CELL CULTURE, DIFFERENTIATION AND EXPERIMENTAL DESIGN

3.3.1SH-SY5Y

Exponential growing human neuroblastoma SH-SY5Y cells (Ross et al., 1983), were cultured with Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) GlutaMAX™ supplement (Invitrogen) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Euroclone) and 25μg/ml gentamicin (Sigma) (growth medium), in humidified atmosphere of 5% of CO2 in air at 37 °C. Cultures were maintained by subculturing 900000 cells into 25 cm² flasks (Sarstedt) every 2 days (once they reached 90% confluence). Cell differentiation was induced by treating cells with all-trans-retinoic acid (RA, Sigma) at 10 M concentration and lowering the FBS in the culture medium to 2% (differentiation medium) 24 h after seeding. In undifferentiated control samples, Dimethyl sulfoxide (DMSO) was added as equivalent amount (in which RA is dissolved). In experiments with peptides added to culture medium, cells were seeded in a 24- well plate (15000 cells/well) coated with gelatine (Sigma, porcine skin 0.005% in H2O milliQ)/poly-L-lysine (Invitrogen, 1g/ml) solution. 24h after cell seeding (day 0), the growth medium was replaced by the differentiation medium; then, 24h after RA induction (day 1) peptides were added to the culture medium to asses if they can influence cell viability/proliferation and promote/inhibit neurite elongation. Peptides are not present in control samples. Cell viability and proliferation were assessed at all time points (days 0, 1, 2), while neurite lengths were measured 24 h after the peptides addition (day 2). In experiments with peptides adsorbed onto the substrate, peptides dissolved in gelatine solution were adsorbed onto the bottom of a 24-well plate prior to cell seeding (15000 cells/well). Culture wells coated with poly-L-lysine were used as control. 24 h after cell seeding (day 0), the growth medium was replaced by the differentiation

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medium. Cell viability and proliferation were assessed at three time points (days 0, 1 and 4) or four time points (days 0, 1, 4 and 7) while neurite lengths were measured at day 4. In order to determine the percentage of adsorbed peptides, the fluorescein isothiocyanate (FITC)-conjugated version of peptides were used to coat well bottoms. Briefly, peptides were dissolved in a gelatine solution and incubated in a 24-well plate; then the fluorescence was measured with a plate reader (Ascent Fluoroscan, excitation 485 nm, emission 538 nm). Three hours after incubation, wells were washed 2 times with PBS and fluorescence was measured again in order to calculate the percentage of peptides adsorbed onto the wells. In experiments with cells seeded onto the scaffolds, PLLA and CNT- PLLA sheets were cut into round slices with 13 mm diameter to be well suited for positioning in 24-well plates. After sterilization by UV irradiation, PLLA and CNT- PLLA scaffolds were incubated for 3h with FBS and then cells (15000/well) were seeded onto their surfaces. 24 h after cell seeding (day 0), the growth medium was replaced with differentiation medium. Culture wells coated with poly-L- lysine were used as control. Cell distribution (between scaffold and well bottom) analysis was performed at day 0, cell viability and proliferation were assessed at days 0 and 2 and neurite lengths were measured at day 2. In experiments with cells growing onto CNT-PLLA scaffolds and treated with peptides, the CNT-PLLA scaffolds were incubated for 3 h with FBS and then cells (15000/well) were seeded on their surfaces. 24 h after cell seeding (day 0), the growth medium was replaced with differentiation medium and 24 h after RA induction (day 1) the peptides (1M L1-A or LINGO1-A) were added to culture medium. Culture wells coated with poly-L-lysine were used as control. Cell viability and proliferation were assessed at days 0, 1 and 2, while neurite lengths were measured at day 2. In experiments with cells seeded onto the electrospun scaffolds, fibre covered coverslips were removed from the paper support to be well suited for positioning in 24-well plates. After sterilization by UV irradiation, ePLLA and eCNT-PLLA scaffolds were incubated for 3h with FBS and then cells (15000/well) were seeded onto their surfaces. 24 h after cell seeding (day 0), the growth

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medium was replaced with differentiation medium. Culture wells coated with poly-L-lysine were used as control. Cell distribution (between scaffold and well bottom) analysis was performed at day 0, cell viability and proliferation were assessed at days 0 and 2 and neurite lengths were measured at day 2.

3.3.2H

(

CMC

)

hCMCs were isolated by ficoll density gradient separation from human donor peripheral blood and gently provided by prof. Di Liddo group (Department of Pharmaceutical and Pharmacological Sciences University of Padua). In experiments with cells seeded onto the scaffolds, CNT-PLLA sheets were cut into round slices with 13 mm diameter to be well suited for positioning in 24-well plates. After sterilization by UV irradiation, CNT-PLLA scaffolds were incubated for 24h in the culture medium. Then cells (12000/well) were seeded onto scaffold surfaces in complete growth medium (DMEM/F12 supplemented with 16.5% of heat-inactivated FBS and 50 U/ml penicillin - 50 g/ml streptomycin). Cells were maintained in humidified atmosphere of 5% of CO2 in air at 37 °C. 24 h after cell seeding (day 1), the growth medium was replaced with differentiation medium (DMEM/F12 supplemented with 2% of heat-inactivated FBS, 50 U/ml penicillin - 50 g/ml streptomycin and RA 10 M). In undifferentiated control samples, DMSO was added as equivalent amount (in which RA is dissolved). Cells were maintained in culture for five days after cell seeding and analyses were performed at the following time points: cell proliferation, days 1, 3 and 5; morphological observations, day 1; qPCR experiments, days 1, 3 and 5; immunofluorecence, day5. In experiments with peptides added to culture medium, cells were seeded in 24-well plates (12000 cells/well) in complete growth medium. 24h after cell seeding (day 1), the growth medium was replaced by the differentiation medium; then, 24h after RA induction and the lowering of FBS (day 2) peptides (1 M concentration) were added to the culture medium to asses if they can influence cell proliferation and differentiation. Peptides are not

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present in control samples. Cell proliferation was assessed at days 1, 3 and 5 while qPCR experiments were performed at day 3 and 5. In experiments with cells seeded onto the eCNT-PLLA scaffolds, fibre covered coverslips were removed from the paper support to be well suited for positioning in 24-well plates. After sterilization by UV irradiation, eCNT-PLLA scaffolds were incubated for 24h in the culture medium. Then cells (12000/well) were seeded onto scaffold surfaces in complete growth medium. 24 h after cell seeding (day 1), the FBS concentration was reduced to 2%. Morphological observations and qPCR analyses were performed at day 1.

3.4 ANALYSIS OF CELL VIABILITY/PROLIFERATION AND