Felgendreff Philipp, Schindler Claudia, Mussbach Franziska, Xie Chichi, Gremse Felix, Settmacher Utz, Dahmen Uta
Department of General, Visceral and Vascular Surgery, University Hospital Jena, Jena, Germany.
Research Program "Else Kröner-Forschungskolleg AntiAge", Jena University Hospital, Jena, Germany.
Heliyon. 2021 Feb 13;7(2):e06129. doi: 10.1016/j.heliyon.2021.e06129. eCollection 2021 Feb.
Biological organ engineering is a novel experimental approach to generate functional liver grafts by decellularization and repopulation. Currently, healthy organs of small or large animals and human organs with preexisting liver diseases are used to optimize decellularization and repopulation.However, the effects of morphological changes on allo- and xenogeneic cell-scaffold interactions during repopulation procedure, e.g., using scaffold-sections, are unknown. We present a sequential morphological workflow to identify murine liver scaffold-sections with well-preserved microarchitecture.
Native livers (CONT, n = 9) and livers with experimentally induced pathologies (hepatics steatosis: STEA, n = 7; hepatic fibrosis induced by bile duct ligation: BDL, n = 9; nodular regenerative hyperplasia induced by 90% partial hepatectomy: PH, n = 8) were decellularized using SDS and Triton X-100 to generate cell-free scaffolds. Scaffold-sections were assessed using a sequential morphological workflow consisting of macroscopic, microscopic and morphological evaluation: (1) The scaffold was evaluated by a macroscopic decellularization score. (2) Regions without visible tissue remnants were localized for sampling and histological processing. Subsequent microscopical examination served to identify tissue samples without cell remnants. (3) Only cell-free tissue sections were subjected to detailed liver-specific morphological assessment using a histological and immunohistochemical decellularization score.
Decellularization was feasible in 33 livers, which were subjected to the sequential morphological workflow. In 11 of 33 scaffolds we achieved a good macroscopic decellularization result (CONT: 3 scaffolds; STEA: 3 scaffolds; BDL: 3 scaffolds; PH: 2 scaffolds). The microscopic assessment resulted in the selection of 88 cell-free tissue sections (CONT: 15 sections; STEA: 38 sections; BDL: 30 sections; PH: 5 sections). In 27 of those sections we obtained a good histological decellularization result (CONT: 3 sections; STEA: 6 sections; BDL: 17 sections; PH: 1 section). All experimental groups contained sections with a good immunohistochemical decellularization result (CONT: 6 sections; STEA: 5 sections; BDL: 4 sections; PH: 1 section).
Decellularization was possible in all experimental groups, irrespectively of the underlying morphological alteration. Furthermore, our proposed sequential morphological workflow was suitable to detect tissue sections with well-preserved hepatic microarchitecture, as needed for further repopulation experiments.
生物器官工程是一种通过去细胞化和再细胞化来生成功能性肝移植的新型实验方法。目前，小型或大型动物的健康器官以及患有肝病的人类器官被用于优化去细胞化和再细胞化过程。然而，在再细胞化过程中，形态变化对同种和异种细胞 - 支架相互作用的影响尚不清楚，例如使用支架切片时。我们提出了一种顺序形态学工作流程，以识别具有保存良好微结构的小鼠肝支架切片。
使用SDS和 Triton X - 100对天然肝脏（对照组，n = 9）和实验诱导病变的肝脏（肝脂肪变性：STEA，n = 7；胆管结扎诱导的肝纤维化：BDL，n = 9；90% 部分肝切除诱导的结节性再生性增生：PH，n = 8）进行去细胞化，以生成无细胞支架。使用由宏观、微观和形态学评估组成的顺序形态学工作流程评估支架切片：（1）通过宏观去细胞化评分评估支架。（2）定位无可见组织残留的区域进行采样和组织学处理。随后的显微镜检查用于识别无细胞残留的组织样本。（3）仅对无细胞组织切片使用组织学和免疫组织化学去细胞化评分进行详细的肝脏特异性形态学评估。
33个肝脏成功进行了去细胞化，并接受了顺序形态学工作流程。在33个支架中的11个中，我们获得了良好的宏观去细胞化结果（对照组：3个支架；STEA：3个支架；BDL：3个支架；PH：2个支架）。显微镜评估筛选出88个无细胞组织切片（对照组：15个切片；STEA：38个切片；BDL：30个切片；PH：5个切片）。在其中27个切片中，我们获得了良好的组织学去细胞化结果（对照组：3个切片；STEA：6个切片；BDL：17个切片；PH：1个切片）。所有实验组都包含免疫组织化学去细胞化结果良好的切片（对照组：6个切片；STEA：5个切片；BDL：4个切片；PH：1个切片）。
所有实验组均可行去细胞化，无论潜在的形态学改变如何。此外，我们提出的顺序形态学工作流程适用于检测具有保存良好的肝脏微结构的组织切片，这是进一步再细胞化实验所需要的。
