1. 6-well Transwell plate
2. BD collagen sollution (cat# 354236) (If use dried collagen, refer to method for the preparation of collagen solution) (4oC)
3. 10X DMEM, sterile (4oC)
4. 1N sterile NaOH
5. autoclaved Distilled H2O
6. 50ml Falcon centrifuge Tubes
7. sereological pipets (sterile)
8. Pipetters and tips
-Keep all reagents and operation sterile-
9. Larminar flow hood
10. Centrifugate (optional)
11. Ice bucket
12. Surgical blades
13. Water bath
14. 60^oC Oven
15. Leica Microtome
16. Staining apparatus and solutions

1. Place collage, 10X DMEM, 1N NaOH and sterile H₂0 on ice.
2. To a 50ml centrifuge tube, add H₂O, 10X DMEM, 1N NaOH and collagen sollution as in table 1 and mix carefully by pipetting up and down and avoid producing bubbles.

table 1. Calculation of collagen gel components

1. Add 1.5ml/well for 6-transwell plate. spread evenly by gently shaking. incubate at 37^oC for at least 10 min.
2. trypsinize cells by standard precedure. Count cells and allocate certain amount cells (fibroblast cells: 500K/well; Tumor cells: 200K/well) into fresh sterile tubes, pellet to remove trypsin and EDTA.
3. Resuspend cells in 2 ml regular media and put onto collagen gel (matrix).
4. Fill the lower camb with 3 ml media after incubation for 1-3 hours.
5. Incubate at regular condition and change media periodically as needed (usually every 2-3 days).
6. Terminate culture at the 6th day by replacing media with 10% formalin solution and fix gel overnight.
7. Cut gel with surgical blade from Transwell into plastic container with 10% formalin and send to Procurement Center for embedding.
8. Cut two sections per gel at 5 um using microtome and stain.
9. Observe under microscope and take pictures.