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Arab W, et al.

was made from the stock solution. Briefly, gelatin 2.5 Biocompatibility Studies
(porcine skin type A, Sigma) was dissolved at 30 mg/
mL in Milli-Q water under constant stirring at 40 °C, 2.5.1 MTT Assay
then autoclaved. After that, 30 mg/mL of Alginic acid
sodium salt powder (Sigma) was dissolved in the gelatin Biocompatibility studies were carried out in 96-well
plates. C2C12 were seeded at a density of 10,000 cells/
solution. The resultant alginate-gelatin solution was then well and incubated overnight in the complete growth
ionically crosslinked by calcium chloride (CaCl , 150 medium. The cell culture medium was replenished,
2
mM, Sigma) for 5 min. Finally, calcium chloride was and different concentrations of peptide solution were
removed and corsslinked alginate-gelatin was washed added to the wells. The wells without peptides were
with phosphate buffer saline. used as positive control. After 24 h incubation at 37 °C,
a colorimetric MTT assay was performed to determine
2.2 Scanning Electron Microscopy Analysis cell viability according to the manufacturer’s protocol.
Briefly, 10% MTT reagent was mixed with fresh serum-
The hydrogels were dehydrated using gradient ethanol: free medium and added to each well including positive
30, 50, 70, 90 and 100% was used for dehydration, control wells. The plate was incubated at 37 °C between
each step for 15 min. Further dehydration was done 2–4 h, then, 100 μL of DMSO was added to each well
twice in 100% ethanol for 15 min each. The dehydrated to dissolve the insoluble crystals of formazan. Finally, a
samples were dried for 20 min in 1:2 solution of plate reader (PHERAstar FS, Germany) was used to read
hexamethyldisilazane (HMDS): 100% ethanol, the absorption of individual wells at 540 nm.
followed by 20 min incubation in a fresh solution of 2:1 2.5.2 Cytotoxic Assay
HMDS:100% ethanol and then 20 min in 100% HMDS;
the last step was repeated twice. Finally, the samples Mouse myoblast cells were seeded and treated with
were left in a fume hood overnight with the container peptides according to the protocol described above. After
24 h incubation, 50 µL from the incubated medium with
cap left loose. The samples were mounted onto sample a different concentration of peptides and alginate-gelatin
holders using conductive tape, and then sputter-coated were transferred to a new 96-well plate followed by
with Iridium 5 nm and Gold/Palladium 3 nm. The adding 50 µL of cytotoxic reagent and incubated for 30
coated samples were then examined with field emission min in the dark. Stop solution was added, and the release
scanning electron microscopy system (FEI Nova of lactate dehydrogenase (LDH) was quantified at 490
Nano630 SEM, Oregon, USA). nm using a plate reader (PHERAstar FS, Germany).
2.3 Cell Culture 2.5.3 3D Cell Proliferation Assay

Mouse myoblast cells (C2C12) were cultured either CellTiter-Glo Luminescent 3D cell viability assay was
®
in a T175 or T75 culture flask in complete DMEM performed to quantify ATP production in 3D hydrogels
media (10% fetal bovine serum, and 1% penicillin/ which reflect the number of metabolically active cells.
streptomycin). The cells were incubated in a humidified After each time point, an equal amount of CellTiter-
®
incubator with 95% air and 5% CO2 at 37 °C. The cells Glo luminescent reagent was added to the 3D cultured
were subcultured by trypsin at approximately 80% hydrogels and mixed for 2 min to digest the hydrogels
confluence. The culture media was replenished every and then incubated for 25 min. Finally, the luminescence
48 h. was recorded using a plate reader (PHERAstar FS,
Germany).
2.4 3D Culture of Myoblast Cells in Peptide
Hydrogels 2.5.4 Live/Dead Staining
Live/Dead fluorescence staining is a standard fluo-
Mouse myoblast cells were encapsulated in peptide rescence imaging method used to visualize live cells
hydrogels in glass bottom dish (Nunc, 12 mm). Peptide (green) and dead cells (red). After each time point, the
solutions CH-01 (4 mg/mL) and CH-02 (3 mg/mL) were cell culture media was removed and incubated for 30
added to the plate at 40 μL/dish. Mouse myoblast cells min in the dark in DPBS solution containing live/dead
(30,000 cells/dish) that re-suspended in 2× PBS were fluorescence staining (2 µM calcine for live cells (green)
gently mixed with peptide solutions. Gelation occurred and 4 µM ethidium homodimer-1 for dead cells (red).
within 3–5 min, and subsequently, the culture medium Finally, the staining solution was removed and washed
was added to the dishes. On day 2, 4 and 8, the 3D cell with fresh DPBS [33] . An inverted confocal microscope
viability assay, live/dead assay and cytoskeletal staining (Zeiss LSM 710, Germany) was used to observe and
were performed. image the live and dead cells.

International Journal of Bioprinting (2018)–Volume 4, Issue 2 3