XENOPUS TISSUE CULTURE *

40 mM HEPES

9.532 g/litre adjusted to pH 7.9 with NaOH and autoclaved.

10x APBS

KCL	2. g
NaCl	60. g
Na2HPO4 (anhydrous)	0.73 g
KH2PO4 (anhydrous)	0.2 g
glucose	20. g
	____
Final volume	1 litre

Dissolve in nearly 1 litre. Weigh a further 0.6 g of Na2HPO4 and use this to buffer the solution to pH 7. The Na2HPO4 dissolves very slowly so be careful not to overshoot. Dilute an aliquot 1:10 and check that the pH is 7.45-7.55. Make up to 1 litre and autoclave. Dilute 1:10 with sterile water before use.

Xenopus Complete Medium

Dulbecco's MEM	500 ml
Foetal calf serum	70 ml
40 mM HEPES	140 ml
Antibiotic-antimycotic	14 ml

Store at 4oC.

Dulbecco's minimal essential medium; Store 4oC

Foetal calf serum; Store -20oC

40 mM HEPES; Store at R/T

Antibiotic; Store -20oC

Antimycotic; Store -20oC

Amphibian phosphate buffered saline; Store at R/T

Trypsin-EDTA (10x Stock Gibco), Dilute in APBS; Store -20oC

The cells should be sub-cultured about once a week. The cultures should be checked under the microscope for infection. The old medium is then poured off and the flasks are rinsed twice with 1x APBS (about 20 ml for the 250 ml flasks and 40 ml for the 650 ml flasks). Pipette 1x Trypsin-EDTA (diluted from 10x stock into 1x APBS) into the flasks. 2-3 ml of diluted trypsin is sufficient for a 250 ml flask and 5-6 ml is enough for a 650 ml flask. There should be enough liquid to just cover the surface of the flat.

The flats are left for 30 min to 1 hour with occasional shaking until the cells can be seen to have detached from the bottom.

Ideally the cells should be sub-cultured into a new flat. However, the bulk of the cells are usually pipetted off and the remainder are diluted with growth medium. In theory, 10% of the cells should be kept, in practice it is better to keep 3% or less if culturing will be continued in the same flask. 20 ml of growth medium is added to 250 ml flat or 50 ml to a 650 ml flat.

The cells are grown at 18-26oC in the dark. Unless the flats are kept in a CO2 incubator they should be kept tightly closed to maintain the correct pH.

With growth at lower temperatures the cells may not need subbing once a week. In this case, the old medium should be removed and replaced by fresh each week.

Trypsinise a 70% confluent culture and place the cells in a 50ml sterile tube. Take an aliquot and count the cells in a haematocytometer. Spin the cells down at 1,000 rpm for 5 min. in Super Minor centrifuge. Transfer to hood and pour off the trypsin. Resuspend the cells in freezing medium (Dulbecco's MEM with 20% FCS and 10% DMSO - use the ultra-pure DMSO kept in the -70o freezer for this). Aim at a concentration of about 1 x10-cells/ml. Place 1 ml aliquots in freezing vials taking care to keep the lid and thread free of liquid. The freezing medium should have been pre-cooled and the resuspended cells should be placed on ice for 30 min. The vials are then packed in a polystyrene box and placed in the -70oC freezer overnight. They may then be transferred to the liquid nitrogen store.

Put 40 ml of growth medium containing 10% FCS in a 50 ml sterile tube and warm to 25o. Remove the frozen cells from the liquid nitrogen store cautiously, placing the vial in plastic box or bottle until it is known whether the vial will explode (liquid nitrogen can force its way into the vial and then expand explosively as it warms). After a moment or two place the vial in a beaker of water at 25o. Shake constantly until the culture is thawed. Dry the vial and spray thoroughly with 70% alcohol. Wipe and place in the hood. Using pre-flamed artery clamp and forceps, unscrew the cap and pour the contents of the vial into the medium. Mix thoroughly, spin the cells down (1000 rpm, 5 min) and resuspend in fresh medium in order to remove the DMSO. Place the resuspended cells in a flat and incubate.

Cell Factories have a surface area of about 60 times a large flask and contain 1.8l medium. They can be seeded with cells from between 3 and 6 70% confluent large flasks. For convenience two to four Factories are used together. Once the initial seeding is made, Factories can be used to produce cells for 6 or more growth cycles as follows. To save on medium and serum, some medium is reused, the amount depending on the condition of cells and growth rate. Note, we have tried to reduce the need for fetal calf serum using various mixture of this and other sera, including supplemented sera. No other combination has worked for the cell lines we have. Reeder lab. does have a cell which will grow on one of the supplemented sera.

1. Empty contents of cell factory into container. Pour 250 ml 1X trypsin (in 1X APBS) into factory and leave for approx. 15 min. Shake occasionally. Repeat with second cell factory.

2. Either empty containers of used medium completely (to second container and then to outside via double outlet) or retain a certain percentage to supplement new medium. DO NOT forget to retain 1l of used medium per two cell factories to inhibit trypsin at step 7.For each 2 cell factories retain 1.3l for 64% medium replacement or 1.8l for 50% replacement. Repeat step 1. for the remaining two cell factories.

3 Prepare media for 2 cell factories;

a) all new:-
2500 ml	DMEM
350 ml	FCS
700 ml	Hepes pH 7.9
70 ml	Pen-Strept
7.5 ml	Fungizone
36 ml	Glutamine (optional)
b) 64% new:-
1600 ml	DMEM
225 ml	FCS
450 ml	Hepes pH 7.9
50 ml	Pen-Strept
5 ml	Fungizone
25 ml	Glutamine (optional)
1300 ml	Used Medium
c) 50% new:-
1250 ml	DMEM
175 ml	FCS
350 ml	Hepes pH 7.9
35-50 ml	Pen-Strept
3.75-5 ml	Fungizone
18-25 ml	Glutamine (optional)
1800 ml	Used Medium

4. When cells have detached, pour 25ml of trypsinised cells into sterile container and as soon as convenient transfer to new medium for reseeding the two cell factories. Empty the rest of the trypsinised cells from the two cell factories into a 1 litre centrifuge bottle. Wash each cell factory with 250 ml 1X APBS and pool into the centrifuge bottle and centrifuge 2000 r.p.m. at 4oC for 10 min. (Sorvall RC3C).

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5. Place 1.8 litres complete reseeded media into each cell factory.

6. Repeat steps 2-5 for the remaining two cell factories. Note that if four factories are used, it is more convenient to work with all four in parallel.

7. Discard S/N and resuspend cells into 1 litre complete used medium and place into a single 1 litre centrifuge bottle. Centrifuge as above.

8. Discard S/N and resuspend pellet into 200 ml 1X APBS and pour into four 50 ml Sarstedt. Centrifuge as above but for 5 min..

9. Discard S/N and resuspend pellet into 50 ml 1X APBS and centrifuge as above.

10. Freeze cells at -160oC in 50ml tube with pinhole in cap or continue with prep. as below.