This study evaluated whether cell sheets containing a network of lymphatic endothelial cells (LECs) promoted lymphangiogenesis after transplantation in vivo. Cell sheets with a LEC network were constructed by co-culturing LECs and adipose-derived stem cells (ASCs) on temperature-responsive culture dishes. A cell ratio of 3:2 (vs. 1:4) generated networks with more branches and longer branch lengths. LEC-derived lymphatic vessels were observed 2 weeks after transplantation of a three-layered cell sheet construct onto rat gluteal muscle. Lymphatic vessel number, diameter and depth were greatest for a construct comprising two ASC sheets stacked on a LEC/ASC (3:2 ratio) sheet. Transplantation of this construct in a rat model of femoral lymphangiectomy led to the formation of functional lymphatic vessels containing both transplanted and host LECs. Further development of this technique may lead to a new method of promoting lymphangiogenesis.

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Normothermic machine perfusion (NMP) has not yet been established as a technique for preserving organs for a day. A key contributing factor to the same is that the perfusing solutions cannot circulate continuously and evenly in the organs. Here, we conceived a method of applying intermittent air pressure from outside the organ to assist its circulatory distribution during perfusion. We used a perfusion culture system while applying external pressure to culture rat kidneys and compared the circulatory distribution in the kidneys, changes in tissue morphology due to injury, and perfusate filtration. The intermittent pressurization (IMP) (−) group showed markedly poorer circulation on the upper side compared with that in the lower side, alongside histological damage. On the other hand, the IMP (+) group showed improved circulation in the upper side and had lesser histological damage. Furthermore, the IMP (+) group maintained the ability to filter perfusate for 24 h. In transplantation medicine and regenerative medicine research, this method has the potential to contribute to more efficient organ preservation and more functional tissue regeneration in the future.

Organ transplantation is a definitive treatment for endocrine disorders, but donor shortages limit the use of this technique. The development of regenerative therapies would revolutionize the treatment of endocrine disorders. As is the case for harvested organs, the ideal bioengineered graft would comprise vascularized endocrine tissue, contain blood vessels that could be anastomosed to host vessels, have stable blood flow, and be suitable for transplantation into various sites. Here, we describe a transplantable endocrine tissue graft that was fabricated by ex vivo perfusion of tricultured cell sheets (islet β-cells, vascular endothelial cells, and mesenchymal stem cells) on a vascularized tissue flap of in vivo origin. The present study has three key findings. First, mild hypothermic conditions enhanced the success of ex vivo perfusion culture. Specifically, graft construction failed at 37°C but succeeded at 32°C (mild hypothermia), and endocrine tissue fabricated under mild hypothermia contained aggregations of islet β-cells surrounded by dense vascular networks. Second, the construction of transplantable endocrine tissue by ex vivo perfusion culture was better achieved using a vascular flap than a muscle flap. Third, the endocrine tissue construct generated using a vascular flap could be transplanted into the rat by anastomosis of the graft artery and vein to host blood vessels, and the graft secreted insulin into the host’s circulatory system for at least 2 weeks after transplantation. Endocrine tissues bioengineered using these techniques potentially could be used as novel endocrine therapies.