DC2010 isolation of oral cancer cells and carcinoma associated fibroblasts

PROTOCOL FOR ISOLATION OF PRIMARY ORAL CARCINOMA ASSOCIATED FIBROBLASTS

'explant technique'

Preparations

Be sure you have:

• washing medium: DMEM with 2% AB/AM (for 50 ml add 49ml DMEM medium and 1 ml Ab/AM)
• culture medium for isolating fibroblasts: DMEM medium (Sigma) + 10% FBS (InVitrogen) + 1% AB/AM InVitrogen),
• FBS (InVitrogen)
• Sterile tissue forceps (delicate).
• New scalpel blades no.22.
• 10 ml plastic pipettes.
• 6 and 10 cm diameter dishes (Nunc)
• 6 and 96 wells dishes

Preparing the dish with explants

1. Collect and transport the sample at +4C in 5ml DMEM medium + 2% AB/AM (in a 15 ml centrifuge tube). Keep the sample at 4ºC, (it can last. 4 days) till processing.
2. Transfer the sample in a 6 cm dish with 5ml of washing medium.
3. Cut out with a scalpel the bleeding or necrotic areas. NB: do it very thoroughly! Do not keep odd tissue.
4. Wash the sample (should not be bigger than 0.25 cm2; if it is, cut it in 0.25 cm2 pieces) 2x in 10 ml washing medium. NB: wash it very thoroughly! Try to get rid of al traces of blood.
5. Transfer the sample in a new 6 cm dish and add some drops of medium on it. NB: keep the tissue moist at all times!
6. Cut the sample in very small bits (approx. 2-4mm2) with a scalpel NR.22.
7. Transfer the small bits of tissue/explans in a 10 cm dish and place them at distance (around 1-2 cm diameter around each explant).
8. Let the dish with the explants in the hood for 2-5 min so the medium around the explant evaporates a bit and gets the explant to stick to the dish. NB: check it carefully so you donot dry the explants.
9. Add 10 ml culture medium from one side of the dish (keep the dish tilted towards the side you add medium) and cover all explants gently with medium.
10. Place the dish in the incubator.

‘Cleaning’ the CAF cultures from tumor cells

1. Check the growth from the explants and the morphology of the cells outgrowing from explants.
2. Mark the explants with outgrowth of fibroblasts.
3. Scrape away the explants with the outgrowth of cells with epithelial (cobble stone) morphology.
4. Repeat this procedure at each 2-5 days.
5. You can also try selective trypsinization and further grow the cells that have detached in the first 1 min of trypsinization. NB: Unfortunately, most likely the cultures will be mixed at further passages so characterize them by FACS before using them in any experiments.
6. To select clean CAF cultures we use serial dilution. We do this in two ways:
   1. trypsinize the cells for 1 min only and resuspend the cells in 20 ml culture medium in a 10 cm dish. Under the microscope pick one cell with a 10ul pipette and transfer it in a well of a 96 well plate in fibroblast culture medium. Do this for as many wells as you manage, ideally half of a 96 wells plate. Assess the morphology of the cells growing in the wells after 5-7 days and select only the wells with cells with fibs morphology for further propagation. I usually mix the cells then from 5-10 wells so I do not get a clonal population.
   2. FACS can be done to clean the CAF cultures from human tumor cells by using ESA Ab (we are using CD326 /EpCam-APC from Miltenyi Biotec (cat. No 130-091-254) and then serial dilution of sorted cells. We sort the negative CD326-APC cells and seed them either as 500-1000 cells in 6 wells or as 1-10 cells in 96 wells. After 7 days we check the morphology of sorted cells growing in each well and select for further growth only the wells with cells of fibroblast morphology.

NB: Before experiments check again their purity by FACS after staining with CD326-APC.