Developmental Biology
A subscription to JoVE is required to view this content.
You will only be able to see the first 2 minutes.
The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.
If that doesn't help, please let us know.
Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues
Chapters
Summary January 10th, 2016
We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.
Transcript
The overall goal of this protocol is to establish primary fibroblast cultures from the ears and tail of mice. This method can help answer key questions in the cell biology field, such as the difference between healthy and cancer cells. The main advantage of this study is that we can generate fibroblast from ears that were stored in medium at room temperature for up to 10 days before isolating the cells.
Though this method can provide insight into mouse biology, it can also be applied to other mammalian animal organisms such as rats and rabbits. Before collecting the tissues, load 10 centimeter culture dishes with 10 milliliters of complete medium. Then, after the mouse has been euthanized, proceed with using sterilized instruments to collect the tissue samples.
Collect ear tissues with about a one centimeter radius, and collect about five centimeters of tail tissue. Transfer the tissues to a conical tube loaded with 40 milliliters of 70%ethanol. Let them soak for five minutes, and then air-dry them in an open 10 centimeter culture dish for five minutes in a culture hood.
After the tissues dry, transfer them onto the media-loaded plate. Now, snip the hair off all the tissues using scissors, and then cut the tissues into pieces smaller than three millimeters. Transfer the diced tissue to cryotube vials, and then fill the vials with collagenase D pronase solution, to the 1.8 milliliter mark.
Now incubate the tissues at 37 degrees Celsius, with shaking at 200 rpm for 90 minutes. At the hood, load a dish with 10 milliliters of complete medium. Over the dish, place a 70 micron cell strainer, and after the incubation, load the tissue digestion into the strainer.
Now, grind the tissues in the strainer using a plunger. Do this for at least five minutes. Occasionally shake the strainer into the medium to wash out dissociated cells.
It is important that the digested ear and tail tissues are ground out well in order to cull out a good number of cells. Also be sure to shake the cell strainer after grinding the tissues. This will wash out additional cells from the strainer.
Next, collect the cell suspension in a 15 milliliter conical tube. Then, rinse the strainer with 10 milliliters of medium, and add the rinse solution to the collection. Now, wash the cells.
Firstly, spin the cells down in a refrigerated centrifuge. Secondly, remove the supernatant. And thirdly, suspend the cells in 10 milliliters of complete medium.
Repeat the wash once more, and finish the procedure with the cells in complete medium, ready for culturing. To culture the cells, first, transfer the cell suspensions to 10 milliliter culture dishes. Then, add 10 microliters of amphrotericin B solution to each culture.
Now, incubate the cells for three days. On the third day, replace the medium with fresh complete medium with amphrotericin B.Soon after, the cultures will reach 70%confluency. At this point, remove the medium and rinse the culture with five milliliters of 1X PBS.
Then, replace the PBS with two milliliters of 1X trypsin EDTA solution, and incubate the cells for five minutes. After the brief incubation, gently tap the dishes and add six milliliters of fresh complete medium to each dish. Now, pipette the released cells from the dish to two 15 milliliter conical tubes.
Then, spin the tubes down for five minutes at 450 Gs in a refrigerated centrifuge. Resuspend the pellets in five milliliters of complete medium and measure the cell density of the suspensions. Now, feed the cells to 10 centimeter dishes at 200, 000 per dish.
Incubate these cells for three to four days until they reach 70%confluency, and then repeat the process of collecting, and replating the cells at a specific density. They can be grown this way for up to 25 days. Three days after the cell extraction, there was significant debris in the culture before exchanging the medium.
The cell debris is highlighted with white arrows. The scale bar represents 100 microns. New medium significantly reduced the cell debris.
Three day old cultures produced from tissue that had been stored at room temperature for 10 days demonstrated that the fibroblasts are fairly hardy. From such tissues, 70 to 80%fibroblast confluency was achieved after five to six days. The cells were labelled with a marker for fibroblasts, vimentin, seen in red.
DAPI was used to label the nuclei, which is seen in blue. The scale bar is 10 microns. The uniform staining suggests that the fibroblast culture is pure.
The described protocol routinely got this result. After watching this video, you should have gotten this ending of how to quickly and reliably establish primary fibroblast cultures. While attempting this procedure, extra attention should be given during the extraction of fibroblasts from the tissues, as insufficient grinding of the tissue will result in lower cell numbers.
Related Videos
You might already have access to this content!
Please enter your Institution or Company email below to check.
has access to
Please create a free JoVE account to get access
Login to access JoVE
Please login to your JoVE account to get access
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Please enter your email address so we may send you a link to reset your password.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Your JoVE Unlimited Free Trial
Fill the form to request your free trial.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Thank You!
A JoVE representative will be in touch with you shortly.
Thank You!
You have already requested a trial and a JoVE representative will be in touch with you shortly. If you need immediate assistance, please email us at subscriptions@jove.com.
Thank You!
Please enjoy a free 2-hour trial. In order to begin, please login.
Thank You!
You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free.
To get started, a verification email has been sent to email@institution.com. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.