Decellularization protocols

Previously published protocols used several decellularizing agents including detergents and enzymes such as TX, SDS, sodium deoxycholate (SDC), and CHAPS [24, 25]. In this study, the decellularization process with different combinations of detergents was carried out using a perfusion peristaltic pump (Peristaltic Pump P-1, Pharmacia Biotech, Sweden) at a 1.5 mL/min rate. Figure 1A depicts the decellularization protocols of the present study in detail. Briefly, vessels were decellularized by either detergent perfusion or shaking in the detergent solution. In the perfusion group, vessels were perfused with 50 mL of 0.5% SDS or 1% TX or a combination of 0.25% SDS and 0.5% TX for 16 h at room temperature. In the shaking group, the vessels were shaken at 37 °C in 50 mL of a detergent solution including 8 mM CHAPS, 1 M NaCl, and 25 mM EDTA for 2 h. Next, the tissues were washed by shaking in the PBS solution for 30 min, and then they were shaken at 37 °C in a solution of 1.8 mM SDS, 1 M NaCl, and 25 mM EDTA for 2 h. Afterward, the scaffolds were perfused with 0.1% peracetic acid for an hour and then rinsed with sterile PBS containing antibiotic/antimycotic. A cocktail was utilized to neutralize the acid and remove the residual detergent components from the vascular scaffold. The decellularized vessels were then stored in sterile PBS at 4 °C for further investigations including the evaluation of DNA, collagen, and GAG content as well as histological analysis. Moreover, all the experiments performed in the present study were carried out in three independent runs [13].

Schematic protocol indicates decellularization process using SDS, TX, and CHAPS detergents. Perfusion process was performed at room temperature and lasts 16 hours, while shaking process was performed at 37 °C (A) Gross view of NRA and DRA tissue decellularized using 0.25% SDS + 0.5% TX is shown in Fig. 1 indicating tissue transparency following the decellularization process (B). NRA: Native Rat Aorta, DRA: Decellularized Rat Aorta, SDS: Sodium Dodecyl Sulphate, TX: Triton X-100, CHAPS: 3-[(3-cholamidopropyl)

To investigate the efficacy of the decellularization process, samples were fixed in 10% formalin solution for 24 h. The paraffin-embedded samples were stained with hematoxylin and eosin (H&E), Masson’s trichrome for distinguishing collagen, and orcein for the determination of elastic fibers according to the standard protocols before being examined under a light microscope (Olympus BX53, Japan).

To evaluate the quality of decellularization, the total DNA content of native and decellularized vessels was measured. Firstly, the samples were homogenized in the lysis buffer provided in the DNA extraction kit. Total DNA content was extracted according to the manufacturer’s instructions, and the DNA concentration was quantified using a spectrophotometer (ND-1000, Thermo Fisher Scientific, USA) at the 260 nm absorbance wavelength and was normalized according to the wet tissue weight. The results are expressed as ng/mg wet weight of samples.

A colorimetric assay was employed to assess the collagen content of native and decellularized samples through the measurement of hydroxyproline [26]. Concisely, homogenized samples were incubated with cupric sulfate, sodium hydroxide, and hydrogen peroxide at 80 °C for 5 min and then were cooled. Afterward, sulfuric acid and ρ-dimethylaminobenzaldehyde in 1-propanol were added to the samples, which were then incubated at 80 °C for 30 min, and absorption was read at 560 nm using a spectrophotometer (Epoch, BioTek, USA). Moreover, the hydroxyproline level was directly determined using a hydroxyproline standard curve and normalized using wet tissue weight.

Native and decellularized samples were first digested in papain overnight at 65 °C to assess the GAG content using the GAG assay kit (Kiazist, Iran). Then, the samples were centrifuged (Sigma 3-16PK, Germany) at 8000 g for 15 min, the protein precipitant solution was added to the supernatant, and the samples were centrifuged again at 8000 g for 15 min. Subsequently, a GAG reagent was added to the samples and total absorption was measured at 560 nm using a spectrophotometer (Epoch, BioTek, USA). Moreover, the chondroitin sulfate standard curve was used to directly determine the GAG content and the total tissue GAG content was normalized using wet tissue weight.

To characterize the topography of the luminal surface, native and decellularized tissues were freeze-dried (Eleya, Japan), mounted on aluminum stubs, and coated with a thin layer of gold. The analysis of morphology and sample structure was performed at a voltage of 15 kV by FESEM (TESCAN MIRA3, TESCAN, the Czech Republic) [27].