To explore the effects of different culture plates coating time with human type Ⅰ collagen on the cell morphology, adhesion, proliferation, migration rate and cell cycle of cultured human primary epidermal cells.

Fresh human skin tissue was taken from Department of Urology Surgery, Fourth Medical Center of PLA General Hospital, then the human type Ⅰ collagen solution with a volume fraction of 1% was placed. Dishes were coating with collagen solution and divided into 10 s group, 1 min group, 5 min group, 15 min group, 30 min group, and 0 s group according to the different coating time. Then epidermal cells were dissociated from skin tissue by dynamic digestion with trypsin, inoculated in culture plates with different collagen coating time and cultured in a cell culture incubator with 5% CO₂ at 37 ℃ with epidermal culture medicum (CnT-Pr medium). Then cell morphology was observed under interted phase contrast microscope. Cell adhesion and proliferation were detected using cell counting kit-8 (CCK-8). Cell migration was observed by scratch assay, and cell cycle was detected using flow cytometry. Data were processed with analysis of variance and LSD-t test.

(1)Cell quantity and morphology: there was no significant difference in the morphology of epidermal cells within each group over time. The size of cells in each group were relatively uniform, spherical or eye-shaped, and the number of cells gradually was increasing. The number of cells in 0 s group was less than that of the other 5 groups and more unattached cells were found in the 0 s group than in the other groups. (2)Cell adhesion: 24 hours after human primary epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, 1 min group, 5 min group, 15 min group, 30 min group and 0 s group was 0.28±0.07, 0.30±0.05, 0.33±0.06, 0.34±0.07, 0.36±0.05, and 0.16±0.02. The difference in absorbance among the 6 groups was statistically significant (F=4.640, P=0.014). There were no significant differences between any 2 of 5 groups (the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group) (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the difference were statistically significant (t=2.640, 3.059, 3.584, 3.889, 4.187; P=0.021, 0.010, 0.004, 0.002, 0.001). (3) Cell proliferation: the human primary epidermal cells proliferation curve showed that the proliferation of cells in each collagen-coated group was similar, which was significantly higher than that of cells in the 0 s group. Two days after human primary epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group was 0.41±0.05, 0.41±0.02, 0.46±0.06, 0.49±0.08, 0.53±0.12 and 0.09±0.04. The difference in absorbance among the 6 groups was statistically significant (F=16.050, P<0.05). There were no significant differences between any 2 of 5 groups (the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group) (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the differences were statistically significant (t=0.323, 0.323, 0.374, 0.401, 0.44; with P values below 0.05). Four days after human primary epidermal cells were inoculated, the difference in absorbance among the 6 groups was statistically significant (F=9.816, P=0.001). There were no statistical differences among the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the difference were statistically significant (with P values below 0.05). Six days after primary human epidermal cells were inoculated, the absorbance value of epidermal cells in the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group was 2.76±0.20, 3.03±0.17, 3.03±0.16, 3.18±0.17, 3.33±0.26 and 0.53±0.25. (4) Cell migration: 12 hours after scratching, the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (15.60±4.11)%, (18.26±6.79)%, (18.09±6.97)%, (18.00±4.70)%, (17.40±5.97)%, (4.05±1.71)% respectively. Twenty-four hours after scratching the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (36.33±5.63)%, (38.45±11.97)%, (42.36±14.40)%, (41.96±10.78)%, (44.04±12.28)%, (9.17±3.28)%. Thirty-six hours after scratching the cell migration rates of the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group and the 0 s group were (73.71±17.90)%, (62.33±12.45)%, (69.79±20.82)%, (81.89±18.05)%, (73.49±22.89)%, (11.62±2.64)%. Overall comparison among groups showed statistically significicant difference (F=9.914, P<0.05). Further comparison between the groups showed that there were no statistical differences between the 10 s group, the 1 min group, the 5 min group, the 15 min group and the 30 min group (with P values above 0.05). The 0 s group was compared with the 10 s group, the 1 min group, the 5 min group, the 15 min group, the 30 min group respectively, and the differences were statistically significant (with P values below 0.05). (5) Cell cycle: the results of cell cycle test showed that 5 days after human primary epidermal cells were inoculated , there were no significant differences of the cell proportions of G1, G2 and S phase in cell cycle among the 6 groups (with P values above 0.05).

Type Ⅰ collagen coating culture plates play an important role on the culture of human epidermal cells and different culture plates coating time showed no significant effects on that.