PMC

Y-27632 treatment improves the procurement of primary prostate epithelial cells. Cells from human prostate specimens were cultured in the presence or absence of Y-27632. Numbers of epithelial cells generated from 10,000 cells during 10 days of culture are shown (n = 4, Mann–Whitney U-test).

Keratinocytes treated with Y-27632 maintain the growth potential after cessation of Y-27632 at serial passage. (A) Schematic depiction of Y-27632 treatment during keratinocyte serial passage. Thick solid arrows indicate time frames when Y-27632 was used. Thin dashed-line arrows indicate time frames when cells were cultured in the absence of Y-27632. (B) CFE in the first round (days 0–14) in the presence or absence of Y-27632; measured on day 14 (n = 4; Mann–Whitney U-test). (C) CFE in the second round (days 14–28) in the absence of Y-27632 for all samples of “no Y,” “Y × 6” and “Y × 14”; measured on day 28 (n = 4; Mann–Whitney U-test). (D) Colonies of the second round (days 14–28, in the absence of Y-27632); fixed and stained on day 28. A representative set of four independent experiments summarized in (C) is presented. Numbers of cells seeded at the passage on day 14 are shown on the top.

Y-27632 treatment during the first 6 days of primary culture is critical for increased CFE. (A) Human skin specimens and primary keratinocytes were treated with Y-27632 during tissue processing, subsequent 14-day culture, or both. Thick solid arrows indicate time frames when Y-27632 was used. Thin dashed-line arrows indicate time frames when specimens or cells were not exposed to Y-27632. CFE is increased only if Y-27632 is present during the cell culture period. Wells in each row are triplicates. (B) Primary keratinocytes were treated with Y-27632 over various time frames during the 14-day culture. Thick solid arrows indicate time frames when Y-27632 was used. Thin dashed-line arrows indicate time frames when cells were cultured in the absence of Y-27632. Y-27632 treatment for the first 6 days (sample e) increases CFE equally well as the treatment for the entire culture period (sample b). Enlarged images of colonies in samples b, d, and e are shown on the right. Note larger colony sizes in samples d and e compared to those in sample b.

Y-27632 treatment increases colony-forming efficiency (CFE) and cell output of epidermal keratinocytes freshly isolated from skin specimens. (A) Keratinocyte colonies formed in 14 days of culture either without (top row) or with 10 μM of Y-27632 (second to fourth rows). This is a representative experiment of eight independent sets. Three wells in each row represent triplicates within the single set. (B) Fifty times higher CFE induced by Y-27632 treatment for 14 days (p = 1.0 × 10⁻⁹; n = 8; two-tailed t-test). Error bars indicate standard deviation. (C) Keratinocyte colonies after treatment with Y-27632 at 0, 5, 10, and 20 μM for 14 days. (D) The number of cells generated out of 1000 cells by 0, 6, or 14 days of Y-27632 treatment during 14 days of culture (n = 4; Mann–Whitney U-test).