Surfactant-Free Decellularization of Porcine Auricular Cartilage Using Liquefied Dimethyl Ether and DNase.

The most common decellularization method involves lipid removal using surfactant sodium dodecyl sulfate (SDS) and DNA fragmentation using DNase, and is associated with residual SDS. We previously proposed a decellularization method for the porcine aorta and ostrich carotid artery using liquefied dimethyl ether (DME), which is free from the concerns associated with SDS residues, instead of SDS. In this study, the DME + DNase method was tested on crushed porcine auricular cartilage tissues. Unlike with the porcine aorta and the ostrich carotid artery, it is important to degas the porcine auricular cartilage using an aspirator before DNA fragmentation. Although approximately 90% of the lipids were removed using this method, approximately 2/3 of the water was removed, resulting in a temporary Schiff base reaction. The amount of residual DNA in the tissue was approximately 27 ng/mg dry weight, which is lower than the regulatory value of 50 ng/mg dry weight. Hematoxylin and eosin staining confirmed that cell nuclei were removed from the tissue. Residual DNA fragment length assessment by electrophoresis confirmed that the residual DNA was fragmented to less than 100 bp, which was lower than the regulatory limit of 200 bp. By contrast, in the uncrushed sample, only the surface was decellularized. Thus, although limited to a sample size of approximately 1 mm, liquefied DME can be used to decellularize porcine auricular cartilage. Thus, liquefied DME, with its low persistence and high lipid removal capacity, is an effective alternative to SDS.

Tensile Strength of Porcine Aorta Decellularized with Liquefied Dimethyl Ether and DNase.

In a previous report, we proposed a method for decellularizing porcine aortas by removing lipids from the aortas using liquefied dimethyl ether (DME) instead of the conventional sodium dodecyl sulfate (SDS). This is followed by DNA fragmentation with DNase. In the current work, the physical properties of porcine aortas decellularized using the DME method are evaluated by tensile strength tests. Conventional SDS decellularized aortas are typically swollen, rupture very easily, and have poor elasticity. By contrast, DME-treated samples are found to be less elastic. However, the maximum stress required for rupture is greater than that for the original aorta. These results indicate that decellularization with DME and DNase increases the maximum stress that can be withstood. Reduction of elasticity may derive from the appearance of temporary C=N bonds due to Schiff-base reactions that occur during the lipid removal process by liquefied DME, and methods to avoid this are desirable.