Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds

Extracellular matrix (ECM) is a complex structure composed of bioactive molecules representative of the local tissue microenvironment. Decellularized ECM biomaterials harness these biomolecules for regenerative medicine applications. One potential therapeutic application is the use of vocal fold (VF) specific ECM to restore the VFs after injury. ECM scaffolds are derived through a process of decellularization, which aims to remove unwanted immunogenic biomolecules (e.g. DNA) while preserving the composition of the ECM. The effectiveness of the decellularization is typically assessed at the end by quantifying ECM attributes such as final dsDNA content. However, batch-to-batch variability in ECM manufacturing remains a significant challenge for the standardization, cost-effectiveness, and scale-up process. The limited number of tools available for in-process control heavily restricts the uncovering of the correlations between decellularization process parameters and ECM attributes. In this study, we developed a technique applicable to both the classical batch method and semi-continuous decellularization systems to trace the decellularization of two laryngeal tissues in real-time. We hypothesize that monitoring the bioreactor's effluent absorbance at 260 nm as a function of time will provide a representative DNA release profile from the tissue and thus allow for process optimization. The DNA release profiles were obtained for laryngeal tissues and were successfully used to optimize the derivation of VF lamina propria-ECM (auVF-ECM) hydrogels. This hydrogel had comparable rheological properties to commonly used biomaterials to treat VF injuries. Also, the auVF-ECM hydrogel promoted the down-regulation of CCR7 by THP-1 macrophages upon lipopolysaccharide stimulationin vitrosuggesting some anti-inflammatory properties. The results show that absorbance profiles are a good representation of DNA removal during the decellularization process thus providing an important tool to optimize future protocols.