3.2 Selection of optimum SilMA hydrogel
3.2.1 Culturing and Identification of BMSCs
BMSCs have a characteristic of adherent, so that the non-adherent cells
were removed when the culture medium was changed. The typical spindle
shape of isolated BMSCs were presented by optical microscope image in
Fig 4A1 and A2. Flow cytometry analysis
in Fig 4A3 revealed BMSCs matched the characteristics of
MSCs that CD29 (98.5%), CD44 (99.0%) and CD90 (99.4%) were highly
expressed and few cells were positive in CD34 (0.93%). The results
indicated that BMSCs could be passaged stably in vitro and maintain
their phenotypes.
3.2.2 Culturing and morphologic observation of autologous tracheal
epithelia
Optical microscope image of autologous tracheal epithelia was shown in
Fig 4B. On the fifth day of culture, the cells have already adhered to
the culture flask, became larger, and became polygonal. The cells
gathered into clusters, which were like paving stones (Fig 4
B1 and B2). Cells reached 90%
confluence at day 13 (Fig 4 B3 and B4).
The cells grew vigorously and uniformly, and more large flat and
polygonal cells can be observed, which were densely distributed like
paving stones.
3.2.3 Cell proliferation
As described above, the SilMA hydrogel with
concentration
of 10%, 15% and 20% were prepared and used in CCK-8 test. At the
third day, the 10% SilMA hydrogel was degraded, which was consistent
with the result of degradation test. Therefore, Fig. 5A only showed the
results of the concentration of 15% and 20%. The cells attached to the
surface of the hydrogel continued to proliferate during the test,
especially at the fifth day. Compared with conventional culture, there
is still a certain gap in the number of cells, but the hydrogel with
concentration of 20% is more suitable for cell growth than 15% and the
difference was statistically significant.
3.2.4 H&E staining analysis of 3D co-culture
In order to select the appropriate concentration ulteriorly and evaluate
the growth of cells in the hydrogel, 3D co-culture experiments were
conducted. Same as section 3.2.3, the 10% SilMA hydrogel was degraded.
As a result, Fig. 5B
represented
the H&E staining of 15% SilMA and
Fig.
5C represented 20%. SilMA hydrogel with 20% concentration possessed a
more compact microstructure and smaller pore size. After 7 days of 3D
co-culture, the number of alive cells decreased significantly, which was
different from the result of 2D co-culture in section 3.2.3. However,
the gratifying result was that under the same field of vision, more
cells survive in the 20% SilMA hydrogel and the difference was
statistically significant. The results were further quantified and
plotted into a column chart, as shown in Fig. 5D.