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For the most part, many tumors, particularly from rodent tissue, can be put into culture after careful dissection and mechanical mincing of the tumor with scissors. The B16
transplantable mouse melanoma is a good example. The method described below can be used for any relatively friable tumor type. Tumors with a great deal of connective tissue [such as the Engelbreth-Holm-Swarm (EHS) fibrosarcoma] will most likely require more extensive digestion to detach the cells from the matrix. This can be accomplished by increasing the concentration of enzymes, adding different enzymes such as elastase, and/or increasing the duration of treatment with enzymes. One can minimize damage to cells contained in such "tough" tumors by treating the tissue several times in succession, removing and washing the free cells after each treatment to minimize enzyme damage to the released cells, and combining the cells obtained at the end.
Setting Up a Primary Culture From a Tumor
Materials for Dissection
1. B16 tumor about 5 mm in diameter (Jackson Labs, Cold Spring Harbor) (this comes packaged subcutaneously in a C57BL6 mouse)
2. Scissors, straight, 9 inch; curved, 9 inch; straight, 4-inch dissecting (iris); curved, 4-inch dissecting (iris)
3. Forceps, straight, 4 inch; curved, 4 inch
4. 70% ethanol in beaker (for soaking instruments)
5. F12"CDME medium, 25"C50 ml in sterile conical 50-ml tube with gentamycin (25 ^g/ml)
6. 60-mm plastic tissue culture dishes, sterile
7. Pasteur pipettes, 9 inch, sterile
8. Rubber Pasteur pipette bulb, 2 ml (or pipettor)
Materials for Culture (all materials are sterile)
1. Tissue culture dishes: ten 60 mm or five 100 mm
2. F12"CDME, with 25"C50 ^g/ml gentamycin added
3. 5 and 10 ml pipettes
4. Fetal bovine serum
Procedure for Dissection (this should be done outside the tissue culture room)
1. Kill the animal by CO2 asphyxiation or by cervical dislocation.
2. Rinse the animal briefly in 70% ethanol.
3. Hold up the skin adjacent to the tumor and make an incision.
4. Carefully excise the tumor from the surrounding stroma and place in a 60-mm dish.
5. Remove any excess fatty tissue and necrotic tissue. Also try and remove all excessive fibrotic connective stroma. Place the tumor in a sterile 50-ml conical tube and transport it to a tissue culture hood.
Procedure for Tissue Culture
1. Place the tumor in a 60-mm dish and moisten with a few drops of medium with a Pasteur pipette.
2. Using at first a 9-inch pair of scissors and forceps (sterilize all instruments by soaking in a beaker of ethanol for 20 min), chop the tissue into small chunks.
3. With a straight iris scissors, cut the chunks into smaller pieces.
4. With a curved iris scissors, mince the tissue further until there are no discrete tissue pieces observable with the naked eye. It is important to keep the tissue moist with medium, but not so diluted that chopping the tissue becomes impractical.
5. At this point, the tissue pieces should pipette easily with a 5-ml pipette.
6. Add about 2"C3 ml medium at a time to the tumor tissue and carefully pipette this suspension into a 50-ml conical tube.
7. Carefully resuspend the tissue pieces in a 50-ml conical tube, and QS with medium to 50 ml.
8. Spin down the tissue suspension by bringing the centrifuge up to speed (800 rpm) and immediately bringing it down again. This gets rid of excessive debris in the supernatant without allowing it to pack down in the tissue pellet.
9. Remove the supernatant and repeat steps 7 and 8.
10. At this point, if there is not a significant amount of fibrous tissue, the pellet can be resuspended in 10 ml F12"CDME and added to each of five 100-mm dishes (1 ml/dish) or ten 60-mm dishes (2 ml/dish). Note: If the tissue pieces are small enough to pass through the bore of a 1- or 2-ml pipette, then use this to pipette individual aliquots into each dish. If using a larger pipette, continue to aliquot only 1"C2 ml/dish at a time, as drawing up 10 ml of this suspension and attempting to disperse it will result in a disproportionate number of tissue pieces in each dish, as the tissue tends to migrate to the miniscus end of the pipette.
11. Add F12"CDME medium to a total of 5 ml in a 60-mm dish or 10 ml in a 100-mm dish, taking into account the addition of 5% serum. Add 5% fetal bovine serum.
12. Place in 37jaC, 5% CO2 humidified incubator.
13. After 24 hr, observe the plates under a phase contrast microscope.
14. If the cells appear to be attached to the plate and spreading out from the tissue, remove the medium and transfer it with any suspended tissue to a new dish. Add fresh medium with serum to the original plate.
15. If the medium appears quite yellow (acidic), wash the supernatant by centrifugation at 900 rpm for 3"C4 min, remove the supernatant, and resuspend the pellet in fresh F12"CDME with 5% serum in a new dish.
16. If there is much fibroblast growth, reduce the serum to 0.5% and add insulin (10 ^g/ml) and transferrin (10 ^g/ml).