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All cell culture reagents were obtained from Sigma unless otherwise stated. Sterile disposable plasticware (e.g. tissue culture flasks, centrifuge tubes and pipettes) were purchased from Falcon. Fibroblast cell lines were established from a skin biopsy, cultured amniocytes from amniotic fluid and cultured chorionic villi cells from chorionic villi biopsy of patients. The fibroblasts investigated in this project were supplied either as frozen ampoules, which had been stored in liquid nitrogen or as growing cells. Cultured chorionic villi, amniocytes and lymphoblastoid cells were grown by the Enzyme

Patient Sex Material available Notes

EW female fibroblasts Severe - died within the first year of life.

DC male fibroblasts,

plasma

Severe - died age 11 weeks (Clayton gf a/., 1992) CB female fibroblasts, amniocytes,

amniotic fluid, plasma

Severe - died within the first year of life. Prenatal diagnosis attempted. (Clayton gf a/., 1993)

JB male fibroblasts Severe- died age 3 months

(Hutchesson et fl/., 1995)

KT female fibroblasts Severe - died age 6 months

(Clayton et a/., 1992) VB female lymphoblastoids,

plasma

Not severe - alive, age 10 years sister o f TB

TB male lymphoblastoids,

plasma

Not severe- alive, age 12 years brother of VB

NH male fibroblasts, ascites fluid, plasma

Severe- died age 3 months (Charlwood e ta /., 1996, 1997)

Table 2.1 CDGS type 1 patients investigated in this study.

Patient Samples Length of treatment Galactose-l-phosphate (weeks) (pmol/g)

G1 fibroblasts N/A N/A.

LV fibroblasts N/A N/A

TR plasma 0 4.0 38 0.31 NA plasma 0 2.84 3 1.09 BR plasma 6 0.34 RE plasma 42 0.22 SO plasma 0 8.75 TI plasma 50 0.15 NI plasma 42 0.5

Table 2.2: Galactosaemic patients investigated in this study.

2.2.1 Tissue culture medium

2.2.1.1 H am ’s FIO with fetal calf serum

Ham’s FIO medium (500 ml) was supplemented with 70 ml o f fetal calf serum (Imperial Laboratories), 5 ml of penicillin/streptomycin (1 mg/ml) and 73 mg of L-glutamine and sterilised by filtration through a 0.22 p.m Millex-GV Millipore filter. The medium was stored at 4°C for up to 1 month.

2.2.1.2 H am ’s FIG with SATO mixture

SATO mix was added to Hams FIO medium instead of fetal calf serum when serum-free medium was required.

SATO mix

SATO mix wks composed of 1.3% (w/v) BSA rat-ocyte 4 ,4 .4 mM putrescine, 20 mM thyroxine, 20 mM triiodothyroxine, 90 mM progesterone and 0.1 mM sodium selenite in water. The solution was sterilised by filtration through a 0.22 |im Millipore filter and stored at -2(fC .

Ham’s FIO medium (100 ml) was supplemented with 2 ml of insulin (0.5 mg/ml), 1 ml of transferrin (10 mg/ml), 2.22 ml of SATO mix, 0.5 ml of 2(X) mM glutamine and 1 ml o f penicillin/streptomycin (1 mg/ml) and sterilised by filtration through a 0.22 jxm Millipore filter. Insulin and transferrin were dissolved in sterile water and stored at -20^C. The complete medium was stored at 4°C for up to 1 week.

2.2.2 Cell culture conditions

Fibroblasts, cultured chorionic villi cells and amniocytes were grown as monolayers in tissue culture flasks that had a surface area of either 25 cm^ or 75 cm^. Normally the cells were grown in Ham’s FIO culture medium supplemented with fetal calf serum, but for secretion experiments serum-free medium (Ham’s FIO with SATO) was used. 10 ml of medium was added to a 75 cm^ flask and 4 ml of medium was added to a 25 cm^ flask. Flasks were incubated at 37^C in 5% (v/v) CO2.

2.2.2.1 Mycoplasma detection

Fibroblasts, cultured chorionic villi cells and amniocytes were routinely screened for mycoplasma contamination and affected flasks treated with Mycoplasma Removal Agent (MRA, ICN) at 1 ml per 100 ml cell culture medium for 5 days. Cells were grown for 4 days on a glass coverslip in a cell culture dish (25 cm^) containing cell culture medium. The cells were fixed by adding 5 ml of methanol: acetic acid (1:3) for 2 min. This solution was decanted and 5 ml of methanol: acetic acid (1:3) was added for a further 5 min. This was repeated twice more, and then the cells were left to air-dry. The coverslips were stained by incubation with 50|ig/ml DAPI in PBS for 30 min. They were rinsed twice with water and mounted onto a microscope slide. The mycoplasma were visualised under a fluorescent microscope with a 53/44 barrier filter and a BG-3 exciter filter at a magnification of 500 X.

2.2.3 Reconstitution of cells stored in liquid nitrogen

Ampoules containing fibroblasts were removed from liquid N2 and thawed at 37°C. The cells were centrifuged at KXX) x g for 5 min. and the medium was removed by aspiration. The cells were then re-suspended in 4 ml of cell culture medium and transferred into a 25 cm^ flask.

2.2.4 Trypsinisation of cells

Trypsin bu ffer 6 g/1 NaCl, 3 g/1 sodium citrate, in sterile water pH 7.8 Trypsin solution 0.125% (w/v) trypsin in trypsin buffer

D M SO -m edium 750 |xl DMSO in 10 ml Ham’s FIO with fetal calf serum

The trypsin buffer was sterilised in an autoclave at 120^C for 30 min, and the working trypsin solution and DMSO medium were filtered through a 0.22 |im Millipore filter. AH of the above solutions were stored at 4®C.

When the fibroblasts, cultured amniocytes or cultured chorionic villi cells had reached confluency, as judged by phase contrast microscopy, they were trypsinised for

harvesting, sub-culturing or storage in liquid nitrogen. The medium was removed by aspiration and 4 ml of trypsin buffer was added. The flask was rinsed and the trypsin

buffer removed. Trypsin solution (2 ml) was added to the flask which was incubated at 37°C for 10 min. For sub-culturing, the detached cells were transferred into two new flasks and all three flasks were fed with 10 ml o f cell culture medium. For harvesting or storage in liquid nitrogen, the detached cells were transferred to a sterile centrifuge tube. The flask was washed out with 2 ml of medium and this was also added to the tube. The flask was then discarded. The cells were centrifuged at 1000 x g for 10 min. The

medium was then removed. Harvested cells were washed twice with phosphate buffered saline (PBS) and after the final wash 50 pi of water was added and the cells were stored at -20^C or -70®C. Cells for storage in liquid nitrogen were re-suspended in 2 ml of DMSO-medium and transferred into an ampoule, which was stored overnight at 4°C and placed in the liquid nitrogen cylinder the following morning.

t

2.2.5 Collection of ^-Hexosaminidase activity secreted into fibroblast

cell culture medium

Fibroblasts were grown to confluence under routine cell culture conditions (section 2.2). The cells were washed in PBS and incubated in serum-free medium (Ham’s FIG +

SATO, section 2.2.1). Immediately after the addition of the medium a 0.5 ml aliquot was withdrawn from each flask and stored at -20°C. After 5 days, the spent medium was removed and centrifuged at 1000 x g for 5 min. The supernatant was stored at -20PC. The cells were harvested from the flask by trypsinisation and pelletted (section 2.2). In the mannose supplementation experiments, 5 mM mannose was added to the cell culture medium and fibroblasts treated as above. P-Hexosaminidase activity was measured in the cell culture medium (ICX) pi- undiluted) as described in section 2.7.2.1 and 2.1.22, and related to the protein concentration of the fibroblast pellet.