Cell Culture Techniques

2.2.1.1 Synchronised Culture - Peripheral Blood Lymphocytes

Cultures from a synchronised method will yield a high mitotic index resulting in less condensed chromosomes, and more near prophase and prometaphase stages; this method is essential for high resolution banding. The collection of whole blood was done into a sterile Lithium heparinized tube. Under aseptic conditions, 0.5-lml peripheral blood was cultured in a 50ml flask containing Iscoves modified Dulbecco's medium supplemented with foetal calf serum (appendix 5.3.1) and stimulated with 200|ll phytohaemoglutinin (PHA). The culture was incubated at 37°C for 48-72 hours and shaken twice a day to resuspend the cells. 0.2ml of thymidine (30mg/ml) was added to each culture and incubated for 17 hours or overnight. Thymidine will block cell growth at the G 1/S phase transition. The cells will be stimulated to growth and division by the addition of 0.2ml deoxycytosine (0.3mg/ml) for a further 5-6 hours incubation. 0.2ml colcemid (10|ig/ml), a mitotic spindle inhibitor, was added and incubated for 20 minutes. The cell culture was mixed and transferred into two 10ml centifuge tubes and harvested following a standard protocol.

2.2.1.2 Suspension Culture - Lymphoblastoid Cell Lines

Lymphoblastoid cell lines generally grow in the medium without attaching to the surface of the culture flask. This type of cell culture is called a suspension culture. Frozen cells stored in liquid nitrogen were thawed immediately in a 37°C waterbath. The lid of the vial was loosened, then reclosed before putting into the waterbath. The cells were transferred to a sterile 15ml centrifuge tube and 10ml of prewarmed RPMI medium supplemented with fetal calf serum (appendix 5.3.2) was added dropwise and mixed slowly to allow the cells to recover. The cells were spun at SOOrpm for 5 minutes. The medium was removed and the cells resuspended in firesh medium and

incubator gassed with 5% CO2. The culture flask was placed upright throughout the

incubation. The cap of the flask was left slightly loose to allow the CO2 access to the

cells. As the cells began to grow in clumps the medium turned yellow. The spent medium was removed by centrifugation and the cells were fed with fresh medium once every 2 days. When the culture flask was thick with clumps of cells, some were transferred to a centrifuge tube. ISjll colcemid (lOjlg/ml) was added to a 5ml cell culture and incubated at 37°C for 1 hour prior to harvesting. anc\ s-brcmodeov-juricitoi

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2.2.1.3 Monolayer Culture - Anchorage Dependent Cell Lines

Some established cell lines are anchorage dependent and adhere to the surface of the culture flask. These cells grow as monolayer cultures, although some form multilayers, mostly of fibroblast and epithelial cell types. The culture flask was maintained in a horizontal position to provide a large surface area for cell growth. Frozen cell lines were thawed in a 37° C waterbath and transferred into a sterile 15ml centrifuge tube containing DMEM medium supplemented with fetal calf serum (appendix 5.3.3). The cells were spun at SOOrpm for 5 minutes, the supernatant removed and the cell pellet was resuspended with 5ml medium. The cell suspension was transferred to a 50ml culture flask and incubated in an 8% CO2 incubator at 37°C

with the cap loosened. The cell culture was checked, at least once a day, with an inverted microscope for signs of attachment, flattening and proliferation of the tumour cells. The medium was changed once every 2 days or more or less frequently depending on the growth rate of the cells.

2.2.1.4 Subculture of Monolayer Cells by Trypsinisation

Subculturing requires dislodging the cells from the culture flask using an enzyme solution and then washing them to remove the enzyme. Cultures were subcultured when areas of tumour cell growth become very confluent. The medium was removed and the cells washed with Hanks balanced salt solution followed by versene solution. A few drops of 2.5% trypsin in versene solution were added to the flask, just enough

to cover the layer of cells. The flask was incubated at 37°C for 3 minutes. The flask was shaken gently to optimise cell dissociation. The trypsin action was inactivated by adding 2ml DMEM medium supplemented with fetal calf serum. The cell suspension was distributed to two or more new flasks and incubated. The cells were harvested when the secondary cultures reached a confluent stage. 7.5pi colcemid (lOpg/ml) was added to the flask and incubated for 2 hours at 37°C prior to harvesting.

2.2.1.5 Harvesting and Chromosome Slide Preparation

Following colcemid treatment the cells in suspension were spun at lOOOrpm for 5 minutes. The supernatant was removed and the cell pellet was resuspended gently in warm hypotonic solution (0.075M KCl) to prevent cells from rupturing. The mixture was incubated for 15-20 minutes at 37°C. Fixative solution (3 parts methanol to 1 part glacial acetic acid) should be prepared fresh before use. A few drops of chilled fixative were added and mixed. The cells were spun at lOOOrpm for 5 minutes, the supernatant removed, leaving a small amount just above the pellet. The centrifuge tube was tapped gently to resuspend the cell pellet. 10ml fresh fixative was added and the cell suspension was kept in the fridge at 4°C for 30 minutes. The cells were spun at lOOOrpm for 5 minutes. This step was repeated twice, before slides were made.

SHdes were cleaned and soaked in methanol with a few drops of concentrated HCl, A few drops of fresh fixative were added to the cell pellet to give a slightly opaque suspension. Using forceps, a dry clean slide was held horizontally and 1-2 drops of cell suspension was dropped on to the slide and dried rapidly. The cell density and chromosome spreading was checked under a phase contrast microscope with the lOX objective. If the cell density was too high a few more drops of fixative were added to the cell suspension. If the cell density was low the cell suspension was spun down and the cell pellet resuspended in a smaller amount of fixative. If the chromosomes were trapped within the cytoplasm, the slide was treated with 50%

glacial acetic acid before air drying. Slides for banding were aged for 2-3 days at room temperature or overnight at 56-60°C prior to banding. Slides for FISH analysis were dehydrated in 70%, 90% and 100% ethanol series and stored refrigerated at 4°C untü required.

2.2.1.6 Giemsa-Trypsin Banding

Giemsa-bands or G-bands were obtained by digesting the chromosomes with a proteolytic enzyme, trypsin (appendix 5.3.4). Chromosome slides were aged for 2 days at room temperature and incubated in 2XSSC at 60°C for 1 hour. The slides were washed in 0.9% saline, treated with 0.025% trypsin (Koch Light Ltd.) in Hanks balanced salt solution for 10 seconds at 10°C, washed in saline and stained in 10% Giemsa (BDH) in buffer pH 6.8. The slides were rinsed in buffer pH 6.8, air dried and viewed under the bright field microscope. The trypsin treatment varied from one slide to another depending on the origin of cells obtained for chromosome preparations and had to be checked individually for banding morphology. Photomicrographic prints of G-banded chromosomes were used to illustrate ideograms and karyotypes. For photography and printing details see appendix 5.3.5.

2.2.1.7 Freezing of Cells and Storage

Tissue culture cells were stored using cryoprotective agents to prevent them from any damage caused by freezing. The cell suspension was transferred to a centrifuge tube and spun at lOOOrpm for 5 minutes. The excess medium was removed and the cell pellet mixed and resuspended with 1.5ml freezing medium (appendix 5.3.6.) and transferred to a 1.5ml ampoule as 1ml aliquots. The freezing medium, dimethylsulfoxide, DMSO, is harmful to growing cells and once added to the cell suspension, the tubes should be kept on ice before transferring to liquid nitrogen. Cells stored in liquid nitrogen showed a high survival rate over a few years.

2.2.1.8 Smear Preparation from Solid Tumours

A rapid and direct method for cytological preparations of well preserved, thin layered interphase nuclei was obtained from smears of solid tumour. A small piece of fresh resected tissue from a solid tumour (about 0.5mm^) was placed on one end of a clean dry microscopic glass slide and spread it out with a second slide as though making a wet blood film. This was later modified to a 'touch' technique. The slides were fixed immediately for about 30 seconds in a solution of equal volumes of acetone and ethanol and 0.05% IN trichloroacetic acid. The slides were air-dried and stored in the fridge at 4°C untü required for FISH.

2.2.1.9 Extended DNA Fibre Preparation from Agarose-Embedded Cells.

High molecular weight DNA was prepared by embedding purified lymphocytes from fresh blood in low melting point (LMP) agarose. The isolation of lymphocytes was performed from diluted whole blood (v/v) in PBS using lymphocyte separation medium (Flow Laboratories). The cells were washed twice in PBS and the cell density determined with a haemocytometer. About lO^/ml cells was resuspended in 1% (v/v) LMP molten agarose (appendix 5.3.8). The block formers were cleaned in O.IN HCl, rinsed in distilled water and dried. The mixture was added into the block formers, 100 jll/block and allowed to set at 4°C for 5-10 minutes. The agarose blocks were transferred into a universal container containing ESP buffer (appendix 5.3.8) predigested with proteinase K. The mixture was incubated at 50°C for 24 hours. The agarose blocks were washed twice with ESP, then twice with prewarmed TE buffer. To inactivate the proteinase K the blocks were washed 3 times for 30 minutes with freshly prepared phenylmethylsulfonylfluoride, PMSF (40mg/ml) in isopropanol, at 50 °C. The TE buffer was removed and replaced with 0.5M EDTA buffer. The blocks were stored in 0.5M EDTA at 4°C untü required. Extended DNA fibre was prepared by placing a smaU piece of agarose block on a polysine glass slide and warmed on a

heating plate. Using a second glass slide the molten agarose was immediately spread like a wet film. The slides were air-dried and stored at 4°C.

2.2.2 Preparation of DNA from YACs and Plasmids