Aug 20, 2025

Public workspaceFibroblast culture from rodent/lagomorph ear

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvmb3kog3p/v1
  • Ashley Seifert1,2,
  • Ebenezer Aryee1,2,
  • Ajoy Aloysius1,2
  • 1University of Kentucky;
  • 2Department of Biology
Ashley Seifert
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvmb3kog3p/v1
Protocol Citation: Ashley Seifert, Ebenezer Aryee, Ajoy Aloysius 2025. Fibroblast culture from rodent/lagomorph ear. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmb3kog3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 14, 2025
Last Modified: August 22, 2025
Protocol Integer ID: 224712
Keywords: ASAPCRN, fibroblast, acomys, mouse, rat, rabbit, ear, fibroblast culture from rodent, primary fibroblasts from the ear pinnae, fetal fibroblasts from the ear bud, fibroblast culture, lagomorph ear this protocol, fetal fibroblast, fibroblast, lagomorph ear, head skin of rodent, primary fibroblast, seifert lab, ear pinnae, rodent, involving animal, rattus, lagomorph, ear bud, head skin, institutional animal care
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020495
NIH
Grant ID: R01AR070313
Abstract
This protocol describes the procedure for isolating and culturing either primary fibroblasts from the ear pinnae or fetal fibroblasts from the ear buds and head skin of rodents and lagomorphs, including Acomys, Mus, Rattus, and Oryctolagus, used in the Seifert Lab.

Note that any protocol involving animals should be approved by your Institutional Animal Care and Use Committee (IACUC) before use.
Protocol materials
ReagentIsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405
Reagent4 mm biopsy punchSklarCatalog #96-1146
Reagent1.5 mL Eppendorf tubes
ReagentFBS (HyClone)
ReagentAntibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062
ReagentDMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV
ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651
ReagentFalcon® 70 µm cell strainerCorningCatalog #352350
Troubleshooting
Anaesthetize/euthanize animal appropriately
Note
For Acomys and Mus, we anaesthetize with Concentration3 % (v/v) ReagentIsoThesia (Isoflurane) SolutionHenry Schein Animal HealthCatalog #029405 at Amount1 µL oxygen flow rate


Primary fibroblasts
Follow the following procedure for primary fibroblasts from adult animals
Harvest tissue from the center of the left and right ear pinna with a Reagent4 mm biopsy punchSklarCatalog #96-1146

Place tissue from both ears for each animal into a single Reagent1.5 mL Eppendorf tubes

Enzymatically dissociate the tissue first with a with 1:1 trypsin (0.25%)-dispase mix for 1 hour at 37oC. Dice up tissue with a razor and transfer tissue into a new eppendorf tube with 1X HBSS. Spin at 2000 rpm for 7 minutes. After that take out the HBSS and add collagenase IV (2.5 mg/ml) and incubate at 37oC for 1 hour.
Remove the enzyme, add 1X HBSS and mechanically dissociate cells by filtering through a ReagentFalcon® 70 µm cell strainerCorningCatalog #352350 , with the gentle nudge of a syringe (3-10ml) plunger.

Pellet the suspension by spinning at 2000g x 7 minutes at 26oC.
Resuspend pellet in ReagentDMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV supplemented with 10% ReagentFBS (HyClone) and 1% ReagentAntibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062

Culture at Temperature37 °C under 3% O2 and 5% CO2 in ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651

Passage at ~80% confluency. Count cells using a hemocytometer and seed 300,000-500,000 cells in a fresh ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651
Embryonic fibroblasts
Follow the following procedure for fetal fibroblasts from embryos from timed-pregnant females after euthanasia
Dissect the ear buds and head skin from the embryo

Place tissue into Reagent1.5 mL Eppendorf tubes

Enzymatically dissociate cells with 0.25% trypsin for Duration00:10:00 at Temperature37 °C

Neutralize the enzyme in complete growth medium containing serum
Mechanically dissociate the tissue using a 20 gauge needle to obtain a single-cell suspension
Culture cells in ReagentDMEM and Hams F-12 50/50Fisher ScientificCatalog #MT10090CV supplemented with 10% ReagentFBS (HyClone) and 1% ReagentAntibiotic/Antimycotic (100X)Thermo Fisher ScientificCatalog #15240062 at Temperature37 °C under 3% O2 and 5% CO2 in ReagentTissue culture dish (10 cm)Merck MilliporeSigma (Sigma-Aldrich)Catalog #Z707651