SOLIDTUMORCULTURESFORCHROMOSOMEANALYSIS

I. Purpose:

A. Samples of solid tumors or lymph nodes may be sent from patients with cancer. These samples should be processed directly and also set up for short term culture of tumor cells. Care must be taken to avoid over-growth of normal fibroblast cells that are also present in the tumor specimen.

II. Culture Procedure:

A. Aseptic technique must be used when setting up the cultures, preferably under a laminar flow hood. Label a 15 ml centrifuge tube for the direct harvest, and 3 T-25 flasks and 1 100 mm petri dish for short term cultures with the patient number, patient name, type of tissue, and date. Flasks should also be labeled (using greek letters) to identify primary cultures when flasks are sub-cultured; this allows for identification of cultural artifacts and psuedo vs real mosaicism.

B. Cultures are grown in complete Nutrient Mix F-10. Media should be fresh (less than 4 days old) and be prewarmed and at pH 7.0 - 7.5. All cultures are incubated in a wet, 5% CO2 incubator at 37 C.

C. Using forceps transfer tumor to the petri dish, if the sample is very large cut off a section. Add 3 ml of media to the dish. Using scissors, cut tumor into pieces about 5 mm X 5 mm. Holding a corner of the tumor with forceps, mash it using the end of a sterile 5 ml pipet to free the tumor cells from the tissue. Mash each piece of tumor thoroughly. Transfer 1 ml of cell suspension to the labeled tube for direct harvest, and 1 ml to each of 2 T-25 flasks for short term culture (1 should be harvested after 24 hours). Add 2 ml fresh media to the tissue in the petri dish. Using scissors cut the tissue into small bits. Transfer 1 ml to the last flask for short term culture, leaving the rest in the petri dish. Add 2 ml of fresh media to each of the flasks and the centrifuge tube, add 4 ml of media to the petri dish. Incubate the flasks checking for growth after the third day. 
Tumor cells tend to grow as non-adherent cells although they may grow in close association with fibroblasts. Cultures should be harvested as soon as possible to minimize the occurance of in-vitro chromosome abnormalities that may not reflect the true karyotype of the tumor. Cultures are passaged by adding an equal volume of fresh media to the flask. When the volume becomes large, transfer half to a new flask. As soon as the cultures are growing well they should be harvested. Transfer half the culture to a new flask, add an equal volume of fresh media and harvest the next day.

III. Harvest Procedure:

A. Add 30 mcl of colcemid working solution to the tube or flask. Incubate for 45 minutes. Transfer cell suspension to a 15 ml centrifuge tube if necessary.

B. Centrifuge the tubes for 6 minutes at 150 x g (900 rpm in the Sorvall GLC-2B centrifuge).

C. Aspirate and discard all but 0.3 ml of supernatant. Resuspend the cell pellet by mixing by hand, do not use a mechanical vortexer as this may break the cells. Add 3 ml of prewarmed hypotonic solution and mix. Incubate the tubes at 37 C for 8 minutes.

D. Add 2 drops of fix to the tubes. Centrifuge the tubes for 6 minutes at 150 x g.

E. Aspirate and discard all but 0.3 ml of supernatant. Mix by hand to break up the cell pellet. 

F. Slowly add 2 ml of fixative (1 pasteur pipetful) letting it run down the side of the tube so that it layers on top of the cell suspension. Mix rapidly by hand so that all the cell suspension is fixed evenly. Wash down sides of tube with another pipetful of fix. Let sit at room temperature for 15 - 20 minutes.

G. Centrifuge for 6 minutes at 150 x g.

H. Aspirate and discard all but 0.3 ml of supernatant. Resuspend the cell pellet. Add 2 ml of fixative and mix, wash down sides of the tube with 2 ml fix. Let sit at room temperature 15 - 20 minutes.

I. Repeat steps G,H 1 or 2 times until the pellet is clean and white. The culture is now ready to make slides. For best results, slides should be made the same day as the harvest.

IV. Slide Preparation:

A. See procedure for regular lymphocyte cultures.

B. If possible, stain one slide without trypsinization in order to check for the presence of dmins.

V. Solutions:

Colcemid working solution: 10 mg Colcemid in 250 ml dH2O, sterile filtered, store at 4 C.

Complete Nutrient Mix F-10: 100 ml Nutrient Mix F-10, 20 ml Fetal calf serum, 1 ml Penicillin/Streptomycin solution, 1 ml L-Glutamine solution, store at 4 C.

Fixative: 30 ml Methanol and 10 ml Glacial Acetic Acid,
prepared fresh.

Hypotonic solution 0.075 M KCl: 2.8 g Potassium chloride dissolved in 500 ml dH2O.

VI. Reagents:

Colcemid: GIBCO cat# 890-3014. 10 mg bottle. 

Fetal calf serum: HYCLONE/STERILE SYSTEMS INC. cat # A-1111-D. defined fetal bovine serum 100 ml bottle, store frozen.

Glacial Acetic acid: AMERICAN SCIENTIFIC PRODUCTS cat# 9508-2. Acetic acid, Glacial ACS (Aldehyde free), 500 ml bottle.

L-Glutamine: GIBCO cat#320-5030. L-Glutamine solution 100X 
(200 mM), 20 ml bottle, store frozen.

Methanol: AMERICAN SCIENTIFIC PRODUCTS cat# 3016-1. Methyl alcohol anhydrous AR (ACS)(Absolute) Acetone free, 500 ml bottle.

Nutrient Mix F-10: GIBCO cat#430-1200. Nutrient Mixture F-10 (HAM) powdered form, prepared to instructions, 10 x 1 liter package.

Penicillin/Streptomycin: GIBCO cat# 600-5140. Penicillin/Streptomycin solution, 
10,000 units/ml / 10,000 mcg/ml, 20 ml bottle, store frozen.

Potassium Chloride: COLUMBUS CHEMICAL INDUSTRIES, INC. ACS granular, 500 g lots.