Fast, robust and effective decellularization of whole human livers using mild detergents and pressure controlled perfusion

Human whole-liver perfusion-decellularization is an emerging technique for producing bio-scaffolds for tissue engineering purposes. The native liver extracellular matrix (ECM) provides a superior microenvironment for hepatic cells in terms of adhesion, survival and function. However, current decellularization protocols show a high degree of variation in duration. More robust and effective protocols are required, before human decellularized liver ECM can be considered for tissue engineering applications. The aim of this study is to apply pressure-controlled perfusion and test the efficacy of two different detergents in porcine and human livers. To test this, porcine livers were decellularized using two different protocols; a triton-x-100 (Tx100)-only protocol (N = 3) and a protocol in which Tx100 was combined with SDS (N = 3) while maintaining constant pressure of 120 mm Hg. Human livers (N = 3) with different characteristics (age, weight and fat content) discarded for transplantation were decellularized using an adapted version of the Tx-100-only protocol. Decellularization efficacy was determined by histology and analysis of DNA and RNA content. Furthermore, the preservation of ECM components was assessed. After completing the perfusion cycles with detergents the porcine livers from both protocols were completely white and transparent in color. After additional washing steps with water and DNase, the livers were completely decellularized, as no DNA or cell remnants could be detected. The Tx100-only protocol retained 1.5 times more collagen and 2.5 times more sGAG than the livers decellularized with Tx100 + SDS. The Tx100-only protocol was subsequently adapted for decellularizing whole-organ human livers. The human livers decellularized with pressure-controlled perfusion became off-white in color and semi-transparent within 20 h. Livers decellularized without pressure-controlled perfusion took 64–96 h to completely decellularize, but did not become white or transparent. The addition of pressure-controlled flow did remove all cells and double stranded DNA, but did not damage the ultra-structure of the ECM as was analyzed by histology and scanning electron microscopy. In addition, collagens and sGAG were maintained with the decellularized ECM. In conclusion, we established effective, robust and fast decellularization protocols for both porcine and human livers. With this protocol the duration of decellularization for whole-organ human livers has been shortened considerably. The increased pressure and flow did not damage the ECM, as major ECM components remained intact.

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