Nov 09, 2023
Human primary fibroblast culture
- Ching-Chieh Chou1,
- Judith Frydman1
- 1Department of Biology, Stanford University
External link: https://doi.org/10.1038/s41556-025-01623-y
Protocol Citation: Ching-Chieh Chou, Judith Frydman 2023. Human primary fibroblast culture. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3edklk5/v1
Manuscript citation:
Chou, CC., Vest, R., Prado, M.A. et al. Proteostasis and lysosomal repair deficits in transdifferentiated neurons of Alzheimer’s disease. Nat Cell Biol 27, 619–632 (2025). https://doi.org/10.1038/s41556-025-01623-y
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 08, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 90642
Keywords: ASAPCRN, Fibroblast, protocol for human primary fibroblast culture, human primary fibroblast culture, culture
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This is a protocol for human primary fibroblast culture.
Troubleshooting
Preparing culture medium
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
Thawing, subculturing and freezing fibroblasts
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.
After the content is thawed, immediately transfer and slowly add to a 15 mL conical tube containing 5 mL of cell culture medium.
Centrifuge at 200xG for 5 min to pellet the cells.
Remove the supernatant, add 10 mL of fresh culture medium for culturing cells in a 10-cm dish.
Note
If the cell numbers are low, recommend to first culture the cells in a well of a 6-well plate filled with 2 mL of fresh culture medium to improve cell survival and growth.
Replace medium every 3-4 days. Fibroblasts should be confluent after one week.
For subculturing, rinse the dish with sterile 1x PBS to remove all complete medium before splitting.
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.
Neutralize trypsin-EDTA activity by adding the complete media.
Gently pipette to resuspend the cells and transfer the cell-containing medium to a 15 mL conical tube.
Centrifuge at 200xG for 5 min to pellet the cells.
Remove the supernatant, add fresh culture medium and split the cells at 1:3 ratio.
Replace medium every 3 to 4 days. Fibroblasts should be 80 to 90% confluent after one week and ready for experiments.
Note
Avoid subculture at low-density as it will age the cells.
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).
Note
Recommend freezing cells at a high concentration.
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.
For long-term preservation, transfer the cryogenic vial from a -80°C freezer to a liquid nitrogen tank.