The healing of cutaneous wounds requires an organized but complex sequence of several biological and molecular events. The control of this process is far from being attained by the wide range of approaches that have been developed and even clinically used. We have previously shown that human adipose stem cells (hASCs) cell sheet constructs have an impact on epidermal morphogenesis and on promoting neotissue vascularization[1]. The characteristic adhesive nature of the extracellular matrix (ECM) of these cell sheets was shown to be key for the observed host response but little is known about the influence of its composition. Thus, the goal of this work was to address this question by analysing in detail the components of the ECM of hASCs cell sheets.

Cells-self ECM from both hASCs and human dermal fibroblasts (DFbs) (used as control) were obtained by cell sheet engineering after decellularization using several protocols: freeze-thaw (FT) cycles, Triton x-100 with NH₄OH (TB) and a combination of both protocols. All the procedures were followed by DNase treatment. Decellularization effectiveness was assessed after DAPI (4′,6-diamidino-2-phenylindole) and haematoxylin/eosin (HE) staining. DNA and protein quantification were obtained by PicoGreen and Bradford assays, respectively. Efficient decellularization was achieved with different cycles of FT and with the combination with TB. The use of TB showed similar results but treated samples were more fragile. Protein quantification revealed higher values for cell sheets treated with freeze-thaw, disclosing a greater preservation of ECM components without the use of detergents. These results demonstrated the possibility to effectively decellularize cell sheet from hASCs and preserve their ECM proteins.

Acknowledgements: This research was supported by ERC Consolidator Grant – ECM_INK (ERC-2016-COG-726061), MicroLiver Project (PTDC/BBB-ECT/4317/2014), the European Union for The Discoveries Centre for Regenerative and Precision Medicine (H2020-WIDESPREAD-2014-1-739572) and the Gene2Skin Project (H2020-TWINN-2015-692221).

1.           Cerqueira, M.T., et al., Human adipose stem cells cell sheet constructs impact epidermal morphogenesis in full-thickness excisional wounds. Biomacromolecules, 2013. 14(11): p. 3997-4008.