**5. Conclusions**

This study demonstrates that cryopreservation did not affect the capacity of SVF to spontaneously create an in vitro prevascular network in the absence of angiogenic growth factor supplementation in 3D conditions. Moreover, the angiogenic potential of SVF was further demonstrated after the in ovo implantation of prevascularized sponges resulting in improved vessel recruitment and improved construct integration within the CAM tissue. All together, these results demonstrate that the use of cryopreserved SVF, combined with marine-derived collagen, allows a simplified and cost-efficient method for assistance in the vascularization of TE constructs.

**Author Contributions:** Conceptualization, S.F.-R. and R.P.P.; formal analysis, S.F.-R. and R.P.P.; funding acquisition, R.P.P.; investigation, S.F.-R., G.S.D., C.O., A.M. and T.H.S.; methodology, S.F.-R., A.M., T.H.S. and R.P.P.; resources, M.J. and R.H.; supervision, R.P.P. and R.L.R.; validation, R.P.P.; writing—original draft, S.F.-R.; writing—review and editing, S.F.-R. and R.P.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (Grant agreemen<sup>t</sup> No. 805411); Portuguese Foundation for Science and Technology under doctoral fellowship PD/BD/135252/2017 and individual gran<sup>t</sup> IF/00347/2015; European Regional Development Fund, through INTERREG España-Portugal 2014-2020 under BLUEBIOLAB (0474\_BLUEBIOLAB\_1\_E) project, through Atlantic Area Programme under BLUEHUMAN (EAPA\_151/2016) project and through NORTE2020/PT2020 Programme under ATLANTIDA (Norte-01-0145-FEDER-000040) project.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Hospital de S. João/University of Minho (217/19; CEICVS 008/2019).

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We thank Cármen G. Sotelo (IIM-CSI, Vigo, Spain) for the kind offer of blue shark skin collagen.

**Conflicts of Interest:** The authors declare no conflict of interest.
