Collagen gel containing 3T3 fibroblasts

Contributed by Matt Lewis <hop2kin@yahoo.co.uk>

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

• Pre-chill pipettes and place 12mL rat tail type 1 collagen (>3.8mg/mL) on ice

Everything is kept cold to avoid the collagen solidifying

• 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.
• Using chilled pipette, add the 12mL of collagen gently to the cells and tilt to mix, avoiding bubbles as far as possible.
• Add 10N NaOH to bring the pH up to 7

You may judge the pH visually by the phenol red in the DMEM. Approximately 30–60mL will be required. Use the glacial AcCOOH if necessary but more mixing means more bubbles.

• Pipette 2–2.5mL into each well and incubate O/N
• Add 2mL raft media on top of each matrix.
• Use within 1 week, change media every 2 days.

This method is based, with permission, on an original protocol available here.