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 stimulation in vitro suggesting 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.

Decellularized extracellular matrix (ECM) scaffolds derived from tissues and organs are complex biomaterials used in clinical and research applications. A number of decellularization protocols have been described for ECM biomaterials derivation, each adapted to a particular tissue and use, restricting comparisons among materials. One of the major sources of variability in ECM products comes from the tissue source and animal age. Although this variability could be minimized using established tissue sources, other sources arise from the decellularization process itself. Overall, current protocols require manual work and are poorly standardized with regard to the choice of reagents, the order by which they are added, and exposure times. The combination of these factors adds variability affecting the uniformity of the final product between batches. Furthermore, each protocol needs to be optimized for each tissue and tissue source making tissue-to-tissue comparisons difficult. Automation and standardization of ECM scaffold development constitute a significant improvement to current biomanufacturing techniques but remains poorly explored. This study aimed to develop a biofabrication method for fast and automated derivation of raw material for ECM hydrogel production while preserving ECM composition and controlling lot-to-lot variability. The main result was a closed semibatch bioreactor system with automated dosing of decellularization reagents capable of deriving ECM material from pretreated soft tissues. The ECM was further processed into hydrogels to demonstrate gelation and cytocompatibility. This work presents a versatile, scalable, and automated platform for the rapid production of ECM scaffolds.