Jambar Nooshin Bahram, Tayebi Tahereh, Babajani Amirhesam, Khani Mohammad Mehdi, Niknejad Hassan
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Department of Pharmacology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Cell J. 2023 Jan 1;25(1):35-44. doi: 10.22074/cellj.2022.557600.1081.
Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes.
In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines.
Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold.
The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.
器官移植是终末期肝衰竭的最后治疗选择，但受供体不足的限制。去细胞肝脏可作为具有临床应用潜力的肝脏组织工程的合适基质。优化去细胞过程可获得完全去除细胞成分并保留三维结构的生物基质。本研究旨在通过三种解剖途径评估去细胞效果。
在本实验研究中，通过胆管（BD）、门静脉（PV）和肝静脉（HV）对大鼠肝脏进行去细胞处理；使用化学洗涤剂和酶。通过测量DNA含量、细胞外基质（ECM）总蛋白和糖胺聚糖（GAGs）来评估去细胞效果。通过组织学和免疫组织化学（IHC）染色以及扫描电子显微镜（SEM）检查ECM的保存情况。通过MTT法检测去细胞支架对HepG2和HUVEC细胞系的生物相容性。
通过HV和PV途径去细胞后得到透明支架，细胞完全去除，而BD途径产生的支架不透明，去细胞不完全。苏木精-伊红染色证实了这些结果。PV组和HV组使用1%和0.5%十二烷基硫酸钠（SDS）时DNA损失最大，且HV组DNA含量下降更快。在最后阶段，HV组和PV组分泌的蛋白质明显少于BD组。去细胞后GAGs水平降低，尤其是在PV组和HV组。HV组和PV组的胶原蛋白含量明显高于BD组。IHC和SEM图像显示ECM结构得以保留，细胞成分被完全去除。MTT分析表明去细胞支架具有生物相容性。
结果表明，与PV和BD相比，HV是更适合肝脏去细胞的途径。
