Protocol for 3D Fibroblast Culture in Fibrin Gel

1.  Place Sylgard mold in the centre of a Sylgard coated 35mm dish

2.  Spray 70% ethanol 4 times into the cap of the plate and make sure the ethanol covers the plate

3.  Incubate with the lid facing up in the hood for a minimum of 20 minutes

4.  Aspirate the remaining ethanol

5.  Prepare the fibrin gel

a.    Coat the plates with 0.4 ml of growth media containing 10U/ml thrombin

b.    Add 160ul of 20mg/ml fibrinogen solution drop wise

c.     Mix twice in each direction

d.    Incubate 10 minutes at room temperature

e.    When the gels have formed, continue to next step

6.  Aspirate the media from the cells

7.  Rinse 1 X DPBS and 1 X Trypsin

8.  Add Trypsin and incubate 1-2 minutes in the incubator (shake half way through)

9.  Check cell detachment under the microscope

10.          Add growth media to the floating cells and pipet up and down twice

11.          Transfer the cells to 50ml conical

12.          Count the number of cells

13.          Dilute to 100,000 cells/ml in GM for seeding

14.          Add 1mL to each plate

15.          Incubate 3 days before feeding