Figure 1.
Keratinocytes and fibroblasts in cultures.
3×105 fibroblasts, stained with PHK2 (green), were cultured in 6-well pre-coated plate for 48 hours (1A and 1C), and then 1×106 keratinocytes, stained with PHK26 (orange), were seeded into the wells with (1C) or without (1B) fibroblasts. After 2 days, keratinocytes were also attached on the bottom of the well (1D). On day 10, fibroblasts tended to cover the entire bottom of the well (1E) but keratinocytes did not proliferate very much (1F). In the co-culture, significantly more keratinocytes (round cells) were growing on the fibroblast layer (1G). On day 15, the overgrowing fibroblasts resulted in some cells grew on others (1H), while keratinocytes could still form the skin-like layer on the top of fibroblasts (1I). 1A–1F were under fluorescence microscope (x200 or x400), 1G, 1H and 1I were under regular light microscope (x200), and natural light were used for 1I(x200).
Figure 2.
Effects of different cultures on the growth of keratinocytes (2A) and fibroblasts
(2B). 2A: Keratinocytes were cultured with fibroblasts in common 6-well plates (Kera/Fibr), with Fibr in transwell toseparate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or keratinocytes alone (Kera). Compared to Kera or Fibr//Kera, *P<0.01. Four cultures in each condition. 2B: Fibroblasts were cultured with keratinocytes in common 6-well plates (Kera/Fibr), with keratinocytes in transwell to separate keratinocytes and fibroblasts (Kera//Fibr), with different concentrations of EGF (EGF10: EGF 10 ng/ml, EGF20: EGF 20 ng/ml and EGF40: EGF 40 ng/ml) or fibroblasts alone (Fibr). Compared to the groups without star, *P<0.01. Four cultures in each condition.
Figure 3.
HB EGF, IL-1α and TGFβ1 levels (3a) as well as keratinocyte concentrations (3b) in different cell cultures.
3×105 fibroblasts and 106 keratinocytes, respectively stained with PHK2 and PHK26, were cultured or co-cultured for 5 days. The supernatant was collected (Fig. 3a) and HB EGF, IL-1α and TGFβ1 levels were measured with ELISA. Keratinocytes (K) were harvested and resuspended in 2 ml media, and then were counted by Trypan blue exclusion and Flow Cytometry (Fig. 3b). K/F: keratinocytes were cultured with fibroblasts in common 6-well plates; K: keratinocytes alone; F: fibroblasts alone; In the co-cultures, keratinocytes were cultured with fibroblasts in transwells to separate keratinocytes and fibroblasts (K//F), with fibroblasts transfected with IL-1α siRNA (K/Fa) or TGFβ1 siRNA (K/Fb), or with fibroblasts and anti-HB EGF (K/Fc), anti-IL-1α (K/Fd) or anti-TGFβ1 (K/Fe). *P<0.05 and **P<0.01 compared to the groups without star. Four cultures in each condition. Dotted line shows the keratinocyte seeding level.
Figure 4.
Effects of different cytokines on the growth of Keratinocytes.
Keratinocytes and fibroblasts were respectively stained with PHK26 and PHK2 before co-culture. Keratinocytes were cultured with fibroblasts (K/F), or with 10 ng/ml, 20 ng/ml and 40 ng/ml of HB EGF (K h10, K h20 and Kh40), IL-1α (Ki10, K i20 and K i40) or TGFβ1 (K t10, K t20 and K t40), or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 10 ml after harvested on day 5 following the culture. Four cultures in each condition. Keratinocytes were counted by Trypan Blue exclusion and Flow Cytometry.
Figure 5.
Effects of co-cultures on the growth of Keratinocytes in long term culture.
Keratinocytes were cultured with fibroblasts in common 6-well plates (K/F), with Fibroblasts in transwell to separate keratinocytes and fibroblasts (K//F), with 20ng/ml EGF (K+ EGF), with fibroblasts and anti-HB EGF (K/F + anti-HB EGF), anti-IL-1α (K/F + antiIL-1a) or anti-TGFβ1 (K/F antiTGF β1) or keratinocytes alone (K). 6-well-plates were used in the culture and cells were resuspended in 12 ml after harvested. Four cultures in each condition. Keratinocytes and fibroblasts were respectively stained with PKH26 and PKH2 before culture and counted at the end of each culture (stage) by Trypan Blue exclusion and Flow Cytometry.
Figure 6.
The relationships between cell concentration and HB-EGF, IL-1α, and TGF-β1 concentrations.
Fibroblasts (3.0×105 cells) and keratinocytes (1.0×106 cells) were cultured in 6-well plates. The supernatant and cells that contained both keratinocytes and fibroblasts from 4 wells were harvested at each time point. The cells were then resuspended in 5 ml for counting. The cytokine concentrations were measured by ELISA. (6A) HB-EGF, IL-1α, and TGF-β1 concentrations at different time points. (6B) Total cell concentration (containing both keratinocytes and fibroblasts) at different time points. (6C) The ratio of HB-EGF, IL-1α, and TGF-β1 concentrations over the total cell concentration (×106/ml) at different time points.
Figure 7.
The effects of co-culture on the migration of keratinocytes and fibroblasts.
Keratinocytes (1.0×106 cells) were cultured with pre-seeded 3×105 fibroblasts in common 6-well plates (K/F), with fibroblasts in a transwell to separate keratinocytes and fibroblasts (K//F), or with fibroblasts and anti-IL-1α (K/Fa), anti-TGFβ1 (K/Fb), or anti-HB EGF (K/Fc). Keratinocytes alone (K) and fibroblasts alone (F) were cultured as controls. All cells were treated with Mitomycin C and the fibroblasts in K/Fp were also treated with 1% paraformaldehyde PBS. 2 days after keratinocytes were seeded, the coverslips were transferred to new wells for additional analysis. The 6-well-plates containing cells on coverslips were used for additional analysis. After culturing the cells for 5 days, the coverslips were removed and the cells in the wells were trypsinized and resuspended in 1 ml for counting. Six cultures were analyzed for each condition. Keratinocytes and fibroblasts were stained with PKH26 and PKH2, respectively, before culturing the cells, and then counted by Trypan Blue cell exclusion and flow cytometry after culture. *P<0.05 and **P<0.01 compared to the group without the star. Between *and **, P<0.05.