Whole-liver decellularization of rat liver

Download PDF

Scope of the method

The Method relates to
  • Animal health
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Ratus norvegicus
Type of cells/tissues/organs
Liver

Description

Method keywords
  • liver
  • decellularization
  • matrix
Scientific area keywords
  • basic research
Method description

This method describes the steps to go from a liver to a decellularized matrix. It uses mild and strong detergents to destroy cells and keep the extracellular matrix intact. This matrix can then be used for a variety of purposes, including (but not limited to) repopulation, basis for coating and basic research.

Lab equipment
  • Laminar Air Flow (LAF) ;
  • Peristaltic pump ;
  • Perfusion equipment.
Method status
  • Published in peer reviewed journal

Pros, cons & Future potential

Advantages

Versatile tool: matirx can be used for an array of things.

Challenges
  • Very specific surgery and very dependent on the surgeon;
  • Small differences in cannule placement can have major implications; 
  • Very hard to keep sterile;
  • Time consuming;
  • Remaining SDS is lethal for cells.
Modifications

This method can be modified: cannulation of vena cava and/of arteria hepatica to create an closed environment.

Future & Other applications

This method has a lot of potential, but faces a few big obstacles. If these could be removed, this method is would be a huge step forward.

References, associated documents and other information

References

De Kock, Joery & Ceelen, Liesbeth & De Spiegelaere, Ward & Casteleyn, Christophe & Claes, Paul & Vanhaecke, Tamara & Rogiers, Vera. (2011). Simple and quick method for whole-liver decellularization: A novel in vitro three-dimensional bioengineering tool?. Archives of toxicology. 85. 607-12. 10.1007/s00204-011-0706-1

Other remarks

Material:

  • - Bile cannula
  • - Plastic cannula
  • - Sterile surgical material (forceps, scissors, ...)
  • - Sterile glass petri dish
  • - Sterile injection needles (3-26G3/8)
  • - Sterile drape
  • - Shaving equipment (electronic and/or razor)
  • - Surgical suture (mersilk, 2-0)
  • - Sterile glass recipients
  • - Perlonfilter
  • - Sterivex® filter
  • - Carbogeen (5 % CO2 and 95 % air)
  • - Bidest water
  • - Heparin (5000 IU/ml)
  • - Sedation (e.g. 87.5 mg/kg ketamine and 12.5 mg/kg xylazine
  • - Krebs-Henseleit-buffer (KHB) pH = 7.4 
  • - Krebs-Henseleit-buffer with calcium pH = 7.4 - 70% (v/v) ethanol solution
  • - 1% triton-x solution
  • - 1% SDS solution

Experimental procedure:

  • - Sterilize the perfusion equipment with 70% (v/v) ethanol solution.
  • - Rinse with bidest water.
  • - Sedate the rat (e.g. 87.5 mg/kg ketamine and 12.5 mg/kg xylazine)
  • - Shave the abdomen.
  • - Disinfect the abdomen with 70% alcohol solution.
  • - Make a U-shape incision and put the intestines outside the abdomen.
  • - Put 2 surgical sutures on the bile duct, close the lower suture.
  • - Make an incision in the bile duct and cannulate.
  • - Close the higher suture, fixing the cannula.
  • - Put 2 surgical sutures on the vena porta without closing them.
  • - Put 1 surgical suture on the vena cava inferior without closing it.
  • - Inject 1 ml of diluted Heparin solution (200IU/ml) in the vena saphena medialis.
  • - Close the lower suture on the vena porta.
  • - Make an incision in the vena porta and cannulate with the plastic cannula.
  • - Close the higher suture on the vena porta.
  • - Close the suture on the vena cava.
  • - Excise the liver.
  • - Perfuse the liver with the perfusion equipment (15min KHB, 30 ml/min).
  • - The animal dies of exsanguination.
  • - Perfuse the liver 1 hour with triton-X solution
  • - Perfuse the liver 1 hour with SDS solution
  • - Perfuse the liver 1 hour with Bidest water

Contact person

Paul Claes

Organisations

Vrije Universiteit Brussel (VUB)
Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology
Belgium