TITLE: Human primary fibroblast culture
AUTHORS: Ching-Chieh Chou, Judith Frydman

[DESCRIPTION]
This is a protocol for human primary fibroblast culture.

[STEPS]
SECTION: Preparing culture medium
1. Fibroblast medium @4°C lasts for 1 month. A total of 500 mL:
435 ml DMEM (High glucose, GlutaMAX) (Thermo Fisher Scientific) 
50 ml Fetal bovine serum (FBS) (Thermo Fisher Scientific)
5 ml Penicillin-Streptomycin (Thermo Fisher Scientific) 
5 ml MEM NEAA (100X; Thermo Fisher Scientific)
5 ml Sodium pyruvate (Thermo Fisher Scientific)
0.5 ml beta-mercaptoethanol (Thermo Fisher Scientific)
Sterilized by filtration through a 0.22 µm filter
SECTION: Thawing, subculturing and freezing fibroblasts
2. Remove a cryogenic vial containing the frozen human primary fibroblasts from liquid nitrogen storage and thaw in a 37°C bead bath for a few min. Constantly check the content.
SECTION: Thawing, subculturing and freezing fibroblasts
3. After the content is thawed, immediately transfer and slowly add to a 15 mL conical tube containing 5 mL of cell culture medium. 
SECTION: Thawing, subculturing and freezing fibroblasts
4. Centrifuge at 200xG for 5 min to pellet the cells.
SECTION: Thawing, subculturing and freezing fibroblasts
5. Remove the supernatant, add 10 mL of fresh culture medium for culturing cells in a 10-cm dish.
 

SECTION: Thawing, subculturing and freezing fibroblasts
6. Replace medium every 3-4 days. Fibroblasts should be confluent after one week. 
SECTION: Thawing, subculturing and freezing fibroblasts
7. For subculturing, rinse the dish with sterile 1x PBS to remove all complete medium before splitting.
SECTION: Thawing, subculturing and freezing fibroblasts
8. Add 0.05% Trypsin-EDTA to cover the bottom of the dish. Incubate at 37°C with 5% CO2 for ~5 minutes. Cells should round up and become dislodged.
SECTION: Thawing, subculturing and freezing fibroblasts
9. Neutralize trypsin-EDTA activity by adding the complete media.
SECTION: Thawing, subculturing and freezing fibroblasts
10. Gently pipette to resuspend the cells and transfer the cell-containing medium to a 15 mL conical tube.
SECTION: Thawing, subculturing and freezing fibroblasts
11. Centrifuge at 200xG for 5 min to pellet the cells.
SECTION: Thawing, subculturing and freezing fibroblasts
12. Remove the supernatant, add fresh culture medium and split the cells at 1:3 ratio.
SECTION: Thawing, subculturing and freezing fibroblasts
13. Replace medium every 3 to 4 days. Fibroblasts should be 80 to 90% confluent after one week and ready for experiments. 
 

SECTION: Thawing, subculturing and freezing fibroblasts
14. If want to freeze the cells, after centrifugation (Step 11), remove the supernatant, and add 1 to 2 mL of Recovery Cell Culture Freezing Medium (Thermo Fisher Scientific). 
 

SECTION: Thawing, subculturing and freezing fibroblasts
15. Gently pipette to resuspend the cells and transfer the cell-containing medium to a cryogenic vial. Then store the cryogenic vial in a cryo-freezing container that limits the decrease in temperature at ~1°C per min at -80°C.
SECTION: Thawing, subculturing and freezing fibroblasts
16. For long-term preservation, transfer the cryogenic vial from a -80°C freezer to a liquid nitrogen tank.