2.3 Isolation and culture of cells
2.3.1 Bone marrow mesenchymal stem cells
Bone marrow mesechymal stem cells (BMSCs) were isolated from tibial plateau with whole bone marrow adherent culture method of young New Zealand white rabbit under aseptic conditions accord­ing to our previous protocol.[5, 9] Through this method, non-adherent cells were washed and the culture medium was changed after 48 h for the first time. Thereafter, the culture medium was replaced every 2 days and the cell growth was observed until cells were subcultured at 80% confluence. The 2nd passage BMSCs were obtained to be used in the follow-up experiment, in order to have better differentiation ability. Cell surface markers were examined by flow cytometry.
2.3.2 Autologous tracheal epithelia
The extraction and culture of autologous tracheal epithelia were improved on the basis of the reference.[19] In brief, the experimental rabbits were anesthetized by inhalation of isoflurane (RWD, Shenzhen, China). Then, the mucosal epithelium of trachea was extracted by biopsy forceps in endoscope and digested by 100 U/mL type I collagenase (Absin, Shanghai, China) at 37°C for 12 h. Digested solution was filtered by 200-mesh sieve, followed by centrifugation at 800 rpm for 5 minutes. After the supernatant was removed, and the remnants were collected and seeded in cell culture dish (Corning, New York, USA) containing DMEM-F12 (1:1) (Hyclone, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Clark, USA) and 1% penicillin/streptomycin (Beyotime, Shanghai, China) for 2 h and then the culture medium was transferred to 25 cm2 cell culture flask. The following culture method was the same as BMSCs.
2.4 Selection of theoptimum concentration
2.4.1 Cell Counting Kit-8 test
The 150 μL of sterile SilMA solution was injected to the sterile curing ring, under the 405 nm UV irradiation, to made SilMA hydrogel. Afterwards, SilMA hydrogel on the curing ring was transferred into a 48-well culture plate, 1 mL of the 2nd passage BMSCs suspension was gradually dropped onto the surface of SilMA hydrogel with a density of 5 × 104 cells/piece and incubated for 4 h, followed by transfer to another 48-well culture plate. Then, the culturte medium was exchanged every 48 h, and removed from each well at 1, 3, 5, and 7 day. 200 μL of Cell Counting Kit-8 (CCK-8) (APExBIO, Houston, USA) working solution (dilution ratio: 1: 10 with culture medium under dark conditions) was added to cover the hydrogel. After incubating for 2 h, 100 μL of the supernatant was pipetted to a 96-well plate and the OD450 was measured by a microplate reader (BioTek, Vermont, USA). So that, the adhesive and proliferative effects of cells on SilMA hydrogel of different concentrations could be quantified.
2.4.2 3D co-culture and H&E staining
500 μL of the 2nd passage BMSCs suspension with a density of 5 × 104 cells/mL was added in the sterile SilMA solution. To prepare the hydrogel as described in section 2.4.1, so that BMSCs could be 3D co-cultured with SilMA hydrogel. The hydrogel was cultured with the conventional culture method in incubator (RS232, Thermo Fisher) at 37℃/5%CO2for 7 days. The samples were taken out and fixed in 4% paraformaldehyde (Biosharp, Hefei, China) for 1 h. After fixation, washed in the PBS solution for three times (5 minutes each time), then dehydrated them in gradient ethanol from 30% to 100%, embedded them in optimal cutting temperature (O.C.T.) compound (SAKURA Tissue-Tek, Tokyo, Japan) at 4 ℃ overnight, and successive sectioned them at 6 mm thickness with freezing microtome (Leica, Wetzlar, German). Hematoxylin and eosin (H&E) (Solarbio, Beijing, China) staining was used to evaluate the 3D network structure of SilMA hydrogel and count the alive cells.