Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

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 questions^2,3. 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

1. Prepare complete medium by adding the following components to Roswell Park Memorial Institute (RPMI) 1640 medium: 10% fetal calf serum (FC....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI-1640	HyClone	SH30027.01
Fetal Calf Serum	HyClone	SV30160.03
2-mercaptoethanol	Sigma-Aldrich	M3148
Asparagine	Sigma-Aldrich	A4159
Glutamine	Sigma-Aldrich	G8540
Penicillin/Streptomycin	HyClone	SV30010
Ethanol	Merck Millipore	107017	Absolute for analysis
Collagenase D	Roche Diagnostics	11088866001	From Clostridium histolyticum, lyophilized, non-sterile
Pronase protease	Merck Millipore	53702	From Streptomyces griseus
Tris buffer (pH 8)	1st BASE	1415	Ultra pure grade
0.5M EDTA (pH 8)	1st BASE	BUF-1053	Biotechnology grade
10X Phosphate Buffered Saline (PBS)	1st BASE	BUF-2040-10X4L	Ultra pure grade
Trypsin-EDTA solution 10X	Sigma-Aldrich	59418C-100ML	0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline
Amphotericin B	Sigma-Aldrich	A2492-20ml	250 μg/ml in deionized water, sterile-filtered
Scissors	Aesculap
Forcep	Aesculap	AE-BD312R
0.2 μM syringe filter	Sartorius Stedim	16534
70 μM cell strainer	SPL	93070
Syringe plunger	Terumo	SS+10L
Cryovial tube	NUNC	368362
1.7 ml microcentrifuge tube	Axygen	MCT-175-C
10 cm cell culture dish	Greiner	664160	Cell culture treated dish
15 ml conical bottom tube	Greiner	188271
50 ml conical bottom tube	Greiner	227261
Water bath	GFL	1002
Centrifuge	Eppendorf	5810R
Incubation shaker	Sartorius Stedim		Certomat-BS1
Zeiss Axiovert 25 light microscope	Carl Zeiss AG