Name: Establishing Primary Adult Fibroblast Cultures From Rodents (Video) | JoVE Journal
Uploaded: 2010-10-05T17:00:00.000+00:00
Duration: 11 min 43 s
Description: Learn to isolate primary fibroblasts from rodent skin and lung tissue using collagenase digestion and culture methods. This protocol yields high cell numbers for comparative biology and cell banking research.

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5 minutes

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.

1. Before Starting
<ol>
 <li> Sterilize scissors and forceps with 70% ethanol.</li>
 <li> Place small magnetic stirrer inside a 30 mL beaker, cover with two layers of foil, and autoclave.</li>
 <li> Prepare a 28 Wunsch units/mL stock solution of Liberase Blendzyme 3 in sterile water. Make 0.5 mL aliquots and freeze at -20°C. Thaw a new aliquot before every use. The solution may appear cloudy after thawing. Vortex the solution until it becomes clear.</li>
 <li> Warm up the cell culture media.</li>
</ol>
2. Animal Sample Preparation
Caution: wild animals may contain pathogens such as a rabies virus. Always be aware of open sharps.
<ol>
 <li> Euthanize the animal and place the carcass at 4°C. It is best to use the carcass immediately, however, if this is not practical such as if the animals are collected in the field, the carcasses may be used within 24 hours. After 24 hours the cell yield declines.</li>
 <li> Dissect the animal in the surgical suite or in chemical hood (not in tissue culture hood).</li>
 <li> Underarm area is a convenient site to collect skin samples as underarm skin is thinner, contains less fat, and the fur is less dense. Clean the incision site with 70% ethanol. Make sure the fur is soaked with ethanol. </li>
 <li> Shave the fur around the incision site with a sharp scalpel. Shave a larger area than the desired incision site. Try to minimize cuts to the skin. Spray the area with 70% ethanol and let it dry.</li>
 <li> To collect a skin sample excise a fragment of approximately 1 cm2. Pinch the skin with tissue forceps, and cut with scissors. Try not to cut the fat layer with the skin, as fat interferes with collagenase digestion. To collect the lung samples, wash the chest area with 70% ethanol, and open the skin on the chest with scissors by making a T-shape incision and pulling apart the flaps of the skin. Wash the opened muscle area with 70% ethanol and let dry. Cut the rib cage using sterile forceps and scissors (use bone cutters for larger animals). Use sterile techniques to avoid contaminating internal organs. Do not touch the internal organs with instruments that touched the animal fur. Cut lung fragments of approximately 1 cm2 using sterile forceps and scissors.</li>
 <li> Place tissue fragments in 50 mL tubes with sterile PBS to avoid drying.</li>
 <li> Wash the outside of the 50 mL tubes with tissue fragments with ethanol, and take them to the tissue culture hood.</li>
 <li> Properly dispose of animal carcass.</li>
</ol>
3. Extracting Cells
<ol>
 <li> Transfer the tissue fragments into a 10 cm tissue culture dish using sterile scalpel. Do not transfer too much PBS with the sample.</li>
 <li> Cut the tissue into ~1 mm pieces using two scalpels. Use two blades using a scissor action starting from the center and pulling apart. Keep the tissue balled up, do not cut piece by piece. When the cutting is sufficient the skin resembles putty, it will not separate into pieces but will stretch thin. The lung tissue is easier to cut, and it will separate into tiny pieces.</li>
 <li> Using a scalpel, transfer the cut tissue into sterile 30 mL beaker with sterile stir bar. Wash the plate used for cutting tissue with 10 mL of DMEM/F12 media with 0.14 Wunsch units/mL Liberase Blendzyme 3, and 1X antibiotic/antimycotic, and add the solution to the 30 mL beaker with tissue fragments.</li>
 <li> Cover the beaker with sterile foil, and incubate at 37°C, stirring slowly, for 30 to 90 minutes. The length of the incubation depends on the species and tissue type. Take care not to over-digest the tissue. Best yields are obtained when tissue fragments are still present at the end of the digestion. Skin takes longer to digest than the lung. Skin from large animals takes longer to digest. Check the digestion after 30 min, and then every 10 min. When the skin digestion is complete, the media becomes cloudy and skin fragments separate from each other, and the edges of the pieces become "fuzzy". Lung digestion should be stopped when lung fragments change color from red to white and start forming sticky fibers.</li>
 <li> Pipet the solution containing tissue fragments up and down to break the clumps. Transfer the solution to sterile 50 mL tube. If the fragments move easily through the 10 mL pipette cutting and digestion was done well. Rinse the beaker 3 times with 10 mL of warm DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic and add the media to the 50 mL tube with tissue fragments. Close the 50 mL tube and mix by inversion a few times. The FBS in the media will stop Liberase digestion.</li>
 <li> Spin at 524 g in a swinging bucket tissue culture centrifuge for 5 min. Remove the supernatant. Resuspend the pellet in 10 mL of warm DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic. Pipet the suspension with maximum force to break the tissue pieces.</li>
 <li> Add another 30 mL of DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic, mix and centrifuge at 524 g in a swinging bucket tissue culture centrifuge for 5 min. Repeat one more time to remove the traces of Liberase.</li>
 <li> Resuspend the pellet in 10 mL of DMEM/F12 media with 15% FBS, 1X antibiotic/antimycotic and transfer to a 10 cm tissue culture dish and place in a tissue culture incubator at 37°C, 5%CO2, 3%O2.</li>
 <li> Check the plates every day for fibroblasts and media color. If the isolation was successful, fibroblasts crawl out of the tissue fragments and attach to the plate (Figure 1). The fibroblast start to exit tissue fragments within 2-5 days.</li>
 <li> If the media changes color to yellow, this indicates potential contamination or overcrowding of cells. Examine the plates under the microscope at high magnification. If bacteria, fungi, or worms are present discard the plates (this is rarely an issue with laboratory animals, however may occur when the samples are collected from the wild). If no contamination is present, but the media changed color, this is caused by either too many cells or too many tissue fragments placed in the same dish. If more than 60% of the plate is covered by attached fibroblasts, change the media on the plate and transfer the tissue fragments to a new plate with the new media. If not many fibroblasts attached to the plate, change the media and split the tissue pieces to a 2-4 plates.</li>
 <li> After 7 days, if media had not been changed earlier, change the media and transfer the tissue fragments to a new plate with new media. </li>
 <li> Incubate the cells and tissue fragments for an additional 7 days. By day 14 all viable fibroblasts have exited the tissue fragments.</li>
 <li> After 14 days from the beginning of the cell isolation, discard the old media and tissue fragments, harvest the cells and plate them on a new plate at 5x105 cells/plate EMEM with 15% FBS, 1X Penicillin/Streptomycin, non essential amino acids, and sodium pyruvate. EMEM media will support growth of fibroblasts only and other cell types will die or stop proliferating.</li>
 <li> After the cells reach 80-90% confluence, freeze an aliquot of cells for future use.</li>
 <li> Continue culturing the cells by splitting them at 5x105 cells/plate when the cells reach 80-90% confluence.</li>
</ol>
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 5x105 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).
<img src="/files/ftp_upload/2033/2033fig1.jpg" alt="Mouse tissue microscopy comparison at 4x and 10x magnification, cell structure analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
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.
<img src="/files/ftp_upload/2033/2033fig2.jpg" alt="Cell culture under microscope; study of cell morphology, growth, and differentiation processes." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Mouse lung fibroblasts. 
<img src="/files/ftp_upload/2033/2033fig3.jpg" alt="Cell culture comparison, microscopy image; MSF vs MLF, growing cells, experiment, biological study." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. A field of mouse fibroblasts containing cells in M-stage, photographed at 10X magnification. 
<img src="/files/ftp_upload/2033/2033fig4.jpg" alt="Confluent cell cultures comparison; microscopic image; MSF vs. MLF cells; cell growth analysis." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. A confluent plate of mouse fibroblasts, photographed at 10X magnification.

<ol><li>Seluanov, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distinct+tumor+suppressor+mechanisms+evolve+in+rodent+species+that+differ+in+size+and+lifespan.+Aging+%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging .</a> Cell. 7, 813-823 (2008).</li>
<li>Parrinello, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+sensitivity+severely+limits+the+replicative+lifespan+of+murine+fibroblasts%5BTitle%5D)+AND+%22Nat+Cell+Biol%22%5BJournal%5D)">Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.</a> Nat Cell Biol. 5, 741-747 (2003).</li>
</ol>

No conflicts of interest declared.

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 succe...

<table><tbody><tr><td>DMEM/F12 media</td><td>Invitrogen</td><td>11330-032</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Qualified</td><td>Invitrogen</td><td>01437-036</td><td>The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.</td></tr><tr><td>Antibiotic/Antimycotic</td><td>Invitrogen</td><td>15420-096</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&lt;strong&gt;Liberase TM Research Grade&lt;/strong&gt;</td><td>Roche</td><td>&lt;strong&gt;05401127001&lt;/strong&gt;</td><td>Replacement enzyme.</td></tr><tr><td colspan="4">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.</td></tr><tr><td>EMEM media</td><td>ATCC</td><td>30-203</td><td>The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.</td></tr><tr><td>Feathered #21 disposable, sterile scalpel</td><td>Multiple suppliers</td><td></td><td></td></tr><tr><td>Three Gas Control incubator</td><td>Forma or Heraeus</td><td></td><td></td></tr></tbody></table>

establishing primary adult fibroblast cultures from rodents

Establishing Primary Adult Fibroblast Cultures From 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.

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 ...

Isolating viable fibroblasts from animal skin and lung tissue starts with dissecting skin and lung fragments from an animal carcass. Using sterile technique, the tissue is cut into small fragments and digested with collagenase. The digested tissue fragments are then incubated with rich culture media until fibroblasts exit the tissue.Finally, the tissue pieces are removed and the rich media is replaced with fibroblast growth media. After splitting the cells, aliquots of cells are frozen for future use. Ultimately, a one centimeter squared skin or lung tissue fragment yields 10 to the seventh passage to fibroblasts.And the main advantage of this technique over other methods that do not involve enzymatic digestion of tissue is that it yields high numbers of cells from a variety of animal specimens. This method will help researchers in comparative biology field to efficiently isolate fire bloods from the wide variety of mammalian species. A visual demonstration of this method is critical as the tissue digestion and mincing steps are difficult to learn because they're intuitive and vary depending on the tissue and animal specimen used Before beginning this procedure, sterilized scissors and forceps with 70%ethanol autoclave, a 30 milliliter beaker holding a small magnetic stir inside and thaw a new aliquot of the liase Zyme three solution.Also warm up the cell culture media. When working with wild animals, remember that they may contain pathogens such as the rabies virus. Work carefully and always be aware of open sharps.To begin this procedure, euthanize the animal and place the carcass at four degrees Celsius. It is best to use the carcass immediately or remove the tissue samples within 24 hours after which the cell yield declines. To dissect the animal work in a surgical suite or in a chemical hood, but not in a tissue.Culture hood collect skin samples from the underarm area since the underarm skin is thinner, contains less fat, and the further is less dense. First, clean the incision site with 70%ethanol. Make sure that the fur is soaked with ethanol.Then using a sharp scalpel, shave the fur around the incision site, shave a larger area than the desired incision site. Try to minimize cuts to the skin. After removing the fur, spray the area with 70%ethanol and let it dry.To collect a skin sample, excise a fragment of approximately one centimeter squared. To do so, pinch the skin with tissue forceps and cut with scissors. Try not to cut the fat layer with the skin as fat interferes with subsequent collagenase digestion.To avoid drying, place the tissue fragments in 50 milliliter tubes containing sterile PBS. Next, collect the lung samples. First, wash the chest area with 70%ethanol.Then using scissors. Make a T-shaped incision on the chest skin and pull apart the skin flaps. Wash the opened muscle area with 70%ethanol and let dry.Using sterile forceps and scissors, cut open the rib cage to expose the lungs. To avoid contaminating internal organs, use sterile techniques. For example, do not touch the internal organs with instruments that touched the animal fur.Using sterile forceps and scissors, cut out lung fragments approximately one centimeter squared in size. Place the tissue fragments in 50 milliliter tubes with sterile PBS last properly dispose of the animal carcass. Use ethanol to wash the outside of the 50 milliliter tubes containing the skin and lung tissue fragments.Then transfer the tubes to the tissue culture hood. After obtaining the tissue samples, proceed to extract fibroblasts. First using a sterile scalpel, transfer the tissue fragments from the tubes into a 10 centimeter tissue culture dish.Do not transfer too much PBS with the sample using two scalpels. Cut the tissue into about one millimeter pieces. Use the two blades with the scissor action starting from the center and pulling apart.Make sure to keep the tissue ball up. Do not cut piece by piece. When the cutting is sufficient, the skin resembles putty.It does not separate into pieces but stretches thin. The lung tissue is easier to cut and it does separate into tiny pieces. When cutting is complete, use a scalpel to transfer the cut tissue into the sterile 30 milliliter beaker with the sterile stir bar inside.Wash the plate used for cutting tissue with 10 milliliters of D-M-E-M-F 12 media with 0.14. Once units per milliliter of liase Zyme three and one x antibiotic antimycotic, add the wash solution to the 30 milliliter beaker. Cover the beaker with sterile foil and incubate at 37 degrees Celsius s stirring slowly for 30 to 90 minutes.The length of the incubation depends on the species and tissue type. Take care not to over digest the tissue. Best yields are obtained when tissue fragments are still present at the end of the digestion.Skin takes longer to digest than the lung, and skin from large animals takes longer to digest. Check the digestion every 30 minutes and then every 10 minutes. When the skin digestion is complete, the media becomes cloudy.Skin fragments separate from each other and the edges of the pieces become fuzzy looking. Lung digestion is stopped when lung fragments change color from red to white and start forming sticky fibers pipette the solution containing the tissue fragments up and down to break up the clumps. If the fragments move easily through the 10 milliliter pipette, it is an indication that cutting and digestion were done.Well transfer the solution to a sterile 50 milliliter tube. Rinse the beaker three times with 10 milliliters of warm D-M-E-M-F 12 media with 15%FBS and one x antibiotic antimycotic and add to the 50 milliliter tube with tissue fragments. Close the 50 milliliter tube and mix by inversion a few times.The FBS in the media stops the liase digestion. Next, wash the cells spin at 524 Gs in a swinging bucket tissue culture centrifuge at room temperature for five minutes, remove the supernatant and resuspend the pellet. In 10 milliliters of warm D-M-E-M-F 12 media with 15%FPS one X antibiotic Antimycotic pipette the suspension with maximum force.To break the tissue pieces, add another 30 milliliters of media and repeat the wash. Wash one more time for a total of three washes to remove the traces of lipase. After washing the cells resuspend the pellet in 10 milliliters of D-M-E-M-F 12 media with 15%FBS one X antibiotic antimycotic transfer the cells to a 10 centimeter tissue culture dish place in a tissue culture incubator.After extracting the fibroblasts, allow them to exit the tissue fragments to establish the culture. Check the plates every day for fibroblasts and media color. If the isolation was successful within two to five days, the fibroblasts start to exit.The tissue fragments, the fibroblasts crawl out and attach to the plate. If the media color changes to yellow, this indicates either potential contamination or overcrowding of cells. Examine the plates under the microscope at high magnification.If bacteria, fungi, or worms are present, discard the plates. This is really an issue with laboratory animals. However, it may occur when the samples are collected from the wild.If no contamination is present, check for overcrowding of cells. If more than 60%of the plate is covered with attached fibroblasts, change the media on the plate and transfer the tissue fragments to a new plate. With the new media, if not many fibroblasts attached to the plate, there may be too many tissue fragments placed in the same dish.In this case, change the media and split the tissue pieces to two to four plates after seven days. If the medium had not been changed earlier, change it and transfer the tissue fragments to a new plate. With new media incubate the cells in the tissue for an additional seven days.By by day 14, all viable fibroblasts have exited the tissue fragments. At this stage, discard the old media and tissue fragments. Harvest the cells and plate them on a new plate at five times 10 to the fifth cells per plate in EME and media with 15%FPS one X, penicillin, streptomycin, non-essential amino acids and sodium pyruvate, EME and media will support growth of fibroblasts only and other cell types die or stop proliferating.Let the cells grow until they reach 80 to 90%Confluence freeze an aliquot of cells for future use. Continue culturing the cells by splitting them at five times 10 to the fifth cells per plate. When the cells reach 80 to 90%confluence, normal fibroblasts are large cells with prominent protrusions or lamo.Lip podia fibroblasts grow in a monolayer. A healthy growing culture contains one to 10%cells in M stage recognized as rounded cells elevated over the surface of the plate, but not detached from the plate. Typically, a 10 centimeter dish seated with five times 10 to the fifth cells becomes confluent in three to four days when cells fill a plate, the arrest proliferation in G one stage, a typical 100%confluent plate of fibroblasts contains a tightly packed layer of cells.At 90%confluence. The cells are ready for splitting. Cells can also be maintained on an rested confluent plate for extended periods of time with regular media changes once or twice a week After its development.This technique paved the way for researchers in the field of cell biology to bank and study primary cells from a variety of mammalian species. After wishing this video, you should have a good understanding how to isolate the primary fibrous from animal tissues. Don't forget that specimens from wild animals may contain pathogens.Biosafety level two precautions should always be taken while doing this procedure.

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