1. What medium should be used for culturing CCR fibroblasts?
2. How should a newly received fibroblast cell culture be handled?
3. How is a fibroblast cell line subcultured?
4. How are fibroblast cultures frozen for cryogenic storage?
5. How should fibroblast cell cultures be recovered from cryogenic storage?

1. What medium should be used for culturing CCR fibroblasts?

Alternative media for fibroblasts include: alpha-MEM and Dulbecco's modified MEM.

We add L-glutamine to a final concentration of 2 mM just before use. If long-term storage of cell culture medium is not an issue, commercially prepared medium containing L-glutamine can be used.

CCR does not use antibiotics or antifungals because of the danger of a cryptic infection in a cell repository. An investigator can add antibiotics if desired.

If a cell culture is growing slower than expected, our first approach is to switch to a different lot of pre-tested serum and/or to alter the serum concentration by 5%. Other causes of slow growth include: microbial contamination, too frequent subculture, too low density seeding at subculture, senescence of cell line, change in medium composition, incubator inadequacy in regulating temperature, humidity or CO2.

2. How should a newly received fibroblast cell culture be handled?

See the Web Catalog for details of the culture medium for individual cell lines.

Procedure

1. Wipe culture flasks with a disinfecting solution and place in a 37C incubator overnight with the cell sheet down. Do not remove medium (contains only 5% FBS to slow growth during transport). Observe cell sheet for confluency, morphology of cells and signs of contamination.
2. The next day the flask should examined as above and depending on the confluency of new culture's, the flask may be fed by withdrawing the shipping medium and covering the cells with growth medium (10 - 15% FBS) to a depth of 2mm or subcultured according to the cell count (see: Subculturing Fibroblast Cultures).
3. When subculturing a newly received fibroblast culture, the correct passage number must be determined. If the passage number is noted on the submission sheet or flask, the subcultured flasks should receive the next consecutive passage number.

3. How is a fibroblast cell line subcultured?

Supplies

• 0.53 mM EDTA in HBSS
• 0.04% trypsin/0.53 mM EDTA in HBSS
• Fibroblast Growth medium

Procedure

1. Prepare appropriate volumes of growth medium, EDTA, trypsin and "stop medium" (growth medium with FBS) flasks according to chart.
2. Dispense growth medium into flasks.

1. Remove medium by aspiration.
2. Add EDTA solution to the flask without dislodging the cell sheet and lay the flask cell side down. Cells should be watched closely through an inverted microscope for up to 10 minutes. If the cells begin to round or lift off the flask, the EDTA solution should be removed immediately.
3. Replace the EDTA solution with the EDTA/trypsin solution. Incubate the flasks at 37C for 4 to 7 minutes. Examine the flasks microscopically to make sure the cells begin to round up. The cells should have lifted off the surface after seven minutes. If the cells do not become detached after seven minutes, incubate an additional 1 to 2 minutes.
4. Tighten cap and lightly tap the side of the flask to lift the remaining cells from the flask. Wash the sides of the flask with growth medium (Stop Medium) to inactivate the trypsin. Gently mix cells and medium. Remove an aliquot for a cell count.
5. Seed the flasks according to the following chart:

1. Place flasks in 37C, 5% CO2 incubator, loosen caps if not vented. Check the cultures after a few hours for cell attachment and pH. The time between subcultures will depend on the incubation temperature, cell line, and serum and medium. The majority of mammalian cell lines require subculturing every 3-7 days. If the duration is longer than 5 days, change the culture medium every 3-4 days.

4. How are fibroblast cultures frozen for cryogenic storage?

Supplies

• 0.53mM EDTA in HBSS
• 0.04% trypsin/0.53 mM EDTA in HBSS
• Fibroblast Growth medium
• Fibroblast Freeze medium (growth medium with 10% glycerol or 5% DMSO)

Procedure

1. If cells are to be frozen in 10% glycerol, complete freeze medium may be kept at room temperature until used. Freeze medium prepared with DMSO should be kept refrigerated until used.
2. Each flask that is to be pooled for the freeze (freeze pool) should be examined microscopically for contamination and any unusual growth pattern. One flask should be maintained as a "backup" flask until the viability of the freeze can be checked.
3. Aspirate the growth medium from each flask. Add EDTA to each flask without dislodging cells and incubate at room temperature for 10 minutes. If cells begin to round or the edges of the cell sheet constrict, remove EDTA immediately.
4. Replace the EDTA solution with the EDTA/trypsin. Incubate the flasks at 37C for 4 to 7 minutes. Examine the flasks microscopically to make sure the cells begin to round. The cells should have lifted after seven minutes. If the cells do not become detached after seven minutes, incubate an additional 1 to 2 minutes.
5. Once the cells have lifted, add an equal or greater amount of growth medium to each flask to inactivate the trypsin. Gently triturate and then transfer cell suspension from all flasks and pool cells in a centrifuge bottle. Maintain the centrifuge bottle in ice while pooling flasks.
6. Remove an aliquot of the freeze pool, count the cells and calculate the total viable cells in the freeze pool. Centrifuge the freeze pool at 60-100 x g for 10 minutes at 8-10C.
7. Remove the supernatant and resuspend the cell pellet using gentle trituration in freeze medium at a final concentration of at least 5 x 10⁵ viable cells per ml.
8. Distribute one-ml aliquots of the cell suspension into glass ampoules or plastic cryovials.
9. Seal glass ampules using an oxygen-propane flame. Check each glass ampule for pinholes or glass bubbles formed during sealing by immersion in a methylene blue/ethanol solution at 4C.
10. Freeze the ampules or cryovials at a rate of 1C per minute.
11. Frozen cell stocks are stored in the liquid nitrogen tanks. Glass ampules are submerged in liquid; plastic cryovials are stored in the vapor phase.
12. One ampule or cryovial from every freeze is recovered and cultured to check for viability and sterility.

5. How should fibroblast cell cultures be recovered from cryogenic storage?

Procedure

1. Prepare appropriate recovery medium (see Shipping Sheets for individual cell line).
2. Remove one ampule or cryovial from frozen storage and place immediately in a 37C water bath and agitate vigorously.
3. Once completely thawed, wipe ampule or cryovial with a 70% alcohol sponge. Score the neck of a glass ampule and open utilizing an ampule opener.
4. Remove the contents of the ampule or cryovial using a sterile transfer pipette and place in a T25 tissue culture flask containing 5 ml of the appropriate fresh growth medium for fibroblast cultures.
5. If a cell count is required, mix the contents of the flask gently with a 1 ml pipette and remove 0.2 ml for a 1:5 diluted cell count. Place the flask in the 37C incubator lying cell surface down. Gently swirl the flask to distribute the cell suspension evenly over the flask surface. Adjust the cap to allow appropriate gas exchange (depending on buffering system of the medium). Fibroblast cultures should be refed with fresh medium the day after recovery.
6. Some cell lines recover better if all traces of cryoprotectant are removed by washing and centrifugation. Transfer the contents of ampule or cryovial to a 15-ml centrifuge tube with 3 ml of growth medium. Centrifuge for 5 min at 60-100xg and 10C. Remove supernatant, resuspend pellet, and transfer to a T25 flask with a final volume of 5 ml.
7. Culture as described for subculturing fibroblasts. If cells fail to proliferate after 1-2 weeks, expand the backup flask for a second freeze.