1. Preparation of Media and Reagents
- Preparation of 200x of L-ascorbic acid 2-phosphate stock
- Dissolve 29 mg of L-ascorbic acid 2-phosphate per 5 mL of Dulbecco's Modified Eagle's Medium (DMEM) solution and filter through a 0.22 μm membrane filter. Store as 0.25 mL sterile aliquots at -20 °C.
- Add 0.25 mL aliquot of 200x L-ascorbic acid 2-phosphate stock to every 50 mL of fibroblast media (DMEM with 1% L-glutamine and 10% fetal bovine serum) on the day required, for a final concentration of 0.1 mM of L-ascorbic acid 2-phosphate.
- Preparation of keratinocyte growth media
- Isolation and culture of primary squamous cell carcinoma (SCC) keratinocytes have been described previously. Preparation of keratinocyte growth media is described therein as well.
- Briefly, prepare the media as follows:
300 mL of DMEM and 100 mL of Ham's F-12 supplemented with 10% FBS
0.4 mg/mL hydrocortisone
5 mg/mL insulin
10 ng/mL EGF
5 mg/mL transferrin
8.4 ng/mL cholera toxin
13 ng/mL liothyronine
1x penicillin-streptomycin solution
2. In vitro Construction of Fibroblast-derived Native Matrix in L-Ascorbic Acid 2-Phosphate Supplemented Media
- Seed 200,000 fibroblasts per well in 6-well plates (20,000 cells/cm2) in fibroblast media supplemented with L-ascorbic acid 2-phosphate. Refeed every 2-3 days with 2-5 mL of media. (Note: Refeeding frequency and volume may be adjusted to suit the individual needs of different cells.)
- A thick layer of cells embedded in extracellular matrix will form at the end of 6 weeks, visible to the naked eye (Figure 1). To release this layer from the tissue culture plate, gently scrape the circumference of the matrix with a 1 mL micropipette tip and then push the edges of the matrix towards the center of the well. This layer of cells and matrix (native matrix) should be easily detachable from the plate surface, and now be floating in the media. Spread the native matrix out in the media to avoid it folding up onto itself.
- Let the native matrix float and remodel for 5 days, changing media every 2-3 days. Due to tensile strength and intrinsic remodeling within the matrix, the matrix will contract drastically and become reduced to a smaller, but thicker native matrix, ready to be utilized for invasion assay.
3. Invasion Assay with Tumor SCC Keratinocytes
- Pick up the native matrix gently with blunt forceps and transfer to Nylon net. Once on the Nylon net, spread the matrix gently to lie as flat as possible using a 1 mL micropipette tip and blunt forceps.
- Prepare the sterile clonal cylinders by smearing a small amount of sterile Vaseline on one end.
- Place the clonal cylinders on the native matrix, with the Vaseline side down. This is to ensure a tight seal between the native matrix and the clonal rings.
- Add cSCC cells to the clonal cylinders (250,000 cells in 100 µl of keratinocyte growth media).
- Remove the clonal cylinders after 6 hr when the cSCC cells have settled down on the native matrix.
- Lift the Nylon net with the native matrix and cSCC cells to the air-liquid interface onto bent stainless steel wire mesh support.
- Add keratinocyte growth media supplemented with ascorbic acid until the media level touches the bottom of the native matrix.
- Change media every 2-3 days and harvest at 7 and 14 days, post-seeding of cSCC cells.
