Feeder Layer Tissue Culture

Swiss 3T3 cells (a TRAP positive feeder cell line that grows well in serum containing media such as DMEM/F12 plus calf or fetal calf serum)

Preparing the Swiss 3T3 cells for use as a feeder layer : 1.) Once plate is confluent, treat with 10 g/mL mitomycin C (stock 0.5 mg/mL, o dissolved in H2O) in complete medium for 2 hrs at 37 C. 2.) After treatment, wash plate with pre-warmed Solution A (or serum-free medium) at least 3 times, aspirating after each wash. 3.) Split the 3T3 cells by adding trypsin/EDTA; aspirate after 30-40 sec, incubate another 30 sec at RT, tap the plate to dislodge the cells, and resuspend them in complete medium. 4.) Spin the cell suspension down to obtain a cell pellet. 5.) Pellets can be used as feeder layers immediately, or can be saved for later use. a) For immediate use : - Wash pellet in pre-warmed Sol’n A and spin down - Resuspend in DMEM/F12 - Plate the 3T3 cells 1:3 and mix with an appropriate number of epithelial cells (low density, 250k for a 100mm plate) in DMEM/F12 b) For next day use : - Wash pellet in pre-warmed Sol’n A and spin down - Resuspend in complete medium - Plate 1:3 in complete medium and allow to attach overnight - Wash plate with pre-warmed Sol’n A x2 and put on DMEM/F12 - Plate epithelial cells (low density, 250k for a 100mm plate) c) For later use : - Wash pellet in pre-warmed Sol’n A, - Resuspend 100mm plate in 6ml cryogenic media (90% serum, 10%DMSO) and aliquot into 3- 2ml cryogenic vials - Freeze at –150oC or liquid nitrogen - Use later by thawing a vial into one 100mm dish with complete medium ***Note: The 3T3 cells must be used as feeder layers or frozen down within 2 days of being treated with mitomycin C***

Splitting the feeder layer/epithelial plate: 1.) Once the epithelial cells on the plate are 70-75% confluent, aspirate off medium and wash once with pre-warmed Sol’n A (cells that are too confluent initiate differentiation). 2.) Aspirate off Sol’n A and wash with pre-warmed 0.02% EDTA (0.68mM) in 1x PBS. This wash should be performed fairly rapidly and is done to remove the Swiss 3T3 cells, but leave behind the epithelial cells. The EDTA wash is done ten times by pipetting 10-12mLs directly onto the cells on the outer perimeter of the plate, while rotating the plate. Then using this same EDTA again, do the same for the inner half of the plate cells. 3.) Diagram of 0.02% EDTA in 1x PBS washes:

4.) Aspirate off the EDTA from the previous washes, and repeat the 0.02% EDTA washes for the inner and outer halves of the plate. This makes a total of 40 washes. This EDTA wash protocol is for human keratinocytes, other cells may be less strongly adherent and the number of washes may have to be modified. 5.) Aspirate off the EDTA and wash the plate with pre-warmed Sol’n A x3 still keeping the plate tilted. On the last Sol’n A wash look at the cells under the microscope and verify that all of the Swiss 3T3 are washed away, if they are not then more EDTA washes should be performed. 6.) Trypsinize the epithelial cells using their normal protocol (for keratinocytes trypsin 2 minutes RT, aspirate off the trysin and then incubate for 5 min at 37oC, tap plate, resuspend in DMEM/F12). Epithelial cells should be plated back onto mitomycin C treated feeder layers in low density (250K for a 100mm plate).

***Note: Since Swiss 3T3 cells are TRAP positive, there may be some residual TRAP activity in any samples collected for cell lines that were grown on them.***