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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

Introduction

Primary cells are derived from living tissue and cultured under in vitro conditions. It is generally assumed that primary cells more closely resemble the physiological state and genetic background of the tissue from which they originated than immortalized or tumor cell lines1. For that reason, primary cells represent a useful model for studying biological questions2,3. However, unlike established cell lines that grow indefinitely, primary cells eventually undergo senescence in culture and need to be frequently re-established.

Commonly used primary cells include fibroblasts, epithelial cells, endothelial cells,....

Protocol

Mice were housed in pathogen-free conditions in compliance with the institutional guidelines until euthanization (The Institutional Animal Care and Use Committee (IACUC) guidelines at the National University of Singapore and the National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines).

1. Mice

  1. Order one mouse of the appropriate genetic background. This protocol is based on tissue derived from one C57BL/6 mouse.

2. Preparation of Complete Medium

Representative Results

Extraction of fibroblasts from tissue results in a significant amount of tissue debris (Figure 1). In contrast to tissue debris, fibroblasts adhere to tissue culture plastic surfaces between day 1 and 3 of culture. The medium of fibroblast cultures can be safely changed on day 3 of culture, which should significantly decrease the levels of debris present in the culture (Figure 2). Fibroblasts display an elongated morphology and a clearly visible cytoplasm (Figures 1 and 2

Discussion

Here we provide a simple, inexpensive and fast experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The extraction should result in adherent and rapidly dividing fibroblasts within 3 days post-isolation of the tissue. An important limitation of primary cells is senescence, a permanent growth arrest15. Using the protocol, fibroblast cultures can be passaged for 5 to 6 times before fibroblasts become senescent, indicated by the flattening of cells, increase in size (2-3 ti.......

Acknowledgements

This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

....

Materials

NameCompanyCatalog NumberComments
RPMI-1640HyCloneSH30027.01
Fetal Calf SerumHyCloneSV30160.03
2-mercaptoethanolSigma-AldrichM3148
AsparagineSigma-AldrichA4159
GlutamineSigma-AldrichG8540
Penicillin/StreptomycinHyCloneSV30010
EthanolMerck Millipore107017Absolute for analysis
Collagenase DRoche Diagnostics11088866001From Clostridium histolyticum, lyophilized, non-sterile
Pronase proteaseMerck Millipore53702From Streptomyces griseus 
Tris buffer (pH 8)1st BASE1415Ultra pure grade
0.5M EDTA (pH 8)1st BASEBUF-1053Biotechnology grade
10X Phosphate Buffered Saline (PBS)1st BASEBUF-2040-10X4LUltra pure grade
Trypsin-EDTA solution 10XSigma-Aldrich59418C-100ML0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline
Amphotericin BSigma-AldrichA2492-20ml250 μg/ml in deionized water, sterile-filtered
ScissorsAesculap
ForcepAesculapAE-BD312R
0.2 μM syringe filterSartorius Stedim16534
70 μM cell strainerSPL93070
Syringe plungerTerumoSS+10L
Cryovial tubeNUNC368362
1.7 ml microcentrifuge tubeAxygenMCT-175-C
10 cm cell culture dishGreiner664160Cell culture treated dish 
15 ml conical bottom tubeGreiner188271
50 ml conical bottom tubeGreiner227261
Water bathGFL1002
CentrifugeEppendorf5810R
Incubation shakerSartorius StedimCertomat-BS1
Zeiss Axiovert 25 light microscopeCarl Zeiss AG

References

  1. Fitzpatrick, L. E., McDevitt, T. C. Cell-derived matrices for tissue engineering and regenerative medicine applications. Biomater Sci. 3 (1), 12-24 (2015).
  2. Elenbaas, B., et al. ....

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Primary FibroblastMouse EarMouse TailTissue CultureCell IsolationCollagenase DigestionCell StrainerCell SuspensionCell Washing

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