Orthopaedic Biomechanics, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
BioInterface Science, Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
Xenotransplantation. 2021 Jan;28(1):e12643. doi: 10.1111/xen.12643. Epub 2020 Sep 15.
Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica-based solid-phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in-patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica-based solid-phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA-specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability.
动物组织的去细胞化是获得用于组织工程和器官移植的生物材料的新途径。成功的去细胞化是必需的，因为动物 DNA 会引起炎症反应并含有内源性逆转录病毒，这些病毒可能会传播给患者。成功去细胞化的标准之一是从组织中消化（片段化）和消除（残留量）DNA。DNA 的定量可以通过多种方式完成，但最近已经表明，基于硅的固相萃取方法通常不能完全纯化特定的小 DNA 片段。在去细胞化的背景下，这意味着测量的 DNA 量被低估，这可能会影响处理后的组织用于住院患者的安全性。在本文中，我们回顾了研究人员使用的 DNA 定量方法，并评估了它们对去细胞化后报告的 DNA 含量的影响。我们发现，基于硅的固相萃取后残留 DNA 量的低估可能高达 10 倍。因此，我们建议使用 dsDNA 特异性结合染料（如 Picogreen）直接评估组织裂解物中的 DNA 量，因为它们在检测小片段方面具有更高的准确性，并且易于使用且广泛可用。
