Catalytic Synthesis and Characterization of Biodegradable ...

Chapter 4
Cell viability (%) = (Asample / Acontrol) × 100 (1)
where Asample was the absorbance of the polymers extracted solutions treated cells and Acontrol
was the absorbance of the untreated cells. Each experiment was done in triplicate and
repeated a minimum of three times.
Cell Adhesion and Spreading
RSC96 cells (rat Schwann cell) were used to investigate the cell adhesion and viability of
the several materials. The cells were cultured in cell culture flasks with DMEM medium
supplemented with 10% fetal calf serum in a humidified incubator at 37 and 5% CO2.
The PLA, PTAm-g-PLA2 and PTAm were dissolved in CHCl3 (10 mg/ml), and the
solution was cast onto glass slides. The coated slides were treated under vacuum for 48 h to
remove the trace of CHCl3, followed by sterilizing by ultraviolet radiation for 2 h.
Approximately, 30,000 RSC96 cells were seeded on the cover-slip coated with PLA,
PTAm-g-PLA, PTAm and allowed to grow for 48 hours at 37 o C/5% CO2. RSC96 cells were
routinely grown in DMEM medium containing 10% FBS and antibiotics (100 U/mL
penicillin, 100 μg/mL streptomycin). Subsequently, the cells were fixed in 3%
paraformaldehyde in PBS pH 7.4 for 15 min at room temperature, and washed twice with ice
cold PBS. In order to improve the penetration of the antibody, cells were incubated for 10
min with PBS containing 0.25% Triton X-100. Cells were incubated with 1% BSA in PBST
for 30 min to block unspecific binding of the antibodies. Cells were incubated in the diluted
F-actin antibody (Abcam) in 1% BSA in PBST in a humidified chamber for 1 hour at room
temperature. The solution was decanted and the cells were washed three times in PBS, 5 min
each time. Cells were incubated with the goat polyclonal to Rabbit IgG conjugated with
Rhodamine secondary antibody (Abcam) in 1% BSA for 1 hour at room temperature in dark.
The secondary antibody solution was decanted and washed three times with PBS for 5 min
each in dark. Cells were incubated on 1 μg/ml DAPI (Sigma) for 2 min and rinsed with PBS.
The cells were observed with a Nikon Eclipse TE2000-U fluorescence microscope equipped
with a DXM1200F digital camera.
References
1. E. S. Gil, S. A. Hudson, Prog. Polym. Sci. 2004, 29, 1173.
2. E. G. Fine, R. F. Valentini, R. Bellamkonda, P. Aebischer, Biomaterials 1991, 12, 775.
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