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

Abstract

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.

Keywords: decellularization; extraction; liquefied gas; scaffold; subcritical fluid.

Figure 1

Decellularization procedure of this study using SDS and liquefied DME.

Figure 2

(a) Pig ear before removing auricular cartilage, (b) 10 × 10 × 1 mm sample, (c) 710–1000 μm-sized sample.

Figure 3

Lipid extraction from porcine auricular cartilage using liquefied DME. (a) 10 × 10 × 1 mm samples, (b) 710–1000 μm-sized samples. The three symbols represent individual differences among three samples.

Figure 4

Water extraction from porcine auricular cartilage using liquefied DME. (a) 10 × 10 × 1 mm samples, (b) 710–1000 μm-sized samples. The three symbols represent individual differences among three samples.

Figure 5

Hematoxylin–eosin staining of 10 × 10 × 1 mm sample. DNase treatment was applied for 7 days without degassing as a pretreatment.

Figure 6

Hematoxylin–eosin staining of 10 × 10 × 1 mm samples. (a) Untreated. (b) DME extraction followed by DNase treatment for 7 days with degassing. The images shown below are enlarged images of the area enclosed by the rectangle in the upper right corner of the images shown above.

Figure 7

Hematoxylin–eosin staining of 710–1000 μm-sized samples. (a) Untreated. (b) After lipid extraction using liquefied DME. (c–g) DNase treatment for (c) 1, (d) 3, (e) 5, (f) 7, and (g) 10 days following the DME extraction. The images designated by “-1” indicate the original images, while those designated by “-2” indicate the enlarged images.

Figure 8

Residual DNA amounts in the porcine auricular cartilage upon treatment with liquefied DME. (a) 10 × 10 × 1 mm samples, (b) 710–1000 μm-sized samples. The DNase treatment for 0 days indicates that only lipid extraction with liquefied DME was carried out and no DNase treatment was performed. The red line indicates the regulated value.

Figure 9

Fragments of residual DNA in the samples as detected by agarose gel electrophoresis. (a) Untreated. (b–f) DNase treatment for (b) 1, (c) 3, (d) 5, (e) 7, and (f) 10 days following DME extraction.

Figure 10

FT-IR spectra of porcine auricular cartilage.