在振荡压力灌流器程序大鼠肝脏的脱细胞
Summary
The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.
Abstract
Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.
Introduction
脱细胞和recellularization可以使得能够在体外 1的功能，移植器官的生成。通过除去细胞和抗原物质（ 例如 ，DNA，α-半乳糖表位）从一个器官，非或更少免疫原性外基质（ECM）能够得到。此矩阵可以节省一个器官的三维显微解剖和可作为复育的理想生物基质具有不同的，可能异种来源2的细胞。因此，脱细胞的大鼠肝基质可重新填充与人肝细胞。此人源化微肝脏可以作为离体模型上的疾病（ 如 ，先天性代谢疾病，病毒性疾病或恶性肿瘤）或用于临床前的药物测试3研究。
大鼠肝脏灌注脱细胞几种不同的协议已经公布4-13。在所有协议，脱细胞化是由碱性离子或非离子型去污剂灌注经由插管门静脉实现。尽我们所知，我们是第一批通过选择性灌注经门静脉和/或大鼠肝动脉14报告大鼠肝脱细胞。使不同的血管系统，可在肝脏的选择性灌注可以使更好脱细胞的结果，而且，可以在细胞再增殖中起重要作用。
在这项研究中这里详述，肝脏灌注在一个特制的专用灌流器，振荡压力的条件下，使灌注。这些压力条件模仿肝脏的生理呼吸依赖性灌注： 原位 ，肝脏的隔膜，其变动在呼吸过程中对肝灌注有直接影响的连接函数下挂起。灵感具体地说导致降低隔膜和挤压肝脏，优化肝静脉流出，而到期导致肝脏升高和降低腹内压，优化门静脉流入15。
我们的目的是评估振荡压力条件是否具备通过模仿腹内条件体外对大鼠肝脏灌注脱细胞的同质性产生影响。脱细胞过程的同质性可能是灌注脱细胞低估的因素。所有已知的药物用于肝脱细胞的事业改建为ECM。在灌注不良的地方细胞保持在ECM内，而其它地区已经完全脱细胞。以溶解剩余的细胞中，灌注持续时间或压力必须提高，从而导致更多的改建为良好灌注的区域。因此，洗涤剂脱细胞应均匀器官内分布。
Protocol
Representative Results
Discussion
虽然提出了技术大鼠肝收获和脱细胞是容易复制，也有考虑某些关键步骤：
在制备过程中对肝收获，它避免严重出血是非常重要的，因为它会激活血液凝结，并可能导致在肝脏内的血液凝块的形成。在我们看来，这是有利的，门静脉插管之前直接切开腹部大动脉，以避免通过门静脉灌注过程中通过肝动脉血液流入。一旦肝脏插管并明确灌注，血液凝块的形成是不再是一个问?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to gratefully thank Steffen Lippert, Khalid Aliyev, Korinna Jöhrens and Katharina Struecker for their help during this project.
Materials
| Self built arterial cannula | |||
| Portex Non Sterile Polyethene Tubing | SIMS Portex | REF 800/110/100 | 0,28mm ID 0,61mm OD |
| Portex Non Sterile Polyethene Tubing | SIMS Portex | REF 800/110/200 | 0,58mm ID 0,96m OD |
| Venodrop Safe butterfly catheter | Fresenius Kabi | 3275851 | 21 G |
| portal vein cannula | |||
| Periphereal Venous Catheter | BD | 393224 | BD Venflon Pro 20G |
| three-way stopcock | smiths medical | 888-101RE | |
| surgery | |||
| Cotton Sticks | Hecht-Assistent | 4302 | |
| Cotton Pads | Shaoxing Zhengde Surgical dressing | 13H118-03 | |
| Gauze Bandage | Hubei Haige Medical Instruments | 14388 | |
| Ringer Solution | Fresenius Kabi | 13 HKP022 | 1000ml |
| 10ml Syringe | Braun | 4606108V | 10ml/ Luer Solo |
| 5ml Syringe | Braun | 4606061V | 5ml /Luer Solo |
| Suture (Silk 6/0) | Resorba | H1F | LOT 105001.81 |
| medical drape | Shaoxing Zhengde Surgical dressing | D0613011 | |
| surgical instruments | |||
| needle holder | Geuder | 17570 | |
| micro-forceps | Inox-Electronic | 91150-20 | |
| micro-scissors | Martin | 11-740-11 | |
| micro-forceps | S&T | 112314 | |
| Clamp | Aesculap | BH111R | |
| scissors | F S T | 14501-14 | |
| surgical forceps | Aesculap | BD 557 | |
| Decellularisation | |||
| Respirator | Resmed | 14.24.11.0004 | SmartAIR ST |
| Perfusion Device | Charite, medical engineering laboratory | custome-made device | decellularisation device |
| peristaltic pump ismatec reglo ICC | IDEX | ISM4408 | 4-channel |
| heidelberger extension 75 cm | Fresenius Kabi | 2873 | 75 cm |
| MS/CA pump-segment | IDEX | IS 3510 | MS/CA/click'n'go/POM-C |
| CA 2-stopper tube | Pharmed | BPT NSF-51 | |
| bubble trap | custome-made item | ||
| Luer Lock hose connector | Neolab | No. 02-1887 | |
| Detergents | |||
| SDS pellets | Carl Roth | CN30.4 | 2,5 kg |
| Triton X-100 | Carl Roth | 3051.1 | 10l |
| PBS | Gibco | 14190-094 | DPBS |
| staining | |||
| Eosin 1% | Morphisto | 10177 | |
| Mayer hematoxylin | AppliChem | A4840 | |
| gomori staining | Morphisto | 11104 | |
| AlcainBlue-PAS staining | Morphisto | 11388 | |
| Direct Red 80 | Sigma Aldrich | 365548 |
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