Part I. Preparation for surgery
<ol>
<li>The day before the rat surgery, make certain that all solutions are made, but do not add any enzymes yet. Buffers A, B and the wash buffer should all be filtered for sterility prior to storage. The buffers should all be stored at 4 &deg;C.</li>
<li>6-well tissue culture plates must be plated with 10-20 &mu;g/mL laminin in 2 mL 1XSFM the day before as well, and should be stored at 37 &deg;C in a CO2 incubator.</li>
<li> On the day of the surgery, prepare the surgical instruments by sterilizing the clamp, scissors, and forceps with 70 % ethanol and let them dry on paper towel. </li>
<li>A 1 mL syringe filled with 0.5 mL Heparin Solution (90 U/ml) is made ready. </li>
<li>Now it is good practice to wash the perfusion apparatus by running through 70% ethanol, using the peristaltic pump in reverse. Once finished rinsing with ethanol, rinse the apparatus with double distilled water, and finally rinse with Buffer A. The flow through is collected in a beaker placed in a 37 &deg;C water bath. </li>
<li>To prepare the cleaned perfusion apparatus, fill the right syringe tube with 40 mL of Buffer A and the left syringe tube with 40 mL of Buffer B. </li>
<li>Prime the perfusion tubing to the tip of the catheter by allowing Buffer A to flow through the apparatus. Refill to the buffer A syringe to the 40 mL level. Make sure there are no air bubbles trapped in the tubing after this step. </li>
<li>Now, close the buffer A syringe stopcock valve and repeat the process with Buffer B such that the perfusion tubing is now primed with Buffer B. </li>
<li>Finally, the perfusion apparatus is left with the Buffer B stopcock valve open, but with the flow stopped using the regulator on the IV line. </li>
<li>Discard the buffer flow-though in the collecting beaker.</li>
<li>The last thing to do before the surgery is to add the required enzymes to your stock solution, and then filter the enzyme solution just as the other solutions were filtered the day before. Once filtered, this solution can be left at room temperature.</li>
</ol>
Part II. The Rat heart dissection
<ol>
<li>Following standard protocol euthanize the rat with Isoflurane in a gas chamber. </li>
<li>Sterilize the incision area on the thorax with 70% ethanol. </li>
<li>Open chest using scissors and expose heart.</li>
<li>Inject left ventricle with 0.5 mL Heparin solution (90 U/mL). </li>
<li>Remove heart with large portion of aorta intact, place in ice cold Buffer B. </li>
</ol>
Part III. Myocytes isolation
<ol>
<li>A typical rat heart should yield a high fraction of rod-shaped myocytes (as opposed to dead rounded cells) if the following procedure is properly adhered to. </li>
<li>To begin, clamp aorta to the catheter. The tip of the catheter should not be pushed too far into the heart to ensure good perfusion of the heart through the coronary artery. </li>
<li>Once clamped, tie the aorta to the catheter with a suture.</li>
<li>Next, open the IV line regulator to allow a fast drip rate (~60 drops/min) and allow Buffer B to wash out the heart.</li>
<li>During Buffer B wash, use the small scissors and forceps to remove the atria and any fatty or lung tissue clinging to the heart. </li>
<li>After Buffer B is finished, switch the flow to Buffer A. </li>
<li>While Buffer A is allowed to flow for 5 minutes, several small tasks should be quickly performed. </li>
<li>First, warm the Enzyme Solution in a 37 &deg;C water bath. </li>
<li>Finally, when Buffer A is depleted from the syringe (but still present in the tubing), load 50 mL of the Enzyme Solution in syringe A of the perfusion apparatus </li>
<li>It is now necessary to discard all the flow-through from the collection beaker in the water bath (by pipetting), until the Enzyme Solution perfuses heart. </li>
<li>As soon as the Enzyme Solution perfuses the heart, activate the peristaltic pump which is set up to transfer the enzyme solution from the collecting beaker to replenish syringe A.</li>
<li>Allow the enzyme solution to flow through the heart for 10 min. As the heart digests, it begins to look bloated.</li>
<li>Add 37.5 &mu;L of 0.1 M CaCl2 to the enzyme solution in syringe A to give an effective concentration of 0.1 mM Ca2+. </li>
<li>After 10 minutes increase the concentration of calcium to 0.2 mM by adding an additional 50 &mu;L of 0.1 M CaCl2 to syringe A and let the perfusion proceed for a further 10 minutes.</li>
<li>Cut off the ventricles and transfer to a small sterile beaker containing 20 mL Enzyme Solution.</li>
<li>In the beaker increase the Calcium concentration to 0.4 mM by adding 40 more &mu;L of 0.1 M CaCl2. </li>
<li>Gently mince the heart into 10 or more pieces with a pair of small sterile scissors. </li>
<li>Incubate the beaker for 5 minutes at 37 &deg;C with gentle rocking.</li>
<li>Add 40 mL of 0.1 M CaCl2 to give effective concentration of 0.6 mM Ca2+ and gently triturate the heart pieces with a plastic transfer pipet 3-5 times. </li>
<li>After incubating for an additional 5 minutes at 37 &deg;C, add 40 mL of 0.1 M CaCl2 and gently triturate as before.</li>
<li>Separate digested single myocytes from non-digested connective tissue using filtration through a sterile 500 &mu;m mesh. </li>
<li>Allow the cells to settle in a 50 ml tube for 10 minutes at room temperature.</li>
<li>Discard supernatant with a transfer pipet.</li>
<li>Gently resuspend cells in Wash Buffer #1 and allow the cells to settle for 10-20 min at room temperature.</li>
<li>While the cells are settling, take a small aliquot, and use this time as an opportunity to assess the quality and viability of the cells in suspension under an inverted microscope. A good preparation will have a high proportion (&gt;80%) of rod like cells with crisp striations.</li>
<li>Once the cells have settled, discard the supernatant and gently resuspend cells in Wash Buffer #2. Let the cells settle at room temperature, which should again take 10-20 minutes, and discard the supernatant.</li>
<li>Repeat the washing process once more with Wash Buffer #3, and the cells will be ready for culture.</li>
</ol>
Part IV. Myocyte cell culture
<ol>
<li>Gently resuspend cells in 5% Serum Media. Before transferring the cells to tissue culture plates, wash the plates with 1x SFM. Transfer the cells at a desired density and incubate in a CO2 tissue culture incubator for 3-5 hours.</li>
<li>Switch the media to 1x SFM.</li>
<li>Cells can be cultured for up to four days and used as required for experiments. </li>
</ol>
Part V. Representative Results/Outcome
There should be more than 70% live heart myocytes under inverted microscope when the protocol is done correctly.
<img src="/files/ftp_upload/1308/fig1.jpg" alt="figure 1" />
Table 1: Solution recipe for 1 L 10X Ca2+-free KH Buffer:
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td> </td>
<td>mass</td>
<td>Final Concentration</td>
</tr>
<tr>
<td>NaCl</td>
<td>68.96 g</td>
<td>1180 mM</td>
</tr>
<tr>
<td>KCl</td>
<td>3.58 g</td>
<td>48 mM</td>
</tr>
<tr>
<td>HEPES</td>
<td>59.58 g</td>
<td>250 mM</td>
</tr>
<tr>
<td>K2HPO4</td>
<td>2.85 g</td>
<td>12.5 mM</td>
</tr>
<tr>
<td>MgSO4</td>
<td>3.08 g</td>
<td>12.5 mM</td>
</tr>
</tbody>
</table>
Table 2: Solution recipe for 1 L Buffer A: Add 1.98 g glucose to 100 mL 10X KH buffer and bring to a final volume of 1 L. Adjust osmolarity to near physiology condition (~300) with glucose. Bring to pH 7.4 with NaOH.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td>-</td>
<td>Mass</td>
<td>Final Concentration</td>
</tr>
<tr>
<td>10X KH Buffer</td>
<td>100 mL</td>
<td>100 ml</td>
</tr>
<tr>
<td>Glucose</td>
<td>1.98 g</td>
<td>11 mM</td>
</tr>
<tr>
<td>Bring to 1 L</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>Bring to pH 7.4 with NaOH</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td colspan="3">Adjust osmolarity to near physiological condition(~300) with glucose.</td>
</tr>
</tbody>
</table>
Table 3: Solution recipe for Enzyme Buffers
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td> </td>
<td>mass</td>
<td>Final Concentration</td>
</tr>
<tr>
<td>Collagenase Type II</td>
<td>25 mg</td>
<td>0.05% (w/v)</td>
</tr>
<tr>
<td>Protease XIV</td>
<td>10 mg</td>
<td>0.02% (w/v)</td>
</tr>
<tr>
<td>BDM</td>
<td>0.025 g</td>
<td>5 mM</td>
</tr>
<tr>
<td>Carnitine</td>
<td>0.020 g</td>
<td>2 mM</td>
</tr>
<tr>
<td>Taurine</td>
<td>0.031 g</td>
<td>5 mM</td>
</tr>
<tr>
<td>Glutamic Acid</td>
<td>0.020 g</td>
<td>2 mM</td>
</tr>
<tr>
<td>0.1 M CaCl2</td>
<td>12.5 uL</td>
<td>25 uM</td>
</tr>
<tr>
<td>Buffer A</td>
<td> </td>
<td>Fill to 50 ml</td>
</tr>
</tbody>
</table>
 Table 4: Solution recipe for 25X BDM/Taurine/BSA (B/T/B)
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td> </td>
<td>mass</td>
<td>Final Concentration</td>
</tr>
<tr>
<td>BDM</td>
<td>0.076 g</td>
<td>125 mM</td>
</tr>
<tr>
<td>Taurine</td>
<td>0.094 g</td>
<td>125 mM</td>
</tr>
<tr>
<td>BSA</td>
<td> </td>
<td>0.1% (w/v)</td>
</tr>
<tr>
<td>Buffer A</td>
<td> </td>
<td>Fill to 6 mL</td>
</tr>
</tbody>
</table>
Table 5: Solution recipe for Buffer B and Wash Buffers
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td>Buffer:</td>
<td>B</td>
<td>#1</td>
<td>#2</td>
<td>#3</td>
</tr>
<tr>
<td>Add:</td>
<td> </td>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>0.5 M CaCl2</td>
<td>0.4 mL</td>
<td>0.1 mL</td>
<td>0.125 mL</td>
<td>0.15 mL</td>
</tr>
<tr>
<td>Final concentration of CaCl2</td>
<td>1 mM</td>
<td>1 mM</td>
<td>1.25 mM</td>
<td>1.5 mM</td>
</tr>
<tr>
<td>25X B/T/B</td>
<td>---</td>
<td>2 mL</td>
<td>2 mL</td>
<td>2 mL</td>
</tr>
<tr>
<td>Buffer A to final volume</td>
<td>200 mL</td>
<td>50 mL</td>
<td>50 mL</td>
<td>50 mL</td>
</tr>
</tbody>
</table>
Table 6: Solution Recipe for Culturing Media
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
<tr>
<td>2X Serum-free Media (SFM)</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td rowspan="2"> </td>
<td rowspan="2"> </td>
<td>Final Concentration</td>
</tr>
<tr>
<td>(after dilution)</td>
</tr>
<tr>
<td>Carnitine</td>
<td>0.099 g</td>
<td>10 mM (5 mM)</td>
</tr>
<tr>
<td>Taurine</td>
<td>0.063 g</td>
<td>10 mM (5 mM)</td>
</tr>
<tr>
<td>Creatine</td>
<td>0.075 g</td>
<td>10 mM (5 mM)</td>
</tr>
<tr>
<td>Antibiotic/Antimycotic</td>
<td>0.5 ml</td>
<td>1% (0.5%) (v/v)</td>
</tr>
<tr>
<td>Media 199</td>
<td>45 mL</td>
<td>Fill to 50 mL</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>1X SFM</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td>Final Concentration</td>
</tr>
<tr>
<td>2X SFM</td>
<td>25 mL</td>
<td>1X</td>
</tr>
<tr>
<td>Media 199</td>
<td> </td>
<td>Fill to 50 mL</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>1X 5% Serum Media</td>
<td> </td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td>Final Concentration</td>
</tr>
<tr>
<td>2X SFM</td>
<td>25 mL</td>
<td>1X</td>
</tr>
<tr>
<td>FBS</td>
<td>2.5 mL</td>
<td>5% (v/v)</td>
</tr>
<tr>
<td>Media 199</td>
<td> </td>
<td>Fill to 50 mL</td>
</tr>
</tbody>
</table>

<ol>
	<li>Miriyala, J., Nguyen, T., Yue, D. T., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CaVbeta+subunits,+and+lack+of+functional+reserve,+in+protein+kinase+A+modulation+of+cardiac+CaV1.2+channels.">Role of CaVbeta subunits, and lack of functional reserve, in protein kinase A modulation of cardiac CaV1.2 channels.</a> Circ Res. 102 (7), e54-e64 (2008).</li><li>SX, T. a. k. a. h. a. s. h. i., Mittman, S., Colecraft, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinctive+modulatory+effects+of+five+human+auxiliary+beta2+subunit+splice+variants+on+L-type+calcium+channel+gating.">Distinctive modulatory effects of five human auxiliary beta2 subunit splice variants on L-type calcium channel gating.</a> Biophys. , 84-845 (2003).</li></ol>

A critical step is the speed with which the isolated heart is hung up on the perfusion system. The length of the enzymatic digestion period may be a little different for each rat. The adjustment depends on how soft the heart becomes after the regular period of digestion. The slowly recovery of Ca2+ after enzyme digestion is essential for obtaining Ca2+-tolerant healthy cells.
For guinea pig, the protocol is the same except hyaluronidase is used instead of Protease XIV. ...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S6191</td>
<td>&nbsp;</td>
<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9541</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4034</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P9666</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>63068</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7902</td>
<td>&nbsp;</td>
<tr>
<td>Heparin Sodium Salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4784</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase Type II</td>
<td>&nbsp;</td>
<td>Worthington Biochemical</td>
<td>LS004176</td>
<td>&nbsp;</td>
<tr>
<td>Protease XIV</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P5147</td>
<td>&nbsp;</td>
<tr>
<td>2,3-Butanedione Monoxime (BDM)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B0753</td>
<td>&nbsp;</td>
<tr>
<td>Carnitine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C9500</td>
<td>&nbsp;</td>
<tr>
<td>Taurine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T0625</td>
<td>&nbsp;</td>
<tr>
<td>Glutamic Acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1501</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A3294</td>
<td>&nbsp;</td>
<tr>
<td>Creatine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C3630</td>
<td>&nbsp;</td>
<tr>
<td>Antibiotic</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-009-CI</td>
<td>&nbsp;</td>
<tr>
<td>Media 199</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>11150</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>354232</td>
<td>&nbsp;</td>
<tr>
<td>water bath</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-462-5</td>
<td>&nbsp;</td>
<tr>
<td>variable flow chemical pump</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>15-077-67</td>
<td>&nbsp;</td>
<tr>
<td>6-well culture plate</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

primary culture of adult rat heart myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

Watch this Scientific Journal Video about Primary Culture of Adult Rat Heart Myocytes at JoVE.com

Primary Culture of Adult Rat Heart Myocytes

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

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JoVE Journal

Biology

20.2K ビュー.  Columbia University. 本稿では、成体ラットの心筋細胞を分離、培養に一般的な方法を説明した。コラゲナーゼとプロテアーゼは、単一の心筋細胞を消化して単離するために使用されています。心筋細胞は、ほとんどの実験の要件を満たすこのプロトコルに従って培養した。

ビデオ: Primary Culture of Adult Rat Heart Myocytes

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Isolation and Genetic Manipulation of Adult Cardiac Myocytes for Confocal Imaging

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Adult cardiac myocytes are primary cells that can be isolated from animal hearts and cultured for several days. Within this culture period adenoviral gene transfer can be used to express genetically encoded biosensors (GEBs) or fluorescent fusion proteins. Both approaches allow cellular investigations by means of confocal microscopy.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...