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.