To test for fibroblast cell migration in vitro; I have adapted a commonly used scratch wound assay (Morales et al. 1995; Basu et al.2001) of tissue-culture cell monolayer's to a 6-well/35mM plastic plate format. Reagent concentrations and quantities have been adapted from (Sheryl P. Denker, Diane L. Barber) in addition to the wound assay. The established human transformed keratinocytes cell line; NCTC 12544 (n=2x25cm2), was provided by Dr M.J.Thornton. Cells were passaged and trypsinized; and seeded into the 35mM dishes at a density of 35,000 (3.5 x104) viable cells per dish, grown to confluence in growth media as described in section 2.1.1. Cells were starved overnight in low serum media 2.0ml; 2.0% serum (media containing low percentage of FCS to faster closing of the wound) in DMEM growth medium supplemented with ≥10% FCS (containing growth factors, hormones etc was added to each dish) serum in media added to adhere and allow migration; and the cells were placed in a cell culture incubator (5% CO2, 37°C, humidity tray to permit cell adhesion and avoid evaporation of the media) for 48 hours; grown until confluence or near (≥90%) prior to wound healing. Antibiotics, Penicillin and streptomycin were added to the media in the cell culture hood to improve sterility; as the conditions of which are important for cell tissue cultures are also potentially favorable for bacterial growth). Depending on cell types cells are confluent usually within 4 to 5 days, with a medium change every 2 to 3 days. Once cells were confluent the medium was removed and the cells were mechanically wounded (scratches separated by millimeters, to obtain desired and limited width, scratched at an angle of 30° degrees) using an ordinary paper clip in preference to a pipette tip as this was firmer and were able to scratch a monolayer of cells to simulate a characteristically sized wounds (defined scratch were obtainable) in all pre-designed 35mM 6-well templates (Figure 2). Finally the cells were washed very gently twice with PBS, to remove dislodged cells and debris. At this time point (t=0hr) wound margins were photographed and measured. Some type of cells; strongly cohere, therefore it is very difficult to get a clean line, in this case the remaining cells in the centre of the scratch were detached using the firm tip of the paperclip. All migrating cells were photographed using an inverted Nikon eclipse TS100 microscope at single time points (0, 3, 6, 24, 30 and 48 hours) and varying dose intensities (1nM, 10nM, 100nM and 1000nM) of 17β-Oestradoil and agonists.
2.1.1 Cell counting prior to in vitro cell migration
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The estimation of cell number was carried out by haemocytometry. The total number of viable cells present in the suspension was calculated; as shown in Appendix 2, table 3.
Migration of human keratinocytes, in the presence of 17ï¢-oestradiol
2.2.1 Dose response effects
Initial experiments were carried out on established cell line keratinocytes to compare the effects of ranging concentrations of 17ï¢-(E2), DPN and PPT (1nM, 10nM, 100nM and 1000nM). These concentrations were chosen based on their binding affinities. Oestrogen exerts its wound healing action via ERα and ERβ receptors. The actions of both agonists are significantly improved at increasing concentrations of 17β-(E2), DPN and PTT, as this enhances the binding affinity of these receptors. Therefore varying concentrations of 17β-(E2), DPN and PTT were chosen to quantify their action in cutaneous wound healing (Ashcroft et al., 1997). Stock 17ï¢-(E2), DPN and PTT was required to be diluted at 1:10-folds to varying concentrations. All migrating cells are photographed as described in section 2.1.
Figure 2. Templates used in triplicates for migration assays adapted from Sheryl P. Denker, Diane L. Barber. A standard paperclip (B) with firm tip was used at 30° to stimulate wound cells with limited width according to the pre-designed template (A). A further template (C) with marker line and scratches was used to ensure that the distance between the wound edges was measured at single fixed time points in each dish at each time point, .
The pH of the growth media was required to be kept within control limits around 7.4 to enable cell growth. Cells continually produce substances that may alter the pH of the media. So buffer systems were added to the media including sterility (aseptic technique), and temperature (incubators) to the phenol-red DMEM to monitor the media change. Phenol red can improve growth of cells expressing oestrogen receptors (Bethois, Y. et al, 1986). To prevent the interference of phenol-red, the phenol-red-serum free DMEM test media was used. 2.0ml of phenol red-free, serum-free DMEM supplemented with absolute ethanol vehicle control (0.0001%), 1nM 17ï¢-(E2), 10nM 17ï¢-(E2), 100nM 17ï¢-(E2) or 1000nM 17ï¢-(E2) was added to individual dishes in triplicate. Same method was used to supplement the agonists per wells. Cell migration was assessed after fixed time points. All migratory cells were photographed using inverted Nikon eclipse TS100 microscope.
2.2.2 Time course assay
Always on Time
Marked to Standard
Following these initial experiments, 1000nM of stock 17ï¢-oestradiol concentration was chosen, as this permitted additional concentration when required to be made from the stock concentration at a 10-fold dilution. Therefore, in subsequent assays either 10nM 17ï¢-oestradiol or absolute ethanol vehicle control (0.0001%) was added to 2.0ml phenol red- free, serum-free DMEM. Following the addition of the medium, cells were incubated at 37°C for 48hours, with the distance between the wounded cell edges being measured at fixed time points of 0, 3, 6, 24, 30 and 48 hours. The distances between the wound edges were measured in each dish using photographs at the 6 fixed points (3mm apart) along the length of the wound according to a pre-designed template (Figure 2c). Photographs of all migrating cells were also taken of the central part of the wound, at each of the time points as described in section 2.1. The average distance between the wound edges for the triplicate dishes (derived from a total of 18 measurements, 3 (35mM) 6-well templates under differing concentrations) was then calculated for the differing time points and from this the mean migratory distance at each time point was determined. The diameter across the wells from the photographs was scaled according to the original diameter of the plates (Greiner Bio-One). The mean migratory distances from the photograph reading distance in millimeters (mm) were converted to micrometers (µm). Measurements from the photographs to monitor the closing or healing of the wound were reliable however the measuring the mean migratory distances using the graticule eyepiece from an inverted microscope could have been more accurate but time consuming. An example of the migration rate and the percentage closure were calculated; as shown in Appendix 3. More detailed measurements of all test subjects are presented in Figure 6.
2.3. Migration of keratinocyte cells in the presence of specific ERï¡ and ERï¢ agonists,
Human keratinocyte were passaged and trypsinized following cell counting prior to wounding assay. The cells were grown in 6-well plates for 48 hours and wounded for migration as described in sections 2.1. 2.0ml of phenol-red, serum free DMEM medium; containing each agonists hormone, were added to each dish in triplicate;
Absolute ethanol vehicle control (0.0001%)
10nM PPT (ERï¡-agonist)
10nM DPN (ERï¢-agonist)
The concentration of the specific ER agonists was chosen following review of the data sheets, with consideration of their relative binding affinities (Tocris Bioscience). The cells were then cultured for 48 hours (2-days) and the migratory distance was measured daily and photographs were taken following fixed time points, as described in section 2.2.2.
Imaging Migrating cells
Following dose response and time course assays, all photographs of migrating cells under specific conditions were obtained using an inverted Nikon eclipse TS100 microscope, as described in section 2.1.
For each parameter, the data was calculated in triplicates. Data are presented as the patient mean (n=3) ±SEM. Significance was accepted at *P = <0.05, paired Student's t-test.