Introduction: Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. To mimic the complexity of structures and compositions that characterize a tissue’s own ECM, several ECM-extraction methodologies have been used. However, their outcomes seldomly reflect what is found in native tissue’s ECM. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cell’s own ECM, to be used in the preparation of bioinks.
Methodology: The ECM extraction protocol best fitted for cell sheets was defined considering efficient DNA removal with a minor effect on the ECM produced by cells. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs), obtained as previously described (Cerqueira et al., 2013, 2014) were processed using a customised supercritical system, by varying the pressure of the reactor, presence, exposure time and type of co-solvent. Quantification of the amount of DNA, protein and sGAGs was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. To develop ECM-based bioinks, the extracted ECM was combined with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amount on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Blends with suitable rheological performance were selected for cell encapsulation studies, to assess the influence of bioink composition on cell viability (calcein/Propidium iodide). Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cells was assessed.
Results: The optimised scCO2 protocol allowed efficient removal of DNA while preserving a higher content on ECM proteins and sGAGs than the standard methodologies. Characterization of extract’s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) are distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source.
Conclusions: Overall, this work highlights the benefit of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs.
Acknowledgements: This study was funded by the European Research Council through the ERC Consolidator Grant “ECM_INK”(ERC-2016-COG-726061) and supported by 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) grants.