2.10.1. S kin biopsy samples
Ethical committee approval was obtained prior to the commencement of this study and informed written consent was obtained from patients before the biopsies were taken. Initial studies to establish the methods to be used for handling and growing cells were performed with surgical skin samples from theatre.
2.10.2. S k in biopsy m ethod
Patients were positioned supine and the procedure performed under aseptic conditions. The skin was prepared with an antiseptic cleaning liquid (Tisept from Seton Prebbles) followed by 70% ethyl alcohol. An ellipse of skin (1 cm long, 0.5.cm wide) was excised from the right buttock under local anaesthetic and placed in 'transport medium' (Section 2.14.2). Three sutures were inserted at the biopsy site which were removed by the patient's General Practitioner at 10 days.
2.10.3. E stablishm ent o f fibrobla st cultures (m ethod 1)
The skin was minced to 1 mm^ pieces with crossed scalpels and placed in 10 ml of Ham's F12 medium (without serum) containing 0.125% trypsin (Flow Cat. No. 1689454) and 0.5% collagenase (Sigma Type I) for 4 hours, stirring constantly with a 'Buoy bar' at 37®C. The resulting cell suspension was centrifuged at 1500 r.p.m for 15 minutes, the supernatant aspirated and the cells resuspended in 5 ml Ham's F I 2 medium. Cells were counted in a haemocytometer using a viability stain (acridine orange and ethidium bromide in PBS: Section 2.14.1).
2.10.4. Isolation o f fibroblasts and kératinocytes fr o m a skin sample (m ethod 2)
1. The skin sample was trimmed of surplus fat which was minced and passed to step 5.
2. The skin sample was placed in 10 ml pronase (1 mg/ml) and incubated at 37°C for 2 hours in a 5% CO2, 5% O2 incubator. The mixture was inverted every
30 minutes.
3. The epidermis was peeled away from the dermis using a pair of sterile 190 needles.
4. The fragments of epidermis were further broken up with needles and then placed in the pronase supernatant if necessary. This cell suspension contained the kératinocytes. After centrifugation and resuspension in fresh medium the kératinocytes were counted in a haemocytometer and then plated onto a feeder layer of 3T3 cells according to a standard protocol [96]. Experiments with kératinocytes were carried out in parallel to the fibroblast assays and were performed by Dr Richard Hodgkiss.
5. The dermis was minced with crossed scalpel blades and placed, together with any material from step 1, in 10 ml of 1% collagenase (Sigma Cat. No. CO 130) with a magnetic stirrer at 37°C under aerobic conditions for 1-2 hours until most of the fragments had dissolved. This cell suspension contained the fibroblasts.
6. The cell suspension was centrifuged at 1500 r.p.m for 15 minutes, the
supernatant aspirated and the cells resuspended in 5 ml Ham's F 12 medium + 10 % PCS.
7. The cells were counted in a haemocytometer using a viability stain (acridine orange and ethidium bromide in PBS: Section 2.14.11).
2.11. Assays for measuring fibroblast radiosensitivity
2.11.1. Colony assay
1. A single cell suspension of fibroblasts in Ham's F I2 medium + 10% FCS was prepared from two T75 flasks of plateau phase fibroblasts as described in Section 2.6. The cells were then counted in a haemocytometer.
2. A proportion of the cell suspension (~ 8x10^ cells) was placed in a total volume of 25 ml medium in a spinner vessel and irradiated under stirred
aerobic conditions to a total dose of 30 Gy to produce 'heavily irradiated' cells. The cell suspension was then poured into a 25 ml universal and centrifuged at
1500 r.p.m. for 15 minutes. The supernatant was then poured off and the cells resuspended in 5 ml of fresh Ham's F12 medium + 10% FCS (Section
2.14.1). These cells were then counted in a haemocytometer.
3. The remaining cell suspension of non-irradiated 'live' cells was kept on ice until the heavily irradiated cells were prepared.
4. Fibroblasts were plated into T25 tissue culture flasks in Ham's F12 medium + 10% FCS for a range of different dose points and at either cell density A alone or at both cell densities A and B (Table 2.2). For each flask the total cell number was made up to a total of 20,000 by adding heavily irradiated cells to the 'live' cells. A total of 3-6 flasks was used for each dose point at each cell density, p.b X - r o j dose-rako. ojppfoac. 0 '4 - 8 Qvj )miA..
Table 2.2. Number of cells plated per dose point for fibroblast colony assay.
D o se ( Gy)
No. o f 'live' cells per flask
(Density A)
No. of heavily irradiated cells per flask
(Density A)
No. of 'live' cells per flask
(Density B)
No. of h eavily irradiated cells per flask
(Density B) 0 1000 19000 500 19500 1 1700 18300 850 19150 2 2600 17400 1300 18700 3 6500 13500 3250 16750 4 7000 13000 3500 16500 5 12000 8000 6000 14000
5. The flasks were placed in a 5% CO2, 5% O2 incubator at 37°C with the caps
in the loose position for 3-4 hours to allow cell attachment.
6. The flasks were irradiated under aerobic conditions at room temperature. 7. The flasks were returned to the incubator until the control flasks contained
colonies > 50 cells (~ 14 days).
8. The flasks were stained with crystal violet solution (Section 2.2 mixture 2). 9. Colonies were scored manually with a colony counter. The size of individual
colonies was measured using a light microscope and an eye-piece graticule.
2.11.2. M ultiw ell assay
A single cell suspension of fibroblasts was prepared as previously described (Sections 2.10.3 and 2.10.4) from two T75 flasks of plateau phase fibroblasts. The cells were counted in a haemocytometer. Four 24-well multiwell plates (Costar 3424) were used for each assay. These plates were identical in shape to the 'CAM' coated plates used in the ATCCS assay but were uncoated. The plates were labelled and the cells plated at the same cell densities used in the ATCCS assay (Table 2.1 and Figure 2.1) and placed in a 5% CO2, 5% O2 incubator for 24 hours.
After 24 hours the PBS was aspirated from the first 'spare' well in column 2 of plate 3 (Figure 2.1) and 1 ml of 70% ethyl alcohol was added for 3 minutes to fix the cells and then aspirated. 5 p.Ci of tritiated thymidine (Amersham International Cat. No. TRK 758 ) in 1 ml of culture medium was pipetted into each of the 'background'
wells in column 2 of plates 1 and 2 to stop cell growth. The plates were irradiated under aerobic conditions at room temperature and then returned to the incubator until the high density control wells (4000 cells/well) had reached confluency and there was good growth in the next density (2000 cells/well). This was determined by examination with an inverted light microscope (~14 days). Fixation, staining and analysis of the plates to obtain radiosensitivity measurements was carried out according to the protocol for the ATCCS assay (Section 2.4).