Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues
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.
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.
View the full transcript and gain access to thousands of scientific videos
Overview
This article presents a straightforward protocol for generating primary fibroblast cultures from the ears and tails of mice. The method allows for the use of tissues stored at room temperature for up to 10 days, facilitating research in cell biology.
Key Study Components
Area of Science
- Cell Biology
- Fibroblast Culture
- Mammalian Biology
Background
- Primary fibroblasts are essential for various biological studies.
- The ability to use stored tissues expands research possibilities.
- This method can be adapted for other mammalian species.
- Understanding fibroblast behavior aids in cancer research.
Purpose of Study
- To establish a reliable method for fibroblast culture from mouse tissues.
- To investigate differences between healthy and cancerous cells.
- To provide a protocol that does not require specialized animal training.
Methods Used
- Collection of ear and tail tissues from euthanized mice.
- Use of sterilized instruments for tissue collection.
- Preparation of culture dishes with complete medium.
- Isolation of fibroblasts from tissues stored at room temperature.
Main Results
- Successful generation of primary fibroblast cultures.
- Demonstrated viability of tissues stored for up to 10 days.
- Potential applications in studying mammalian cell biology.
- Insights into the differences between healthy and cancer cells.
Conclusions
- This protocol provides a simple approach to fibroblast culture.
- It opens avenues for research in various mammalian species.
- Further studies can leverage this method for cancer research.
Frequently Asked Questions
Executive Industry Relevance
Establishing primary fibroblast cultures from mouse ear and tail tissues enables biopharma teams to access physiologically relevant cell systems for early discovery and mechanistic studies. The protocol's tolerance for non-sterile, room-temperature-stored tissues increases operational flexibility and sample accessibility, supporting robust target validation and comparative biology. This capability strengthens predictive confidence at key inflection points in the discovery pipeline.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables direct comparison of healthy and diseased cell phenotypes for mechanistic de-risking.
- Supports functional validation of targets in primary, physiologically relevant systems.
- Facilitates hypothesis testing with minimal artifact from immortalized cell lines.
- Improves confidence in early-stage biological findings for portfolio triage.
Screening & Assay Development
- Provides a reproducible source of primary fibroblasts for assay standardization.
- Enables quantitative measurement of cellular responses in a controlled system.
- Supports development of robust, scalable assays for compound evaluation.
- Reduces variability by allowing use of tissues stored under flexible conditions.
Translational & Preclinical Research
- Aligns in vitro models more closely with in vivo biology for translational continuity.
- Facilitates biomarker discovery and validation in disease-relevant systems.
- Supports risk-adjusted advancement decisions by providing physiologically relevant data.
Pipeline & Workflow Integration
This protocol integrates at the interface of early discovery and assay development, providing a foundation for downstream screening and translational research.
- Discovery Biology: Enables hypothesis-driven interrogation of cellular mechanisms in primary systems.
- Screening: Delivers reproducible, quantitative outputs for assay readiness and compound profiling.
- Analytics: Supports measurement of cell density, confluency, and marker expression for comparative analysis.
- Translational Research: Bridges in vitro and in vivo studies by maintaining physiological relevance.
- Enterprise Reuse: Offers a standardized, adaptable protocol for repeated use across projects and teams.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in early-stage research.
- Operational Value: Enhances standardization, reproducibility, and scalability of primary cell culture workflows.
- Strategic Value: Improves go/no-go decision quality and capital efficiency by reducing late-stage biological risk.
- Portfolio Impact: Enables risk-adjusted prioritization and advancement of discovery programs.
Implementation Considerations
- Requires expertise in primary cell isolation and culture techniques.
- Needs access to standard tissue culture instrumentation and analytical tools.
- Demands cross-team standardization for consistent sample processing and data quality.
- Adaptable to other mammalian species with protocol adjustments as supported by the source.
- Cell yield and purity depend on thorough tissue processing and grinding steps.
