Hypothermic and cryogenic preservation of tissue-engineered human bone

To foster translation and commercialization of tissue-engineered products, preservation methods that do not significantly compromise tissue properties need to be designed and tested. Robust preservation methods will enable the distribution of tissues to third parties for research or transplantation, as well as banking of off-the-shelf products. We recently engineered bone grafts from induced pluripotent stem cells and devised strategies to facilitate a tissue-engineering approach to segmental bone defect therapy. In this study, we tested the effects of two potential preservation methods on the survival, quality, and function of tissue-engineered human bone. Engineered bone grafts were cultured for 5 weeks in an osteogenic environment and then stored in phosphate-buffered saline (PBS) solution at 4 °C or in Synth-a-Freeze™ at -80 °C. After 48 h, samples were warmed up in a water bath at 37 °C, incubated in osteogenic medium, and analyzed 1 and 24 h after revitalization. The results show that while storage in Synth-a-Freeze at -80 °C results in cell death and structural alteration of the extracellular matrix, hypothermic storage in PBS does not significantly affect tissue viability and integrity. This study supports the use of short-term hypothermic storage for preservation and distribution of high-quality tissue-engineered bone grafts for research and future clinical applications.