Evaluation of a Decellularization Protocol for the Development of a Decellularized Tracheal Scaffold

Materials and Methods

Animal model. In our study, D/A male rats (n=30) weighing 220-300 g (Experimental Surgery Center and Translational Research, Biomedical Research Foundation of Academy of Athens, Athens, Greece). were used as donors of tracheal allografts. The current protocol for using animals was authorized by the Competent Regional Veterinary Authority (Ref. Number 1152/12-5-2015), in accordance with European Directive 2010/63 and National Legislation on the protection of animals used for scientific purposes (Presidential Decree 56/2013).

Tracheal harvesting and decellularization procedure. The entire length of rat tracheas (n=18) was harvested and initially treated in 1× phosphate-buffered saline (PBS). The decellularization protocol was based on the chemical action of detergents and included solutions of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), sodium dodecyl sulfate (SDS) and α-minimum essential medium supplementation (α-MEM)/fetal bovine serum (FBS) (8,9). For each decellularization cycle, the isolated trachea samples were placed in CHAPS solution (8 mM CHAPS, 1 M NaCl, and 25 mM EDTA diluted at 1x PBS; pH 8) for 22 h at room temperature followed by washing in PBS 1× for 30 min at 4°C. Tracheas were then placed in SDS solution (1.8 mM SDS, 1 M NaCl, and 25 mM EDTA diluted at 1× PBS; pH 8) for 22 h at room temperature followed by washing with PBS 1× for 30 min at 4°C. Tracheas were finally placed in α-MEM/40% FBS at 37°C for 48 h. The above steps were carried out under constant shaking and were repeated three times.

Histological analysis. Histological analysis was performed to evaluate the efficacy of the decellularization protocol. For this purpose, native (n=6) and decellularized (n=6) tracheas were fixed in 10% neutral formalin, paraffin embedded and sectioned into 5 μm sections. The slides were then deparaffinized and stained with hematoxylin and eosin (H&E; Sigma-Aldrich,Inc., St Louis, MO,USA) for nuclear and cellular material. Sections were also stained with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen, Thermo Fisher Scientific, CA, USA) for nuclear material. LEICA DM RA2 (Leica, Wetzlar, Germany) fluorescence microscopy was used in order to evaluate the samples. Tracheal samples were also stained with toluidine blue (Sigma-Aldrich) for sulphated glycosaminoglycan content and Masson's Trichrome (Sigma-Aldrich) for collagen, nuclear and muscle content. Finally, elastic Van Gieson (Sigma-Aldrich) staining was performed for the evaluation of elastin content. Histological samples were evaluated and images were obtained using a LEICA DM LS2(Leica, Wetzlar, Germany) optical microscope. Due to the fact that the tracheaI thickness varies along its length, different parts of the trachea were histologically evaluated as mentioned above, in order to carry out an integrated and reliable analysis.

DNA quantification. DNA quantification was performed in native (n=6) and decellularized (n=6) tracheas. According to the findings of previous studies, a recommended amount of <50 ng dsDNA/mg matrix was considered to be a reliable criterion confirming the effectiveness of the decellularization protocol (10).

Native and decellularized tracheas were weighed and were digested using Proteinase K supplemented with PBS (Sigma Aldrich) and lysis buffer (0.1 M Tris pH 8, 0.2 M NaCl, 5 mM EDTA, 0.4% SDS) overnight at 55°C, followed by DNA isolation. DNA was isolated from the entire trachea using a protocol which is based on solutions of phenol/chloroform/isoamyl alcohol. DNA quantification per milligram dry tissue was performed using Quantifluor dsDNA System E2670 Promega (Glomax; Promega, Madison, WI, USA) according to the manufacturer's instructions.

Cytotoxicity test. Two assays were performed in order to determine the cytotoxicity of the decellularization protocol. The first was a contact test of cells (11) with decellularized trachea and the second was an extract cytotoxicity assay regarding the ATP metabolism of cells. Wharton's jelly mesenchymal stromal cells (WJ MSCs) from human umbilical cord were used in order to perform the above tests due to their efficacy in differentiating into different cell populations such as epithelial or endothelial cells. Fresh umbilical cords were obtained and used after written informed consent from the mothers and were processed in less than 48 hours (12).

For the contact test, decellularized tracheas (n=2) were cut into segments and placed on 6-well plates with WJ MSCs. Different culture procedures were performed with WJ MSCs: i) basic culture medium (α-MEM, 15% FBS, penicillin, streptomycin) as a negative control sample; ii) basic culture medium with decellularized trachea; and iii) basic culture medium with 10% dimethylsulfoxide (DMSO) in order to cause cytotoxicity, as a positive control sample. The samples were incubated at 37°C with 5% CO₂ for 24 hours and cell growth was evaluated by observing morphology and confluency using a LEICA DM LS2 (Leica, Germany) optical microscope.

ATP Colorimetric/Fluorometric Assay Kit (Sigma-Aldrich) was used for the evaluation of metabolic activity of cells which were cultured with digested decellularized trachea. Tracheas (n=4) were digested using 1 ml of α-MEM supplemented with Proteinase K overnight. Proteinase K inactivation was achieved by incubation at 95°C for 5 min. Decellularized tracheas were then placed in a 6-well plate with WJ MSCs and incubated until confluent. Cultures containing only WJ MSCs with α-MEM were used as negative control samples. ATP concentration was determined by phosphorylating glycerol, resulting in a colorimetric (570nm) product proportional to the amount of ATP present.

Statistics. Statistical analysis was performed using GraphPad Prism v 6.01 (GraphPad Software, San Diego, CA, USA). DNA and ATP content were analyzed using Mann–Whitney test. Statistically significant difference was considered when the p-value was less than 0.05. Data are reported as the mean±standard deviation.

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Figure 1.

Histology of native (upper row) and decellularized (lower row) tracheal samples stained with hematoxylin and eosin (A, F), 4’,6-diamidino-2-phenylindole (B, G), toluidine blue (C, H), Masson's trichrome (D, I) and elastic Van Gieson (E, J) after three cycles of decellularization at 20× (C,H) and 40× (A, B, D, E, F, G, I, J) magnification.