View source for 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==
# 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.
# Transfer the sample in a 6 cm dish with 5ml of washing medium.
# Cut out with a scalpel the bleeding or necrotic areas. '''NB: do it very thoroughly! Do not keep odd tissue.'''
# 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. '''
# 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!'''
# Cut the sample in very small bits (approx. 2-4mm2) with a scalpel NR.22.
# 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).
# 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.'''
# 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.
# Place the dish in the incubator.

==‘Cleaning’ the CAF cultures from tumor cells==
# Check the growth from the explants and the morphology of the cells outgrowing from explants. 
# Mark the explants with outgrowth of fibroblasts.
# Scrape away the explants with the outgrowth of cells with epithelial (cobble stone) morphology.
# Repeat this procedure at each 2-5 days.
# 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. '''
# To select clean CAF cultures we use serial dilution. We do this in two ways:
##  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.
## 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.'''

[[Category:Daniela Elena Costea]]
[[Category:Human Oral Cells]]
[[Category:Protocols]]