Ghorbani Fariba, Ekhtiari Masomeh, Moeini Chaghervand Behbood, Moradi Lida, Mohammadi Bahareh, Kajbafzadeh Abdol-Mohammad
Tracheal Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Pediatric Urology and Regenerative Medicine Research Center, Pediatric Center of Excellence, Department of Pediatric Urology, Children's Medical Center, Tehran University of Medical Sciences, No. 62, Dr. Gharibs Street, Keshavarz Boulevard, 1419733151, Tehran, Iran.
Cell Tissue Bank. 2022 Mar;23(1):119-128. doi: 10.1007/s10561-021-09921-z. Epub 2021 Apr 28.
To optimize rabbit kidney decellularization protocol, using sodium dodecyl sulfate (SDS) as a commonly used detergent, a methylene blue based assay was employed for detecting the minimum nontoxic SDS level for future cell seeding. The rabbit kidney tissues were decellularized with the perfusion-based method and underwent several investigations to determine the efficacy of decellularization in preserving the extracellular matrix (ECM) and cell removal. SDS detection was performed by incubating with methylene blue and subsequent extraction with chloroform. MTT (3-(4, 5-dimethylthiazol-2-yr)-2,5-diphenyltetrazolium bromide) assay and SDS release were also evaluated during the entire process. After the first washing cycle, SDS concentration was 0.036, in 500 mL of the washing liquid, which slowly decreased and reached to 0.009 % after at the end of seventh washing cycle. In the 9th cycle, SDS was gradually decreased and reached to 0.003 %. SDS was significantly released after one week of incubation which ceased after ten washing cycles. The results of MTT assay demonstrated that different cells exhibited various sensitivity levels when exposed to serial concentrations of SDS. Human embryonic kidney cells (HEK293) with 0.003 % threshold for cellular toxicity and 87.4 % cell viability were more resistant compared with mesenchymal stem cells with 0.001 % threshold and 85.4 % cell viability. Colorimetric assay with methylene blue is a straightforward and non-invasive method to detect residual SDS present in tissue and can also prevent ECM destruction after several washings for detergent removal from decellularized tissues.
为优化兔肾去细胞方案，以常用去污剂十二烷基硫酸钠（SDS）为研究对象，采用基于亚甲蓝的检测方法来测定未来细胞接种时SDS的最低无毒水平。兔肾组织采用灌注法进行去细胞处理，并进行多项研究以确定去细胞处理在保留细胞外基质（ECM）和去除细胞方面的效果。通过与亚甲蓝孵育并随后用氯仿萃取来进行SDS检测。在整个过程中还评估了MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐）检测和SDS释放情况。第一次洗涤循环后，500 mL洗涤液中的SDS浓度为0.036%，该浓度缓慢下降，在第七次洗涤循环结束时降至0.009%。在第九次循环中，SDS逐渐下降至0.003%。孵育一周后SDS显著释放，在十次洗涤循环后停止。MTT检测结果表明，不同细胞在暴露于系列浓度的SDS时表现出不同的敏感性水平。与间充质干细胞相比，人胚肾细胞（HEK293）对细胞毒性的阈值为0.003%，细胞活力为87.4%，更具抗性，间充质干细胞的阈值为0.001%，细胞活力为85.4%。用亚甲蓝进行比色测定是一种直接且非侵入性的方法，可检测组织中残留的SDS，并且在多次洗涤以从去细胞组织中去除去污剂后，还能防止ECM被破坏。
