Optimizing the Decellularized Porcine Liver Scaffold Protocol

Abstract

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.

Decellularization Techniques for Tissue Engineering: Towards Replicating Native Extracellular Matrix Architecture in Liver Regeneration

“…Among the many techniques listed previously, perfusion-based decellularization is reportedly the most popular approach to decellularizing the liver. This technique involves the delivery of chemical and enzymatic agents such as Triton X-100, SDS, EDTA, and others into the portal or hepatic vein in order to efficiently remove cells and create acellular ECM scaffolds [104][105][106][107]. Certain studies have suggested that the process of freezing-thawing may minimize the quantities of decellularization reagents required; nonetheless, cryoprotectants are recommended to prevent any possible damage to the ECM microstructure [5].…”

“…The DNA content was evaluated with the help of a NanoDrop spectrophotometer based on the measurement of the normalized weight [110,111]. The ECM microstructure was evaluated with the help of scanning/transmission electron microscopy [104,109]. A colorimetric assay was performed to quantify Perfusion-based decellularization has the advantage of pressure-controlled or flow rate-controlled infusion, allowing for more constant distribution of the chemical agents within the organ [110,111,114].…”

“…The DNA content was evaluated with the help of a Nan-oDrop spectrophotometer based on the measurement of the normalized weight [110,111]. The ECM microstructure was evaluated with the help of scanning/transmission electron microscopy [104,109]. A colorimetric assay was performed to quantify the collagen in the ECM based on the detection of hydroxyproline found in the structural protein [111,118].…”

Mesenchymal Stromal Cell on Liver Decellularised Extracellular Matrix for Tissue Engineering

“…The second step of decellularisation was based on the use of SDS, a detergent that lyses cell membranes and causes interruption of noncovalent bonds between ECM structural proteins [ 40 ]. After this procedure, the evaluation of residual materials within the decellularised scaffolds is mandatory to avoid in vitro cytotoxicity or in vivo adverse host responses during the reintroduction of the organ [ 12 , 41 , 42 , 43 ]. In the present study, the presence of nuclear material was qualitatively and quantitatively evaluated after decellularisation, resulting in compliance with the reported criteria [ 29 ].…”

iPSC-derived cells for whole liver bioengineering

“…Decellularization encompasses liver perfusion or full immersion in detergents (e.g., SDS, Triton X-100), which lyse cells without disrupting the tissue microstructure, followed by multiple saline solution washes ( Ahmed et al, 2019 ). Enzymatic (e.g., nuclease, trypsin) or mechanical (e.g., agitation, pressurization, and shear stress) decellularization can be employed alone or in conjunction with detergents to enhance the removal of cell lysis residues ( Faccioli et al, 2020 ).…”