were cultured on each of the carriers. Tenocytes grew stably on the collagen Vitrigel membrane and PET membranes, and a monolayer sheet structure was obtained. Furthermore, the adhesion of the tenocytes to the carriers and the barrier of the prepared tenocyte sheets were tight. Next, each freezing method was examined for the prepared tenocyte sheets. In the vitrification freezing, the tenocyte sheets were detached from each carrier. On the other hand, the slow freezing was able to maintain the adhesive state between the carriers and the tenocyte sheets.

Conclusion: In this study, artificial tendons, or tenocyte sheets, were successfully fabricated and cryopreserved. The addition of "cryopreservation" to the cell sheet manufacturing process maintains the quality of the cell sheet for an extended period, thereby achieving a stable supply. Fundamental medical technology will promote the social implementation and industrialization of transplantation medicine and regenerative medicine.