Generation of Fibroblasts from Human Tissue
ReagentVendorCatalog Number
DMEM Thermo Fisher / Life Technologies 11330057
FBS ClonTech 631101
Gibco™ Penicillin-Streptomycin (10,000 U/mL) Fisher Scientific 15-140-122
MilliporeSigma™ ESGRO Complete™ Gelatin Solution Fisher Scientific SF008
Falcon™ Polystyrene Microplates Fisher Scientific Various
Fisherbrand™ Cover Glasses: Circles Fisher Scientific 2-546-2
Graham-Field™ Single-Use Scalpels Fisher Scientific 08-927-5A
Corning™ Polyethylene Terephthalate (PET) Centrifuge Tubes Fisher Scientific Various
Corning™ TC-Treated Culture Dishes Fisher Scientific Various
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Autoclaved Forceps
  • 10% Bleach Solution
  • 70% Ethanol Solution
  • Sterile, glass, borosilicate pipets (Fisherbrand)
  • Prepare Media:
    DMEM/10% FBS (500 mL):
    • DMEM – 450 mL
    • FBS – 50 mL
    Prior to skin punch delivery:
    1. Obtain circular microscope slides, disposable scalpel, forceps, scissors, 10cm dish
    2. Warm DMEM/10% FBS media (at least 150 – 200ml)
    3. Add 1 mL of gelatin to each well of two 6-well dishes
    4. Fill one 50 mL centrifuge tubes with 25 mL of 10% bleach solution, and another 50 mL centrifuge tube with 50 mL of 70% ethanol solution
    5. Add warm media to two 50 mL centrifuge tubes (~45 mL each)
    Upon Delivery:
    1. Aspirate majority of media off skin tissue, leaving some media to cover the tissue
    2. Pour the tissue sample with a little media onto the 10cm dish
    3. Add ~2-4 mL of fresh media onto the tissue in the dish
    4. Using forceps, grab hold of the tissue and cut away small chunks using the scalpel. Pieces should be small enough to place the cover slip into the well surface and big enough so there is significant tissue to derive the fibroblasts
    5. Aspirate gelatin off 6 well dishes
    6. Transfer pieces using a P1000 Pipetmen into center of a well of the 6-well dish along with 300 uL of media from the plate
    7. Using forcep, pick up circular cover slip and dunk (3-4 times) in ethanol
    8. Wash cover slip by transferring to one of the 50 mL tubes containing warm media and dunking the slip 4 – 5 times
    9. Transfer to the second 50 mL tube of warm media (2-3 dunks) for additional wash
    10. Place cover slip directly on top of skin tissue in the well and gently press down to ensure that tissue remains in place
    11. Once all tissue is covered with slides in each well, gently add 1.5 – 2 mL media onto the microscope slide
    12. Place the dishes in the incubator out of the way, where they won’t be disturbed
    13. Dispose any material that touched the skin punch into the bleach conical
    14. After one week of delivery, add an additional 1 mL of warm media to the wells
    15. After two weeks of delivery, you should observe both fibroblasts and skin cells expanding from the skin tissue
    16. After three to four weeks, the fibroblasts should expand throughout a majority of the dish
    Prior to Passaging:
    1. Obtain forceps, 6cm plate, and desired dish or plate to passage cells onto
    2. Warm DMEM/10% FBS media
    3. Add corresponding volume of gelatin to dishes or plates to passage cells on
    4. Fill one 50 mL centrifuge tubes with 25 mL of 10% bleach solution
    Passaging Fibroblasts from Tissue:
    1. Aspirate media off the tissue and slide within dish
    2. Using forceps, try to pull the microscope slide off the surface of the 6-well dish
    3. Place the microscope slide in the 6cm dish
    4. Add ~1ml of trypsin into the well with tissue and enough trypsin to cover the microscope slide in the 6cm
    5. Place both in the incubator for 5-10 minutes, checking for cell lifting every 2 minutes
    6. Once the majority of the cells begin to lift, vigorously pipet the cells off the slide and the well
    7. Pipet trypsin/cells into 15ml conical
    8. Add enough media to make total volume 10 mL
    9. Centrifuge cells at 200g for 5 minutes
    10. Aspirate gelatin off dishes
    11. Resuspend pellet in desired volume of fresh media and plate cell onto dished
    12. Dispose any material that touched tissue into the bleach conical
    13. Passage at least three times to obtain a pure fibroblast culture
    This protocol was largely based on the methods provided by:
    Vangipuram, Malini et al. “Skin punch biopsy explant culture for derivation of primary human fibroblasts.” Journal of visualized experiments : JoVE ,77 e3779. 7 Jul. 2013, doi:10.3791/3779