Determination of DNA content as quality control in decellularized tissues: challenges and pitfalls.

Abstract

Decellularized organs and tissues are emerging within the field of regenerative medicine to meet the growing demand for organ and tissue transplantation. Quality control of these acellular matrices prior to transplantation is of paramount importance to ensure the absence of an adverse reaction. In particular, thorough evaluation of the DNA content is essential but also poses technical challenges. Therefore, in this study, we compared different methods for quantitative and qualitative evaluation of DNA content in native and decellularized skeletal muscle tissue to identify strengths and weaknesses for each. Histological analysis revealed that Feulgen staining is more sensitive and robust than the commonly used hematoxylin-eosin and 4',6-diamidino-2-phenylindole staining for detection of remaining nuclear material. Furthermore, gel electrophoresis allowed to identify the quality and length of remaining DNA fragments. The results of the quantitative analysis indicated that direct measurement of DNA content in tissue lysates is preferred over silica-based extraction methods, since the latter resulted in the loss of small DNA fragments during extraction. Moreover, a weight loss correction factor should be implemented to take into account the impact of the decellularization on the extracellular matrix. With regard to the detection method, the results revealed that a fluorescence-based approach is more accurate than the use of UV/VIS absorbance. Through combination of the proposed methods, it should be possible to achieve a more standardized evaluation of novel acellular matrices in terms of DNA content and to enhance the predictability of clinical success.