Fibroblast Cell Culture Protocols

Protocol for subculturing and freezing fibroblasts


DMEM- Invitrogen #11960-044 (high glucose without L-glutamine)

15% FBS Fetal Bovine Serum, Hyclone SV30014-03 (from Fisher-Sci)

1% (1x) Penicillin-Streptomycin (Invitrogen#15140-122)

1% (1X) L-glutamine (Invitrogen#25030-081) 200


Trypsin EDTA C .25% (Invitrogen#25200-056)

Hank’s Balanced Salt Solution

HBSS- (Invitrogen#14170 (1X)  (-) calcium Chloride,  (-)magnesium chloride,  (-) magnesium sulfate)

DMSO for Freezing

Cell Culture Grade DMSO (American Bioanalytical #AB03091)

How to handle newly arrived cell cultures

  1. After unpacking, observe the cells under an inverted microscope to make sure they did well during transport. Place flask in a 37C incubator overnight. Do not remove medium.
  2. The next day, depending on the confluency of the cells, the flask may be fed by withdrawing the shipping medium and covering the cells with 5 mlsof new medium (10 – 15% FBS) or subcultured according to the cell count.

Subculturing PRF cell lines

1) Cells should be split when confluent.

2) To split cells (volumes given are assuming cells are in a T25):

A. Using sterile technique, remove media and rinse flask with 2-3 mls of sterile HBSS. Remove and discard HBSS.

B. Add 1 ml trypsin and incubate for 2-3 minutes.  Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float.  If cells are not detached after 3 minutes, you may incubate another 1-2 minutes

C. Tighten cap and gently tip flask from side to side to dislodge all cells.  Flask may be tapped lightly on side to loosen cells if needed.

D. Add 4 mls of new media to flask as soon as cells are floating to inactivate the trypsin.  Rinse down sides of flask several times with the new media to wash all cells off the plastic and in to the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).

E. Using sterile technique, remove an aliquot for counting on a hemacytometer.

F. While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpms.

3) After calculating your cell count, plate cells at 250,000 cells per T 25 filter top flask.

4) Cells should be fed every 2-3 days with fresh growth medium.


Cells should be frozen at no less than 500,000 cells /ml/Corning freezer vial in growth media containing 10% DMSO and 30% FBS and subsequently placed in an isopropanol freezing chamber at -80 degrees C overnight. Transfer to the liquid nitrogen the next day.

For Further Information Please Contact:

Leslie B. Gordon, MD, PhD
Professor of Pediatrics Research Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children’s Hospital, Providence, RI Department of Anesthesia, Children’s Hospital Boston and Harvard Medical School, Boston, MA, Medical Director, The Progeria Research Foundation

Phone: 978-535-2594
Fax: 508-543-0377 

Joan Brazier
Research Study Coordinator
Phone (401) 863-9628
Fax (401) 863-3489