Tissue Engineering and Cell-Populated Collagen Matrices - Methods in Molecular Biology: Extracellular Matrix Protocols - page 277

3.2. Production of Fibroblast Culture
A number of methods exist for the culture of fibroblasts. One convenient
method for the production of these cells from discarded foreskin tissue for use
in organotypic culture is described below (3) .
1.

Wash the foreskin several times with the antimicrobial wash solution.

2. While holding the foreskin with sterile forceps, wash the foreskin for 1 min (no

longer) in 95% ethanol with constant agitation.

3. Immediately remove the ethanol by washing in the antimicrobial solution.

4. Dissect away any subcutaneous tissue and wash the remaining tissue again in

antimicrobial solution.

5. Transfer the foreskin to a 100-mm Petri dish containing antimicrobial solution.

Mince the tissue into 1-3 mm 2 pieces ( see Note 10 ).

6. Place a sterile microstir bar into a round bottom tube and add 5 mL of the

enzyme solution.

7. Transfer the minced tissue and cap the tube. Incubate the tube at 37 ° C with gentle

stirring. After 20 min, vigorously shake the tube.

8. After 30 min, allow the material to settle. Remove as much of the enzyme solu-

tion and discard. Add 4 mL of fresh enzyme solution and continue to incubate as
above, shaking the tube periodically.

9. After a further 30 min incubation, allow the material to settle again and transfer

the supernatant solution to a sterile 15-mL conical tube.

10. Add 5 mL of DMEM-10% NBCS to the 15-mL conical tube containing the

supernatant and centrifuge for 5 min at 600 g . Aspirate the supernatant, resuspend
the pellet in 2 mL DMEM- 10% NBCS and place the tube on ice.

11. Add an additional 4 mL of enzyme solution to the tissue remnants from step 9 .

Repeat steps 9 and 10 .

12. Repeat step 11 until all the tissue is digested and only the stratum corneum of the

epidermis remains.

13. Pool the cell fractions from step 10. Count the cells and determine viability. Add

1X 10 6 cells to each of several T-75 flasks. Add 10 mL of DMEM-10%NBCS to
each flask. Incubate at 37 ° C, 10% CO2. Replace the media every 2-3 d.

14. When the cells are confluent, aspirate off the media and add 5 mL of trypsin/

versene. Incubate the flask at 37 C, 10% CO 2 . Strike flask sides sharply to dis-

lodge the cells. Examine the cells to ensure they have rounded and left the dish.
Add 5 mL of DMEM-10% NBCS to each flask.

°

15. Transfer the solution containing the cells to a sterile 15-mL conical tube and

centrifuge 5 min at 600 g .

16. Discard the medium and replace with 10 mL of DMEM-10%NBCS.

17. Suspend the cells and recentrifuge at 5 min at 600 g .

Count the cells and transfer 1X 10 6 cells to each of several T-150 flasks. Add 20 mL
of DMEM-10%NBCS. Incubate at 37

18.

° C, 10% CO 2 .

19. Cells can continue to be passaged at confluence as described above ( see Note 11 ).