Culture Fibroblasts

General Protocol for the culture of Primary Fibroblasts

All cell culture procedures must be conducted in a bio-safety cabinet.

Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter.

Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival.

Medium:

Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements). Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells and when changing media or splitting cells.

Coating of flasks or dishes:

Coat sterile culture dishes or flasks with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950) for 2 min and then aspirate the excess solution before seeding cells.

Cell recovery from cryovial:

• Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
• Promptly remove the vial and wipe it down with 70% ethanol.
• Transfer cells from the vial to a sterile centrifuge tube. Add 8-10 ml of pre-warmed Cell Biologics Cell Culture Medium. Flush the vial with an additional 0.5-1 ml of medium to ensure complete transfer of cells to the centrifuge tube.
• Centrifuge cells at 200 g for 5 minutes.
• Aspirate the supernatant and resuspend the cell pellet in 6 ml of Cell Biologics’ Cell Culture Growth Medium.
• Add resuspended cells into a T25 flask pre-coated with Gelatin-Based Coating Solution (Cell Biologics, Catalog No. 6950).
• Place the T25 flask in a humidified, 5%-CO₂ incubator at 37°C. Change medium 3-6 hours after thawing cells to ensure >90% cells are attached.
• Also change culture media the following day to remove non-adherent cells and replenish nutrients.
• Change cell culture medium everyday when cells are >60% confluent.
• Cells should be checked daily under a microscope to verify appropriate cell morphology.

Expansion of cultured mouse primary cells: 

• Flush the adherent layer with a 5 ml sterile pipette 3 times to dislodge loosely attached cells.
• Remove and discard the cell culture media from the flask.
• Wash adherent cells 2 times with 10 ml of sterile PBS (1X) without calcium and magnesium to remove nonadherent cells or fraction.
• Remove and discard the wash solution from the flask.
• Incubate cells with warm (37°C) 0.05% Trypsin-EDTA (1X) solution (Invitrogen catalog number 25300) for 1 minute. Use 2.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.0 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 8-10 ml of Cell Biologics Cell Culture Medium supplemented with 5-10 % FBS to a T25 or T75 flask (the FBS will neutralize the trypsin).
• Plate cells in fresh flasks or plates precoated with Gelatin-Based Coating Solution in a humidified, 5%-CO₂ incubator at 37°C. Change medium 3-6 hours after seeding cells to ensure >90% cells are attached.
• Also change culture media the following day to remove non-adherent cells and replenish nutrients.
• Cells should be checked daily under a microscopy to verify appropriate cell morphology.
• Change culture medium every 24-48 hours. Please note that the medium should be changed every day when cells are >60% confluent to remove non-adherent cells and replenish nutrients.
• Pre-wash cells with 1X PBS 2 times whenever replacing the medium.

We recommend splitting primary cells at the follow ratio:

• The recommended split ratio for primary murine cells is 1:2.
• A confluent monolayer of primary cells grown in a T75 flask may be expanded on a 6-well plate ready for use in experiments under the cell culture conditions specified by Cell Biologics.

Procedure for Freezing Cells

Materials:

• 1X Phosphate Buffered Saline (PBS-1X)
• 0.05% Trypsin-EDTA (1X) solution (Invitrogen catalog number 25300)
• Tissue Culture Media
• Cold Freezing Media (10% dimethyl sulfoxide (DMSO), 20% FBS and 70% culture medium)
• Labeled Cryovials
• Confluent cells

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• Flush the adherent cell layer with a 5 ml sterile pipette 3-5 times to dislodge loosely attached cells.
• Remove and discard the cell culture media from the flask.
• Wash adherent cells 2-3 times with 10 ml of sterile PBS (1X) without calcium and magnesium to Remove nonadherent cells or fraction.
• Remove and discard the wash solution from the flask.
• Incubate cells with warm (37°C) Trypsin-EDTA (1X) solution for 1 minute. Use 2.0-3.0 ml of 0.05% Trypsin-EDTA solution when collecting cells from T75 flasks, and 1.0-1.5 ml when using T25 flasks. As soon as cells have detached (the flask may require a few firm gentle taps), add 10 ml of Cell Culture Medium supplemented with 5-10 % FBS to the flask (the FBS will neutralize the trypsin).
• Centrifuge the cell suspension at 200 g for 5 minutes.
• Remove supernatant with sterile Pasteur pipette.
• Quickly re-suspend pellet by adding 1 ml freezing media per vial to be frozen.
• Place Cryovials in Nalgene "Mr. Frosty" freezing container containing100% isopropyl alcohol at -70-80 °C for 24 h.
• Transfer vials to liquid N₂ tank for indefinite storage.

We recommend freezing primary cells at the follow ratio:

• A confluent primary cells grown in a T75 flask may be frozen in 2 cryovials.
• A confluent primary cells grown in a T25 flask may be frozen in 1 cryovial.

Primary cells differ from cell lines in many ways.  Here’s what you should know about primary cells:

1.  Primary cells have a finite life span. They are exactly like the cells that you would find in your own body, so they do die after a certain period of time in culture. The amount of time primary cells survive in culture varies according to cell type.  
2.  Primary cells can be diverse. Cancer cell lines often come from a single patient – for example, HeLa cells came from one woman, Henrietta Lacks. Primary cells, on the other hand, can come from a variety of people. Having a variety of primary cell types from many different donors is especially useful when carrying out early drug testing— it’s important to ensure that the drug is effective for everyone. Before testing the drug in human patients, researchers can use primary cells from different donors to verify that the same effects are observed. 
3. Primary cells can change in culture.  For this reason, and because primary cells do not live forever, Lifeline Cell Technology strongly suggests carrying out experiments on primary cells using earlier passages.
4. Primary cells have not been modified in any way. Except for the enzymatic and/or physical dissociation required for extracting the cells from their tissue of origin, primary cells are not altered in any way. Immortalized cell lines, on the other hand, are often transformed either with cancer genes, viruses or other inducible modifications, which could alter the outcome of experiments.
5. Primary cells are finicky. Cell lines are hardy, mostly because they have to last a long time in culture, and be capable of surviving multiple rounds of cryopreservation and thawing. Primary cells, on the other hand, tend to be more difficult to maintain in culture.