Differential systemic decellularization in vivo to study molecular changes in each vasculature layer in murine models of disease

Abstract

Vascular dysfunction underlies the onset and progression of many life-threatening diseases, highlighting the need for improved understanding of its molecular basis. Here, we present differential systemic decellularization in vivo (DISDIVO), a protocol that enables systemic and independent study of the molecular changes in each vasculature layer in murine models of disease. We describe steps for anesthesia, perfusion surgery, and exsanguination. We then detail detachment and collection of glycocalyx and decellularization and collection of both endothelial and smooth muscle cells. For complete details on the use and execution of this protocol, please refer to Serra et al., Gallart-Palau et al., and Vinaiphat et al.

The authors thank Prof Xavier Dolcet (Basic Medical Sciences Department, University of Lleida) and Laura Casani (IR-Sant Pau - Sant Pau Institute of Biomedical Research, Barcelona) for their help with the protocol revisions. Support for this work was provided by the National Institute of Health/Instituto de Salud Carlos III-ISCIII, Spain (PI22/00443 to X.G.-P.), grant co-funded by the European Union; the Ministry of Science and Innovation-MCIN, Spain, and the National Research Council/ Agencia Estatal de Investigacio´ n-AEI, Spain (PID2020-114885RB-C21 to A.S.), funded by MCIN/ AEI/10.13039/501100011033; the MCIN with funds from the European Union NextGenerationEU (PRTR-C17.I1) and the Autonomous Community of Catalonia ‘‘Biotechnology Plan Applied to Health’’ (EVBRAINTARGET-Y7340-ACPPCCOL007 to X.G.-P. and A.S.), coordinated by the Institute for Bioengineering of Catalonia (IBEC); and the Diputacio´ de Lleida, Spain (PIRS22/03 to X.G.-P.). X.G.-P. acknowledges a Miguel Servet program tenure track contract (CP21/00096) of the ISCIII, awarded on the 2021 call under the Health Strategy Action, co-funded by the European Union (FSE+). A.S. acknowledges a Ramo´ n y Cajal program tenure track contract (RYC2021-030946-I) of the AEI, funded by MCIN/AEI/ 10.13039/501100011033 and by ‘‘ESF Investing in your future.’’ C.L.’s PhD is funded by the European Social Fund for the recruitment of predoctoral researchers (PEJD-2019-PRE/BIO- 16475), M.M.’s PhD is funded by the MCIN-AEI (PR2021-097934), J.A.S.M.’s PhD is funded by the Catalan Research Council-AGAUR (2023 FI-1 00054) and the contributions have also been supported by Diputacio´ de Lleida, Spain ‘‘Ajuts al Talent en Investigacio´ Biome` dica,’’ J.L.’s PhD is funded by AGAUR, Spain (2022 DI 100) and by the company Alge` mica Barcelona S.L. IRBLleida. J.A.S.M., X.G.-P., and A.S. are co-funded by the CERCA Program/Generalitat de Catalunya. Support for this work has also been provided by the Singapore National Medical Research Council (NMRC/OFIRG/0003/2016 to S.K.S.), Canadian Institutes of Health Research Tier 1 Canada Research Chair (to S.K.S.), Canadian Institutes of Health Research Project Grant (to S.K.S.), The Natural Sciences and Engineering Research Council of Canada Discovery Grant (to S.K.S.), Canada Foundation for Innovation grant (to S.K.S.), and start-up research grant from Brock University (to S.K.S.).