Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds.
Monitoring decellularization via absorbance spectroscopy during the derivation of extracellular matrix scaffolds.
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DOI：
10.1088/1748-605x/ac361f
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发表时间：
2021-11-26
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细胞外基质（ECM）是由代表局部组织微环境的生物活性分子组成的复杂结构。脱细胞ECM生物材料利用这些生物分子用于再生医学应用。一种潜在的治疗应用是使用声带（VF）特异性ECM来恢复损伤后的VF。ECM支架通过脱细胞化过程衍生，其目的是去除不需要的免疫原性生物分子（例如DNA），同时保留ECM的组成。脱细胞化的有效性通常在最后通过量化ECM属性如最终dsDNA含量来评估。然而，ECM制造中批次间的差异性仍然是标准化、成本效益和规模化过程的重大挑战。可用于过程中控制的工具数量有限，严重限制了去细胞化过程参数和ECM属性之间相关性的揭示。在这项研究中，我们开发了一种适用于经典的批量方法和半连续脱细胞系统的技术，实时跟踪两种喉组织的脱细胞。我们假设监测生物反应器在260 nm处的流出物吸光度作为时间的函数将提供从组织的代表性DNA释放曲线，从而允许工艺优化。获得了喉组织的DNA释放曲线，并成功地用于优化VF固有层-ECM（auVF-ECM）水凝胶的衍生。该水凝胶具有与治疗VF损伤的常用生物材料相当的流变学性质。此外，auVF-ECM水凝胶在体外脂多糖刺激后促进THP-1巨噬细胞下调CCR 7，表明了一些抗炎特性。结果表明，吸光度曲线是脱细胞过程中DNA去除的一个很好的代表，因此提供了一个重要的工具，以优化未来的协议。
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.