In vitro culture on tissue culture plastic, fibroblasts, or keratinocytes

Co-culture of PBMC T cells with keratinocytes/fibroblasts

Thaw viably frozen keratinocytes and start culturing in 24 well plates with 1ml/well of keratinocyte media. Feed 3 times per week by aspirating all of the media (cells attach to the bottom) and adding 1 ml/well fresh media.

Thaw viably frozen fibroblasts and start culturing in 24 well plates with 1ml/well of fibroblast media. Feed 3 times per week by aspirating all of the media (cells attach to the bottom) and adding 1 ml/well fresh media.

For keratinocytes, 1x10^6 cells will need approximately one week to make confluent sheet. Fibroblasts are much quicker, and they can make confluent sheet within a week from 1x10^5 cells.

Day 0: Stimulation of PBMC T cells

When keratinocytes and fibroblasts become confluent, prepare peripheral blood mononuclear cell (PBMC) T cells from healthy donor blood or leukopak. Isolate PBMC using standard density gradient techniques. Isolate T cells from the PBMC using Miltenyi Pan T cell isolation kit (negative selection, cat # 130-091-156) and start stimulating the T cells with T cell activation beads (prepared from Miltenyi cat # 130-091-441 as per manufacturer’s instructions) with 1 Bead:10 T cells ratio. Cells are activated at a concentration of 1 million T cells in 2 ml of Skin T medium in 1 well of a 24 well plate.
 

Day 1: Set up co-culture

Aspirate media from Keratinocytes and Fibroblasts and replace with Skin T media, 2 ml per well.

Distribute 0.1 million bead activated T cells to each well.
 

Day 3: Feed cells

Gently aspirate ~1 ml of medium from the top of each well, taking care not to disturb the T cells which have settled to the bottom of the well. Replace with 1 ml fresh Skin T medium. 
 

Day 6: Harvest T cells for analysis

Each well will/can provide 2 T cell conditions. Collect T cells from well through gentle pipetting, removing the 2ml of Skin T medium. These will be labeled as “indirect” or “not attached.” After removing the Skin T medium and not attached T cells, add 500 ul of HBSS-HEPES plus 5mm EDTA to each well and return the plate back to the incubator for 5 min or until you can visually discern that the keratinocytes/fibroblasts detach from the bottom of the well. When the keratinocytes/fibroblasts detach, add 1 ml of Skin T medium to each well and pipet up and down 5x. These wells contain keratino/fibro and T cells which were tightly attached to these cells. Collect these cells through filter (70 um mesh cell strainer) and these will be labeled as “attached.” Cells are ready for downstream analysis after centrifugation.
 

Reagents:

Fibroblast medium (500 ml):

DMEM/F12 1:1:  425 ml

15% heat inactivated fetal bovine serum:  75 ml

10 mM Hepes:  5 ml (1M stock)

Penicillin/Streptomycin: 5 ml

L-glutamine: 5 ml

10 ng/ml EGF: 5 ug 

Keratinocyte Medium (500 ml) Gibco/Life Technologies # 17005-042:

Gibco-sfm:  500 ml

Bovine pituitary extract:  one half vial from the kit (about 1ml but it varies)

EGF:  0.2 ng/ml (0.1 ug/500 ml bottle, which is less than the 2.5 ug supplied in the kit)

Penicillin/Streptomycin:  5 ml, if desired

Skin T Medium  (500 ml)

400 ml Iscove’s medium (Corning # 10-016-CV)

100 ml fetal bovine serum

5 ml L-glutamine (Invitrogen # 25030164)

5 ml pen/strep (Invitrogen # 15140163)

1.75 µl 2-mercaptoethanol (Bio-Rad # 161-0710)

Hank’s /Hepes (H/H)

500 ml Hank’s Buffered Saline (Corning # 21-021-CV)

5 ml Hepes buffer 1M (Corning # 25060CI)