Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

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

Decellularization and recellularization may enable the generation of functional, transplantable organs in vitro ¹. By removing cells and antigenic material (e.g., DNA, alpha-Gal epitopes) from an organ, the non- or less-immunogenic extracellular matrix (ECM) can be obtained. This matrix conserves the three-dimensional microanatomy of an organ and can serve as the ideal biomatrix for repopulation with cells of a different, possibly xenogeneic origin ². Thus, a decellularized rat liver matrix could be repopulated with human liver cells. This humanized micro-liver could serve as an ex vivo model for research on diseases (<....

Protocol

Animals were kept at the Facility for Experimental Medicine (FEM, Charité, Berlin, Germany), and all experimental protocols were reviewed and approved by the State Office of Health and Local Affairs (LAGeSo, Berlin, Germany; Reg. No. O 0365/11).

1. Liver Harvesting

1. Pre-surgical Preparation
   1. Use a cork plate for fixation. Put a medical drape on the plate. Using four needles, fix an inhalation mask on the cork plate for intraoperative inhalation narcosis.
2. Prearrangement of the Portal Venous Cannula
   1. Connect a peripheral venous catheter (G 20) to a three-way stopcock. The venous cathete....

Results

The homogeneity and thus the effectiveness of different decellularization protocols were evaluated by macroscopic observation, histological analysis, and analysis of the remaining DNA content within decellularized liver matrices. Furthermore, corrosion casting was performed to visualize the intact microanatomy of livers after decellularization.

Macroscopy

During decellularization, livers become lucent, indicating the removal of cellular content. Livers perfused via .......

Discussion

Although the presented technique for rat liver harvesting and decellularization is easily reproducible, there are certain critical steps to consider:

During preparation for liver harvesting, it is important to avoid severe bleeding because it will activate blood coagulation and may lead to blood clot formation within the liver. In our opinion, it is advantageous to incise the abdominal aorta directly before cannulation of the portal vein to avoid blood inflow via the hepatic artery during perf.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Self built arterial cannula
Portex Non Sterile Polyethene Tubing	SIMS Portex	REF 800/110/100	0.28 mm ID 0.61 mm OD
Portex Non Sterile Polyethene Tubing	SIMS Portex	REF 800/110/200	0.58 mm ID 0.96 mm 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	1000 ml
10 ml Syringe	Braun	4606108V	10 ml/ Luer Solo
5 ml Syringe	Braun	4606061V	5 ml /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	10 L
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