Name: generating primary fibroblast cultures from mouse ear and tail tissues
Uploaded: 2016-01-10T13:00:00
Description: Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

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

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
<ol>
	<li>Order one mouse of the appropriate genetic background. This protocol is based on tissue derived from one C57BL/6 mouse.</li>
</ol>
2. Preparation of Complete Medium</p...

<ol>
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C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. 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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...

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, T cells, B cells, bone marrow-derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC). Fibroblasts are often utilized as primary cell culture model. They offer key advantages over other primary cells. Cell cultures are easily established, readily maintained and require no purification of cells prior to culture. They have rapid initial proliferation and no requirement for specialized medium or activation protocols. Fibroblasts can be efficiently transfected using biological, chemical, and physical protocols4,5. There is a possibility to store ears for up to 10 days at RT prior to establishing cell cultures. Fibroblast cultures are conducive to visualization of cytoplasmic processes and suitable for reprogramming into induced pluripotent stem (iPS) cells6.
Fibroblasts are important cells of the connective tissue that secrete collagen proteins and extracellular matrix7. They provide the structural framework in many tissues8 and play an essential role in wound healing and tissue repair9,10.
Here, we describe a simple and quick (&#60;4 hr) protocol to establish fibroblast cultures from ears and tails of mice11. The protocol requires minimal mouse experience to harvest the tissues (in contrast to other protocols12,13) and can be used to establish cultures from ears stored in medium at RT for up to 10 days.

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SH30027.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asparagine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">107017</td>
			<td>Absolute for analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:140px;">Roche Diagnostics</td>
			<td style="width:119px;">11088866001</td>
			<td>From Clostridium histolyticum, lyophilized, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pronase protease</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">53702</td>
			<td>From Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">1415</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5M EDTA (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-1053</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X Phosphate Buffered Saline (PBS)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-2040-10X4L</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA solution 10X</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">59418C-100ML</td>
			<td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2492-20ml</td>
			<td>250 &#956;g/ml in deionized water, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;">AE-BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;M syringe filter</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 &#956;M cell strainer</td>
			<td style="width:140px;">SPL</td>
			<td style="width:119px;">93070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe plunger</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:119px;">SS+10L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryovial tube</td>
			<td style="width:140px;">NUNC</td>
			<td style="width:119px;">368362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.7 ml microcentrifuge tube</td>
			<td style="width:140px;">Axygen</td>
			<td style="width:119px;">MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm cell culture dish</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">664160</td>
			<td>Cell culture treated dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:140px;">GFL</td>
			<td style="width:119px;">1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:140px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation shaker</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;"></td>
			<td>Certomat-BS1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axiovert 25 light microscope</td>
			<td style="width:140px;">Carl Zeiss AG</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating primary fibroblast cultures from mouse ear and tail tissues

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.

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</strong...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

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.

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 ...

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