This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.
The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.
1. Before Starting
2. Animal Sample Preparation
Caution: wild animals may contain pathogens such as a rabies virus. Always be aware of open sharps.
3. Extracting Cells
4. Representative Results
Normal fibroblasts are large cells with prominent protrusions (lamellipodia) (Figure 2). Fibroblasts grow in a monolayer. A healthy growing culture contains 1-10% cells in M stage, recognized as rounded up cells elevated over the surface of the plate, but not detached from the plate (Figure 3). Typically, a 10 cm dish seeded with 5×105 cells becomes confluent in 3-4 days. The doubling time varies greatly between species, and may be greater for some of the long-lived rodent species1. When cells fill a plate they arrest proliferation in G1 stage. Typical confluent plate of fibroblasts contains a tightly packed layer of cells (Figure 4). When the cells reach 90% confluence they are ready for splitting. If desired, the cells can be maintained on an arrested confluent plate for extended periods of time with regular media changes (once or twice a week).
Figure 1. Fibroblast isolation from mouse skin (A) and lung (B). The media was changed to remove unattached cells and debris prior to taking the pictures on Day 7.
Figure 2. Mouse lung fibroblasts.
Figure 3. A field of mouse fibroblasts containing cells in M-stage, photographed at 10X magnification.
Figure 4. A confluent plate of mouse fibroblasts, photographed at 10X magnification.
Normal primary fibroblasts provide an excellent alternative to established cancer cell lines in biological research. An important advantage of fibroblasts is that they do not carry mutations in oncogenes and tumor suppressors and maintain intact cell cycle checkpoints. This makes normal fibroblasts a preferred system for the studies of cell cycle regulation, DNA repair, and apoptosis. The protocol described here provides a simple recipe for isolation and maintenance of primary fibroblasts. This protocol was used to successfully isolate fibroblasts from over 20 species of rodents.
It is important to maintain sterility at all times when working with cell cultures to avoid contamination. If the laboratory also cultures aggressive cancer cell lines such as HeLa cells, fibroblasts should be handled separately from cancer cells. It is preferred to have a designated hood and incubator for primary cultures.
It is preferable to maintain primary cell cultures at physiological oxygen concentration of 3%. Atmospheric (20%) oxygen shortens lifespan of cultures and increases oxidative stress. Mouse fibroblasts are sensitive to oxidative stress and will senesce or enter crisis within ~14 population doublings when maintained at 20% (atmospheric) oxygen. Mouse fibroblasts can be maintained indefinitely at 3% oxygen2.
Always keep record of the number of population doubling of the cultures. Large species (body mass above 8,000 g) are likely to exhibit replicative senescence, and will have a limited lifespan in culture, even at 3% oxygen1. Cells from smaller species, such as mice and rats, will proliferate indefinitely at 3% oxygen, but may eventually become aneuploid. If possible, begin the experiments using cells at low population doubling.
The authors have nothing to disclose.
We thank Dr. Steven Austad for providing us with the first version of this protocol. This work was supported by grants from the NIH and the Ellison Medical Foundation to V.G. and A.S.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
DMEM/F12 media | Invitrogen | 11330-032 | ||
Fetal Bovine Serum (FBS), Qualified | Invitrogen | 01437-036 | The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts. | |
Antibiotic/Antimycotic | Invitrogen | 15420-096 | ||
Penicillin/Streptomycin | Invitrogen | 15140-122 | ||
Liberase TM Research Grade | Roche | 05401127001 | Replacement enzyme. | |
A note from the authors: Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 – 10 mg, Cat. no. 05401127001 – 100mg) instead. We have switched to this enzyme successfully with no issues. | ||||
EMEM media | ATCC | 30-203 | The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate. | |
Feathered #21 disposable, sterile scalpel | Multiple suppliers | |||
Three Gas Control incubator | Forma or Heraeus |