Media Recipes
1. TIL media
500 ml RPMI 1640 + glutamax
50 ml human AB serum
500 l 2-mercaptoethanol
5 ml HEPES
5 ml Pyruvate
5 ml Glutomax
5 ml PenStrep
Filter sterilize. Media will remain good for ~3 weeks.
2. P815 media
50ml FBS
500 ml RPMI 1640+ glutamax
Filter sterilize. Media will remain good for ~3 weeks.
3. Tumor media
500 ml RPMI 1640 + glutamax
50 ml FBS
500 gentimicin
5 ml HEPES
5 ml insulin/transferrin/selenium
5 ml PenStrep
Filter sterilize. Media will remain good for ~3 weeks.
TIL cultures

TIL are non-adherent, non-pigmented and “tear drop” shaped due to IL-2
activation.

Melanoma tumor cells may be pigmented, are adherent, and larger in size than
TIL. These cells should split out during the first 1-2 weeks of culture in IL-2.

3000 U/ml human IL-2 should be added to the TIL cultures every 3-4 days.
1. Initial culture should be done using flat-bottom 24 well plate.
2. Cells can be split 1:1 when confluent or media color changes.
3. Split into the 24 well plate. When the plate is full, count cells and keep at 1x10 6
cells/ml in 75cm2 flask.
4. Cells will be viable for up to 6 weeks. Exhaustion can be observed as a decrease
in proliferation.
Primary Melanoma Tumor cultures
Melanoma cells are large adherent cells that have a visible cellular membrane under the
microscope. Contaminating fibroblasts look similar but have a more transparent
appearance and a longer morphology. Most melanoma cells will express MCSP-1 but
not CD90 for phenotypic characterization.
Cells are plated at 100,000/ml in T150 or T75 flasks. They can also be plated in 6 well
plates (4ml media) if the counts are low.
Tumors grow at very diverse rates. Usually they will be split when 90% confluent or the
media begins to look orange/yellow.
To split:
Aspirate the supernatant and wash the flask with 1x PBS twice. Add cell dissociation
buffer or 5% trypsin for dissociate the tumor for 5 min at 37degrees. If the cells are in a
flask, you can tap the flask to help dissociate the tumor from the plastic.
Cells are then collected by washing the flask with tumor media and placed in a 50 ml
conical tube, counted, and spun at 1400 rpm for 5 minutes. The tumor can then be
replated or frozen.
P815 cell cultures
P815 cells are non-adherent mastocytoma cells. They rapidly proliferate and do not
respond well to media containing PenStrep or Gentimicin.
They usually need splitting every 2-3 days and are split at 1:10.