Supercritical Extraction of ECM components from Cell Sheets

Supercritical carbon dioxide (scCO2) technology has been recently used in the field of TERM as a method of processing biomaterials. Additionally, this technology has been also applied to decellularize tissues such as aorta, myocardium, pericardium, cornea and adipose tissue. Its great advantage relies on the fact that scCO2, with a critical point at mild conditions (31.1°C and 7.38MPa), is non-toxic, non-flammable, relatively inert and can be removed easily by depressurization. Despite this, scCO2 is apolar and the addition of an entrainer is required to eliminate charged molecules such as phospholipids. CO2-philic detergents are based on detergent alcohols (fatty alcohols with carbon chain length in the range between of C12-C18), rendering these molecules with surfactant-like properties, as well as potential to interact and diffuse with supercritical CO2. Recently, a CO2-philic detergent, Dehypon, was used as entrainer to decellularize articular cartilage, tendon and skin. Based on this, we propose the use of a combination of Dehypon/CO2 as a supercritical fluid with higher potential to decellularize cell sheets. We hypothesised that the use of Dehypon/CO2 will allow using less harmful conditions than those used in traditional detergent-based decellularization methods thus attaining a higher level of preservation of extracellular matrix (ECM) components.

Human dermal fibroblasts (hDFb) and adipose stem cells (ASC) cell sheets were prepared as previously reported (M. T. Cerqueira et al., 2013; M. T. Cerqueira et al., 2014). For decellularization, cell sheets were incubated with 0.1% Triton-X100 and 20 mM NH4OH followed by another incubation with DNase or with 0.2% Dehypon and scCO2. DAPI staining validated the disruption of cell nuclei and subsequent DNA removal from the cell sheets, while preserving the overall matrix ultrastructure, independently of the method used. Total protein quantification using the Bradford assay showed a lower loss of protein when scCO2 technology was used. Importantly the SDS-PAGE electrophoresis confirmed maintenance of the native cell sheet profile and the absence of protein degradation using the scCO2 technology.

Overall, the use of the use of Dehypon/CO2 and the scCO2 technology allowed an improved preservation the cell sheet’s ECM which will allow extending the use of these cell-derived extracts to develop cell-specific tissue models.

Acknowledgments: This study was supported by the FCT/MCTES (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia, e Ensino Superior) through the PD/BD/14301/2018 (DPR); PD/BD/150478/2019 (BD) and the ERC Consolidator Grant “ECM_INK” (ERC-2016-COG-726061).