INVESTIGACIÓN DE MERCADO PARA LA IMPLEMENTACIÓN DE ERP COMO SAAS

2.1.1. Isolation of keratocytes and Epithelium

Rabbit eyes were enucleated after sacrifice by injection o f Pentobarbitone (Rhone Merieux, Harlow Essex) into an ear vein, and the comeas removed with W estcott spring scissors. The Tenets o f the ARVO Statement for the Use o f Animals in Ophthalmic and Vision Research were followed. Comeas were initially incubated in 10 mis o f Antibiotic medium (AM) comprising Dulbecco’s modified Eagle’s Medium (DMEM) - (Sigma -Aldrich Company Ltd, Poole, Dorset, UK ) containing; penicillin (Gibco Life technology. Paisley, Scotland) - (200U/ml), streptomycin (Gibco - 200pg/ml), fimgizone (Sigma - 0.5pg/ml) and 2mM L-glutamine (Sigma) - until use within 24 hrs at 4° C. A Dispase II solution was made up, (Neutral protease - fi*om Bacillus polymyxa grade II - Boerhinger Mannheim, Bell lane, Lewes, East Sussex) 2mg/ml in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma). Comeas were incubated in this solution for 2 to 3 hrs at 37° C.

Using a 6 well plate, 2mls Phosphate Buffered Saline (PBS - Gibco) was placed in each well - allowing one well per comea. A sterile scalpel blade was used to scrape o ff the epithelium into the well - and the remaining stroma was divided up and placed as explants in the bottom o f a small 25cm^ tissue culture flask (Falcon - Marathon, London UK). These were left for 3 to 4 hours until the explants had adhered to the flask. Then 5 mis Keratocyte Growth Medium (KGM) containing DMEM with penicillin (lOOu/ml), streptomycin (lOOpg/ml), Gentamicin (50pg/ml) (Gibco), fimgizone (0.25pg/ml) and 2mM L-glutamine (Sigma) and supplemented with 10% (vol/vol) fetal calf serum (PCS) (Gibco) - was gently pipetted into the flask without disturbing the explants. (Khaw PT, et al, 1992) (Occleston NL, 1996)

2.1.2. Routine maintenance of fibroblast cultures

All flasks containing primary cultures were incubated at 37 ° C, 5%C02 and maintained by changing the growth medium every 3 to 4 days until the cells were nearing confluence. The monolayers were then passaged into new tissue culture flasks (see 2.1.3.). After the first passage, comeal fibroblasts were cultured in KGM containing Newbom calf Serum (NCS)

(Gibco) rather than FCS. The routine changing o f medium and passaging was continued untü passage 6, with frequent storing o f aliquots o f cells in liquid nitrogen for friture use (see section 2.1.4). (Khaw PT, et al, 1992) (Occleston NL, 1996)

2.1.3. Passaging of fibroblast (keratocyte) monolayers

When cell monolayers had become 80- 90% confluent in T25, T75 or T125 tissue culture flasks (Falcon) they were passaged. KGM was aspirated from the flask and the cells rinsed twice in sterile PBS. Cells were incubated for 3 minutes at 37®C, with a 0.15% (vol/vol) phosphate buffered trypsin/ 0.02% (wt/vol) ethylenediamine tetra-acetic acid (EOTA) solution (Gibco). During this process flasks were observed under the inverted microscope for detachment o f cells from the base. Flasks were agitated and re incubated as required until the majority o f the cells had become detached. The enzymatic activity o f this solution was then neutralised with an equal volume o f KGM containing NCS. The solution was transferred to a universal, which was centrifriged at 200g for 8 minutes. The supernatant was discarded and cells resuspended in KGM prior to use in an experiment or to re­ seeding flasks. The cells were seeded into new tissue culture flasks at a split ratio o f 1:3, i.e. one flask o f cells was split into 3 new flasks. (Khaw PT, et al, 1992) (Occleston NL,

1996)

2.1.4. Storage and recovery of cells in Liquid nitrogen

Cultures o f comeal fibroblasts were passaged and the resultant pellet resuspended in 0.5ml o f growth medium. An equal volume o f 10% (vol/vol) dimethyl sulphoxide (DMSG - Sigma)/ KGM was added dropwise with continual mixing. These 1ml aliquots o f cell suspension were then transferred to cryovials (Nunc, Gibco Life Technology, Paisley, Scotland) and stored overnight in the vapour phase o f the liquid nitrogen stores. The vials were then stored in the liquid nitrogen phase until required.

Upon removal from the liquid nitrogen, the contents o f the cryovials were rapidly thawed under hot running water (60^C). The 1ml aliquot o f cell suspension was then transferred to a sterile centrifrige tube and 5ml o f KGM was added dropwise, at a rate o f 1ml per minute, with continual mixing. The cell suspension was then centrifriged at 200g for 8 minutes, the

supernatant discarded and the cell pellet resuspended in KGM and seeded into tissue culture flasks. (Khaw PT, et al, 1992) (Occleston NL, 1996)

2.1.5. Corneal epithelial cell culture techniques

The epithelium (as isolated in 2.1.1.) suspended in PBS was pipetted up and down gently in a 10ml pipette, to break up sheets o f cells, and then transferred to a universal and

centrifuged at 200g for 7 mins. Cells were resuspended in 5mis o f keratinocyte Serum Free Medium (KSF) with supplements, made up as follows:- (To 500ml Keratinocyte serum free medium (Gibco) was added, 2.5pg Human recombinant EGF (Gibco), 25mg Bovine Pituitary Extract (Gibco), Ca CL 15mM (200pF100ml) (Sigma), Penicillin (lOOu/ml) (Gibco), Streptomycin (lOOpg/ml) (Gibco)). This suspension was seeded into a small 25cm^ tissue culture flask (Falcon). Flasks containing primary cultures o f epithelial cells were maintained by changing the supplemented KSF medium every 2 to 3 days until the cells were 60 - 80% confluent. Epithelial cell monolayers were then passaged (see below) into new tissue culture flasks. (TCS Biologicals Ltd, Botolph Claydon, Buckingham, MK18 2LR)

2.1.6. Passaging of Epithelial monolayers

Epithelial cell monolayers were grown until they had reached 60-80% confluence. KSF medium was aspirated and the cells removed from their substratum by incubation for 3 minutes at 37°C with a 0.15% (voFvol) phosphate buffered trypsin/ 0.02% (wt/vol) ethylenediamine tetra-acetic acid (EOTA) solution (Gibco). The enzymatic activity o f this solution was then neutralised with an equal volume o f KSF containing 0.05% (wt/vol) Soybean Trypsin inhibitor (Sigma) and cells centrifuged for 5 min at 200g. The

supernatant was discarded and the cell pellet resuspended in fresh medium. Cells were divided into new tissue culture flasks at a split ratio o f 1:2 and seeded at approximately 4000 cells /cm^. (Protocol followed, TCS Biologicals Ltd, Botolph Claydon, Buckingham, MK18 2LR)

2.1.7. Collagen I coating of culture flasks for dispersion assay

Rat tail tendon collagen (type I) (Upstate Biotechnology-NY) 0.2mg/ml in 0.02M acetic acid (BDH, - Merc, Lutterworth, Leicestershire, UK) was used to coat 75 cm^ tissue culture flasks. Flasks were washed twice with PBS or sterile water before use.