Cell-Embedded Fibrin Constructs

1.  Prepare the fibrinogen solution to a [FINAL] = 20mg/ml

a.    Dilute 4.5g Fibronogen (Sigma F-8360) in 255ml serum/antibiotic-free media

b.    Dissolve slowly at 37 degrees for 4 hours

i.       Swirl gently every 30 minutes

c.     When dissolved, filter through a 0.2Ám filter

d.    Store frozen in 25ml aliquots

2.  Prepare Thrombin solution to a [FINAL] = 200U/ml

a.    Add 0.1% BSA to 50ml serum/antibiotic-free media and mix

b.    Add the 50ml of BSA media to the 10,000 Units of thrombin (Sigma T-3399)

c.   Mix well

d.   When dissolved, filter through a 0.2Ám filter

3.  For 1ml of cell-embedded fibrin you need:

a.    3.3mg Fibrinogen

b.    500K cells

c.     4U Thrombin

4.  Trypsinize and count your cells

5.  Resuspend cells at a concentration of 615K cells per ml of growth media

6.  Put 815Ál of this solution in a sterile eppendorf tube

7.  Add 20Ál of 200U/ml thrombin and mix well

8.  Add 165Ál of 20mg/ml fibrinogen and mix well

9.  Quickly drop 100Ál in the centre of a well

10.          Allow to set in the incubator for 5 minutes

11.          Add media to the cells and culture as normal