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Regenerative medicine has greatly progressed, but tendon regeneration mechanisms and robust in vitro tendon differentiation protocols remain to be elucidated. Recently, tendon explant co-culture (CO) has been proposed as an in vitro model to recapitulate the microenvironment driving tendon development and regeneration. Here, we explored standardized protocols for production and storage of bioactive tendon-derived secretomes with an evaluation of their teno-inductive effects on ovine amniotic epithelial cells (AECs). Teno-inductive soluble factors were released in culture-conditioned media (CM) only in response to active communication between tendon explants and stem cells (CM CO ). Unsuccessful tenogenic differentiation in AECs was noted when exposed to CM collected from tendon explants (CM FT ) only, whereas CM CO upregulated SCXB, COL I and TNMD transcripts, in AECs, alongside stimulation of the development of mature 3D tendon-like structures enriched in TNMD and COL I extracellular matrix proteins. Furthermore, although the tenogenic effect on AECs was partially inhibited by freezing CM CO , this effect could be recovered by application of an in vivo -like physiological oxygen (2% O 2 ) environment during AECs tenogenesis. Therefore, CM CO can be considered as a waste tissue product with the potential to be used for the development of regenerative bio-inspired devices to innovate tissue engineering application to tendon differentiation and healing., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Citeroni, Mauro, Ciardulli, Di Mattia, El Khatib, Russo, Turriani, Santer, Della Porta, Maffulli, Forsyth and Barboni.)