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Protocol

ESTABLISHMENT OF CELL LINES
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PREPARATION OF FRESH TISSUE FOR THE ESTABLISHMENT OF CELL LINES; 1. For solid tissue, dissociate as much as possible into a single cell suspension, or at least small tissue clumps, in DMEM (better-more buffering capacity), or RPMI 1640 media containing antibiotics, L-glutamine and 15% FCS. For body fluids, be it pleural fluid, peritoneal fluid or blood or bone marrow, separate tumor cells from RBC by running over Ficoll Hypaque gradient as follows: 1. Dilute bone marrow or blood 1:3 with media. Use peritoneal or pleural fluid straight. In 15 ml conical sterile tube, layer 3 mL of Ficoll-Hypaque premade solution. Then layer 10 mL of blood or fluid on top. 2. Spin at 2000 rpm, RT in table top centrifuge for 15 minutes. 3. What will result is three visible layers. From the top to the bottom: Top: serum Middle: mononuclear cells and tumor cells Bottom: RBC 4. Remove serum almost to the middle layer, discard. 5. Remove thin, white middle layer containing tumor cells. Pool with other tubes if available. 6. Spin once after adding about 10 mL of media to wash off Ficoll-Hypaque (1250 rpm for 10 min, RT). 7. Coat T25 or T75 flask with pure laminin (if available) 10-100 mg/flask, dissolved in media for about 5 minutes at room temperature. Then add cells to flask without removing laminin. You can use Matrigel according to manufacturer's recommendations. PRIMARY CULTURE METHODS I. MEDIA Insulin (Modified N2 medium or N2E) (Serum free) (Osama El-Badry, personal communication 1988) Transferrin 1 mg/mL Selenium 30 nM Progesterone 20 nM Putrescine (Sigma) 100 mM Add to basal medium of .... 50:50 (v:v) Ham's F12 and Dulbecco-Vogt MEM with: Na2HCO3 1.2 mg/mL HEPES Buffer 15 mM Penicillin 50 m/mL Strep 50 mg/mL L-glutamine 2 mM Outgrowth Medium (Rosenberg) (Laboratory of S. Rosenberg, Personal communication 1988) DMEM 460 mL FCS 25 mL HEPES 1M 5 mL Glutamine 200 mM 5 mL Pen/Strep 5 mL Gentamycin 5 mg/mL 0.5 mL Insulin 5 mg/mL 0.5 mL (in 0.01 N HC1) EGF l0 mg/mL 0.5 mL (dd H20) Choleratoxin loug/mi 0.5 mL Dexamethasone 0.79 mM 0.635 mL (ETOH solvent) Prolactin 0.60 mg/mL 0.835 mL (ETOH solvent) 20% DMEM Complete DMEM 80 mL FCS 20 mL Pen/Strep 0.5 mL Glutamine 0.5 mL Conditioned Media Any one of above media that has been mixed 50% with the same media conditioned 1-3 days by a particular cell type.
II. Tissue Disintegration Protocols A. Collagenase 1A (Sigma C9891) (Freshney, 1987) *Dissolve Collagenase in PBS to 2000 m/mL; set aside on ice *Mince tissue aseptically - in a small amount of HBSS - just enough to keep it moist *Add more HBSS to make a suspension and add the collagenase to make a final solution of 200 m/mL *Incubate at 37°C with shaking *Spin down cells and debris *PBS wash *Spin down and resuspend in growth media or freeze mix B. Collagenase/Dispase (BM 269 638) (Boehringer-Mannheim 1984) (Freshney, 1987) *Dissolve 100 mg enzyme mixture in 1 mL DI water (sterile) *Add 100 mL PBS without Ca++ and Mg++ (pH 7.2-7.8) *Filter sterilize 0.2 mm Enzyme concentrations: Collagenase 0.1 u/mL Dispase II 0.8 u/mL Tissue Disintegration (Aseptic) (Freshney, 1987) *Mince tissue with scissors until they are small enough to go through a 5 mL pipet tip. Use a very small amount of HBSS to keep tissue moist. *Add the collagenase/dispase to the tissue and incubate with shaking at 37°C for 1 hr. Use enough enzyme mix to cover the tissue well. I use about 15 mL mixture/0.5 g tissue. *Spin down the cells and debris at 1200 rpm. Aspirate supernatant. *Resuspend cells in growth media or freeze mix. C. Rosenberg's Procedure (Rosenberg, 1988) *Coarsely mince tissues and put into 500 mL flask to cover bottom. *Add 500 mL enzyme medium. RPMI 340 mL Glutamine 5 mL Pen/strep 5 mL Genamycin 0.5 mL (50 mg/mL final) Hyaluronidase 50 mL (1 mg/mL stock in RPMI Sigma 106518) Collagenase A 50 mL (1 mg/mL stock in RPMI Sigma 103578) DNase 50 mL (0.2 mg/mL stock in RPMI Sigma 104159) *Add sterile stirbar. *Stir overnight at RT or 4 hours at 37°C. *Filter suspension through gauge or screen into 250 mL centrifuge bottles. *Spin 500g (1500 RPMs GSA) for 10 min. at RT. *Resuspend pellet in HBSS, 30 mL. *Pipet ficoll under sample: 10 mL in 50 mL tubes. *Spin at 2000 RPM for 15 min. at RT. *Collect cells at interface. *Wash 2 times with HBSS. *Count viable cells if necessary. III. TISSUE GROWTH CONDITIONS A. Adult Tissues Brain: *Minse the tissue as finely as possible aseptically *Digest with Collagenase/Dispase or no enzyme at all. If no enzyme is to be used, gently aspirate tissues through a 5 mL pipet. This is faster although initial growth is slower at first. *Grow in DMEM with 20% FCS, DMEM, N2E, or outgrowth media Breast: *Mince finely. *Place in 20% FCS, DMEM Kidney: *Mince. *Digest with Collagenase/Hyaluronidase. *Feed with outgrowth medium - 20% FES DMEM will work but cells seem to grow faster in outgrowth. B. Fetal Tissues Fetal tissue culture is much easier than adult tissues as the cells grow much more vigorously. Enzyme treatment is not absolutely necessary but it does lower the overall percentage of fibroblast-like cells and allows quicker growth. I should point out that the tissues should be put into culture as soon as possible. The longer the tissues are kept on ice, the less varied the cell type in the explants become, encouraging fibroblast-like cells to dominate the culture. Also these tissues are very sensitive to the collagenase method so they should not be exposed to this enzyme for longer than is absolutely necessary. Brain: 20% FCS/DMEM: Produces rapid growth with various cell morphologies Outgrowth: Same, but growth is not quite as vigorous Insulin: Mostly floating cells, not many adherent Insulin conditioned: Almost all are floaters Kidney: Collagenase/Dispase 20% FCS/DMEM both Outgrowth confluent in 2 days Insulin did not do too well
Freshney, R.I. (1987) Culture of Animal Cells: a manual of basic technique, Alan R. Liss, Inc., New York, pp.107-126.

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