Collagen gel containing 3T3 fibroblasts (dermal equivalent for raft culture)

• Protocol
• Discussion

Author: Matt Lewis

Ingredients for 6 x collagen matrices in a 6-well plate

1. Roughly 3x10e6 J2-3T3s (a fully confluent T75?)
2. 1.5mL 10x reconstitution buffer
3. 1.5mL 10x DMEM
4. 12mL rat tail type 1 collagen (>3.8mg/mL)
5. 10N NaOH
6. Glacial acetic acid (in case)

Method

1. Pre-chill pipettes, keep collagen on ice
   • The collagen solidifies above 8ºC
2. Mix 1.5mL of 10x DMEM with 1.5mL of 10x reconstitution buffer, keep on ice. Count J2-3T3s and pellet required number in a universal. Add the 3mL of [1:1, 10x DMEM and 10x reconstitution buffer] and swirl to resuspend the cells, keep on ice
   • The J2-3T3s seem to survive this somehow
3. Using chilled pipette, add the 12mL of collagen gently to the cells and tilt to mix, avoiding bubbles as far as possible.
   • Everything is kept cold to avoid the collagen solidifying
   • Avoid bubbles
4. Add 10N NaOH to bring the pH up to 7
   • Judge the pH visually by the phenol red in the DMEM
   • Maybe 30–60mL will be necessary
   • Don’t go too far, use the glacial AcCOOH if you have to, but the more mixing the more bubbles.
5. Pipette 2–2.5mL into each well and incubate O/N
6. In the morning, add 2mL raft media on top of each matrix.
7. Use within 1 week, change media every 2 days.

Buffers

10x DMEM - Dissolve DMEM powder into 0.1 volume of H2O. - Filter sterilise and store at –20ºC in working aliquots. (It looks yellow and doesn’t dissolve completely).

10x reconstitution buffer

• Dissolve 2.2g sodium bicarbonate and 4.8g HEPES in 100mL H2O.
• Filter sterilise and store at –20ºC in working aliquots.