The technical support of L. Rinaldi, S. Sch&#228;ffer, D. Reitz, H. Thomas and A. Weber is gratefully acknowledged. The authors also wish to thank Dr. E. Martinson for extensive proof reading and language editing of the manuscript. The study was supported by University of Giessen Anschubsfinanzierung grant to M. Aslam and D. G&#252;nd&#252;z.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23 1985) and was approved by the local ethics committee of the University of Giessen. Adult male Wistar rats weighing 200 - 250 g were used in this study.
1. Autoclaving
<ol>
	<li>At least one day before the procedure is to be carried out, autoclave all surgical instruments, pipette tips, and Pasteur pipettes to be used in the isolation procedure at 121 &#176;C for 30 min.</li>
</ol>
2. Preparation of Media and Solutions
NOTE: Solutions from steps 2.1-2.4 can be prepared up to a week before the isolation and stored at 4 &#176;C, but prepare the solutions in steps 2.5-2.8 on the day of isolation. The complete recipes of the buffers and media are given in Table 1. Warm all the media and solutions to 37 &#176;C in a water bath before starting the preparation.
<ol>
	<li>Prepare Powell medium by dissolving the chemicals listed in Table 1 in 4.5 L of H2O at room temperature (RT). Adjust the pH to 7.4 with 2.0 N NaOH, and bring the volume to 5.0 L. Sterile filter the solution, and store it at 4 &#176;C.</li>
	<li>Prepare creatine, L-carnitine, and taurine (CCT) medium by dissolving M199 powder medium in 4.5 L of H2O. Add HEPES powder according to Table to the medium and mix for 15 min. Add creatine, carnitine, taurine, and Ara-C according to Table 1. Mix well, adjust the pH with pH meter to 7.4 with 2.0 N NaOH, and bring the volume to 5.0 L. Sterile filter and store at 4 &#176;C.</li>
	<li>Prepare cell culture medium for fibroblasts by adding 10% fetal calf serum (FCS) and 2% penicillin/streptomycin (Pen/Strep) to medium M199. DMEM is equally suitable as M199 and can also be used for fibroblast cell culture.</li>
	<li>Prepare MV2 cell culture medium for endothelial cells by adding all the contents of the supplement mix to the basal MV2 medium according to manufacturer&#39;s instructions. Take a 5 mL aliquot and warm it to 37 &#176;C in a water bath.</li>
	<li>Prepare endothelial cell isolation buffer by dissolving 0.05 g of bovine serum albumin (BSA) in 50 mL of Hanks balanced salt solution (HBSS) and adding 0.5 mL of 200 mM ethylenediaminetetraacetic acid (EDTA) solution to it. Sterile filter this and store it at 4 &#176;C.</li>
	<li>Prepare endothelial cell wash buffer by adding 0.1 mL of 200 mM to 9.9 mL of PBS and store it at 4 &#176;C.</li>
	<li>Prepare collagenase solution by dissolving 27 mg of collagenase in 5 mL of Powell medium and adding 12.5 &#181;L of 100 mM CaCl2 solution to it. Warm it to 37 &#176;C in a water bath.</li>
	<li>Prepare incremental Ca2+ restoration solutions 1, 2, and 3 for cardiomyocytes by adding 12.5 &#181;L, 25 &#181;L, and 120 &#181;L of 100 mM CaCl2 solution to 6 mL, 6 mL, and 12 mL of Powell medium, respectively.</li>
	<li>Pre-coat the cell culture dishes for cardiomyocytes one day before the isolation by adding 1 mL of pre-coating medium containing laminin to each 35-mm cell culture dish. Incubate them in the cell culture incubator at 37 &#176;C overnight.</li>
</ol>
3. Preparation of Magnetic Beads
<ol>
	<li>Place 0.5 mL of the endothelial cell isolation buffer in a 1.5 mL sample tube on ice.</li>
	<li>Add 5 &#181;L (2 x 106) of pan mouse IgG magnetic beads and mix well.</li>
	<li>Place the tube in the magnetic rack for 1 min. The beads will adhere to the wall of the tube towards the magnet. Carefully remove the supernatant with a 1 mL pipette.</li>
	<li>Repeat the washing twice more and finally re-suspend the beads in 50 &#181;L endothelial cell isolation buffer and store at 4 &#176;C until use.</li>
</ol>
4. Preparation of Langendorff Perfusion System
<ol>
	<li>Clean the Langendorff system by perfusion with 2 L of distilled water.</li>
	<li>Circulate 50 mL of Powell medium through the system for 5 min and discard the medium.</li>
	<li>Add 80 mL fresh Powell medium. Continuously perfuse it with &#34;carbogen&#34; by bubbling with a glass Pasteur pipette from the gas cylinder. Let medium recirculate through the system. The system is now ready to perfuse the heart (Figure 1A).</li>
</ol>
5. Dissection of the Rat
<ol>
	<li>Place the rat in a 5 L desiccator under a fume hood and add 1.5 mL of 4-5% isoflurane through the air vent, and close the lid. 
	NOTE: Place the animal on elevated perforated plate inside the desiccator to avoid direct contact of the animal with the liquid isoflurane.</li>
	<li>Wait until the rat loses its lid reflex (usually ~5 min), and then take the rat out of the desiccator. Euthanize the rat by cervical dislocation.</li>
	<li>Cut the abdominal skin and the underlying muscle horizontally in the middle using a pair of sharp scissors. Thereafter, cut the muscle vertically to the sternum of the rat and open the thorax. Remove the heart along with the lungs and immediately place the organs in ice-cold 0.9% NaCl solution.</li>
	<li>Remove the lungs with the help of a pair of scissors and discard them. Clean the heart of any connective tissue, and cut the aorta caudally at the branch of the brachiocephalic trunk.</li>
</ol>
6. Perfusion of the Heart and Digestion with Collagenase
<ol>
	<li>Mount the heart via the aorta in the Langendorff perfusion system (Section 4) using two pairs of thin, curved forceps. In order to ensure that the myocardium is perfused in a retrograde manner, place the end of the cannula of the perfusion system between the first aortic branch and the aortic valve. Fix the aorta using a crocodile clamp.</li>
	<li>Fix the heart with a surgical suture, and remove the crocodile clamp (Figure 1B).</li>
	<li>Open the valve of the cannula to allow the perfusion medium (35 mL) to pass through the heart drop-wise (60 drops/min; 5 mL/min) to wash out any residual blood. Discard any flow-through at this stage. 
	NOTE: Avoid the formation of air bubbles at any stage of the perfusion as this will result in incomplete digestion and failure of the cell isolation procedure.</li>
	<li>Place the hanging heart in the collecting funnel (heart chamber; Figure 1B) and start the peristaltic pump, which will recirculate the perfusion medium from the collecting funnel to the reservoir (Figure 1A).</li>
	<li>Add the collagenase solution (from step 2.8) to the perfusion medium. Perfuse the heart with recirculating collagenase solution for 30 min. 
	NOTE: This step is very critical for optimizing cell yield. The perfusion time depends on the activity of collagenase, which may vary depending upon the batch. Test 3-4 batches of collagenase to find the one with optimal activity; then large amounts of the best-suited batch can be purchased that are sufficient for at least one year for reproducible results.</li>
	<li>At the end of the digestion time, stop the perfusion, remove the heart from the Langendorff system using a pair of scissors, and place it in a glass Petri dish.</li>
	<li>Cut off and discard both atria, and remove any leftover fatty tissues. Carefully peel off the pericardium with the help of two pairs of forceps to avoid contamination of the epithelial cells. 
	NOTE: The pericardium can be further digested with collagenase to obtain pure cardiac epithelial cells if required. A detailed protocol is not given in the present study as this is beyond the scope of the present protocol.</li>
	<li>Cut the heart down the middle with a pair of scissors and put it into the tissue chopper. Mince the heart 2-3 times and re-suspend the minced tissue in 12 mL of the digestion buffer from the Langendorff perfusion. 
	NOTE: Do not chop the heart excessively, as this will result in a higher proportion of dead cardiomyocytes.</li>
	<li>Transfer the minced tissue into 15 mL of collagenase-containing dissociation medium and let it digest in a water bath for a 5-10 min with occasional mixing by re-suspension using a 5 mL disposable plastic pipette.</li>
	<li>At the end of the digestion period, filter the cell suspension through a 100 &#181;m sieve. Discard the tissue debris and undigested material.</li>
	<li>At the end of the Langendorff perfusion, flush the perfusion system immediately with a minimum of 1 L of distilled water. Perfuse the system by recirculating 100 mL of 0.1 N NaOH for 30 min, and finally flush the system with 2-3 L of distilled water. Dry the system by flushing air and cover the opening with para-film.</li>
</ol>
7. Rat Cardiac Cell Isolation
<ol>
	<li>Isolation and culture of cardiomyocytes
	<ol>
		<li>Centrifuge the cell suspension at 25 x g for 1 min (without brakes) to pellet the cardiomyocytes. Cardiomyocytes may also be allowed to settle for 10 min at RT without centrifugation.</li>
		<li>Carefully remove the supernatant containing endothelial cells and fibroblasts with the help of a disposable plastic pipette in a new 50 mL tube.</li>
		<li>Resuspend the cardiomyocyte pellet in Ca2+ 6 mL solution 1 (Step 2.9). Allow 1 min for the cells to adjust to the low Ca2+ concentration environment, and centrifuge the cell suspension again at 25 x g for 1 min (without brakes).</li>
		<li>Discard the supernatant and resuspend the cell pellet in 6 mL Ca2+ solution 2 (Step 2.9). Allow the cells 1 min to adjust to the intermediate concentration of Ca2+, and finally add 12 mL of Ca2+ solution 3 (Step 2.9) to the cells. Mix with the help of a disposable plastic pipette.</li>
		<li>Centrifuge the cell suspension at 25 x g for 1 min.</li>
		<li>Discard the supernatant and resuspend the cells in 20 mL pre-warmed CCT medium. Add 1 mL of the cell suspension to each of 20 sterile 35 mm cell culture dish pre-coated with laminin (Step 2.9) and allow the cells to attach for 2 h in a CO2-free cell culture incubator. 
		NOTE: The cardiomyocytes can also be cultured under CO2 environment; however, in this case media with bicarbonate buffer system should be used.</li>
		<li>After 2 h, replace the medium with new 2 mL of CCT medium to remove any dead cells. 
		NOTE: The cells are ready for any planned experiments and can be cultured for up to 3 days in a typical cell culture incubator (CO2 free) without significant loss of living cells. Typical healthy rod-shaped cardiomyocytes are shown in Figure 2A.</li>
	</ol>
	</li>
	<li>Isolation of endothelial cells and fibroblasts
	<ol>
		<li>Centrifuge the supernatant containing endothelial cells and fibroblasts (from step 7.1.2) at 250 x g for 10 min.</li>
		<li>Discard the supernatant and resuspend the cell pellet in 1.5 mL of endothelial cell isolation buffer in a 2 mL sample tube.</li>
		<li>Add 3 &#181;g (1:500) of mouse anti-rat CD31 antibody to the cell suspension and incubate it at 4 &#176;C for 30 min with end-to-end rotation. 
		NOTE: A titration of the antibody for optimal dilution is advised. This step should be performed at 4 &#176;C as at RT a rapid internalization of antibody by endothelial cells will result in low yield.</li>
		<li>Add the previously prepared (Section 3) pan anti-mouse IgG beads to the cell suspension and incubate for 20 min at 4 &#176;C with end-to-end rotation. 
		NOTE: Alternatively, mouse anti-rat CD31 antibody can be pre-attached to the magnetic beads and then added to the cell suspension. However, this will increase the time of incubation of the beads with the cell suspension and may result in increased non-specific binding of non-endothelial cells and hence higher contamination of other cell types.</li>
		<li>At the end of the antibody incubation time, place the tube containing the cell suspension in a magnetic rack and allow 2 min to settle the magnetic beads attached to endothelial cells to the side of the tube.</li>
		<li>Carefully remove the rest of the cell suspension (containing mainly the fibroblasts) using a 1 mL pipette and add it to a 10 cm cell culture dish containing 10 mL of fibroblast cell culture medium (Step 2.3). Place it in a typical cell culture incubator containing 5% CO2. The procedure continues in Step 7.3.</li>
		<li>Add 1 mL of wash buffer to the tube with magnetic beads containing the endothelial cells.</li>
		<li>Cap the tube, remove it from the magnetic rack, and carefully resuspend the beads by shaking gently up and down 4 - 5 times. Place the tube again in the magnetic rack, allow the beads to settle on the wall of the tube for 1 min, and carefully remove the wash buffer.</li>
		<li>Repeat the washing steps 5 - 7 times to get rid of any contaminating non-endothelial cells.</li>
		<li>Resuspend the endothelial cells attached to the beads in 1 mL of MV2 endothelial cell culture medium, add to a single well of a 12-well plate, and incubate in a CO2 cell culture incubator.</li>
		<li>Allow the cells to attach overnight and change the medium the next day and then every 2-3 days until the cells become semi-confluent (usually 5 - 7 days). 
		NOTE: Typical non-confluent endothelial cells after 3 - 4 days and confluent endothelial cells after 7-10 days of isolation are shown in Figures 3A and 3B, respectively. The cells can then be trypsinized (section 7.4) and seeded into a T25 cell culture flask.</li>
	</ol>
	</li>
	<li>Washing and culture of fibroblasts (continue from step 7.2.6)
	<ol>
		<li>After 1 h of incubation, aspirate the medium and wash the fibroblasts attached to cell culture dish by adding an appropriate volume of pre-warmed PBS. Aspirate the PBS and repeat the step 2-3 times and finally add pre-warmed fresh fibroblast cell culture medium.</li>
		<li>Wash the attached cells again the next day with pre-warmed PBS (as in the previous step) and add pre-warmed fresh medium. Change the medium after every 2-3 days. A typical phenotype of fibroblasts on day 2 of isolation is shown in Figure 4A. 
		NOTE: The fibroblasts become semi-confluent usually within 5-7 days and are then ready to be trypsinized (section 7.4) and used for planned experiments. With this procedure, we usually obtain 95-99% pure fibroblasts. A typical phenotype of fibroblasts on day 7 of isolation is shown in Figure 4B.</li>
	</ol>
	</li>
	<li>Trypsinization of endothelial cells and fibroblasts
	<ol>
		<li>Aspirate the medium from the cell culture dish and add enough pre-warmed PBS to cover the cells.</li>
		<li>Aspirate PBS and add enough trypsin-EDTA solution (0.05% and 0.02%, respectively) to cover the cells and place in the 37 &#176;C incubator for 5 min.</li>
		<li>Resuspend the cells by pipetting the cells up and down with the help of a 1 mL pipette and stop trypsin action by adding 10% FCS.</li>
		<li>Pellet the cell suspension by centrifugation at 250 x g for 10 min at RT.</li>
		<li>Resuspend the cells in an appropriate volume of pre-warmed cell culture medium.</li>
	</ol>
	</li>
</ol>
8. Endothelial Cell Characterization
NOTE: The purity of endothelial cells is determined by Dil-Ac-LDL uptake and immunostaining of von Willebrand factor (vWF). Cells are visualized by fluorescence or confocal microscopy (Figures 3C-3E).
<ol>
	<li>Dil-Ac-LDL uptake by endothelial cells
	<ol>
		<li>Trypsinize the endothelial cells (step 7.4) and culture them overnight on glass coverslips in a 12-well plate (approximately 10 x 104 cells/cm2).</li>
		<li>Aspirate the medium and rinse the cells with 1 mL of pre-warmed HBSS.</li>
		<li>Aspirate the HBSS and add 1 mL endothelial cell culture medium containing 10 &#181;g/mL Dil-Ac-LDL and leave the cells in the 37 &#176;C incubator for 4 h.</li>
		<li>Aspirate the medium and rinse the cells with pre-warmed PBS three times and fix them with 1 mL of 4% paraformaldehyde (PFA; dissolved in PBS). Incubate them with PFA at 37 &#176;C for 20 min. 
		NOTE: PFA is corrosive; always use acid-resistant gloves while handling PFA solution.</li>
		<li>Aspirate the PFA, rinse the cells on coverslip in 12-well plate with 1 mL PBS thrice and incubate them with nuclear staining solution TO-PRO (10 &#181;M) for 1 h at RT. 
		NOTE: If the appropriate filter is available for the microscope, 4&#39;,6-diamidino-2-phenylindole (DAPI; 1 &#181;g/mL) may also be used. In this case incubation time should be reduced to 10 min.</li>
		<li>Rinse cells on coverslip in 12-well plate with 1 mL PBS, aspirate the PBS, and repeat the step three times.</li>
		<li>Take the coverslip out of 12-well plate and mount it on a glass slide with a drop of mounting medium. The cells are ready to be visualized under a fluorescent/confocal microscope. Use an excitation filter of 530-560 nm for Dil-Ac-LDL, 640 nm for TO-PRO, and 340-380 nm for DAPI.</li>
	</ol>
	</li>
	<li>Von Willebrand (vWF) staining
	<ol>
		<li>Culture the endothelial cells as described in step 8.1.1.</li>
		<li>Aspirate the medium and rinse the cells with 1 mL of pre-warmed HBSS; thereafter, fix with 4% PFA as describe in step 8.1.4.</li>
		<li>Aspirate the PFA, rinse the cells on coverslip in 12-well plate with 1 mL of PBS twice, and permeabilize the cells with 1 mL of 0.1% Triton X-100 (in PBS) for 20 min at RT.</li>
		<li>Aspirate the permeabilizing solution and incubate the cells with blocking solution (5% BSA/5% FCS in PBS) for 1 h.</li>
		<li>Incubate the cells with rabbit anti-vWF antibody (1:100) at 4 &#176;C overnight in a humidified chamber. Add one coverslip without the primary antibody as a negative control.</li>
		<li>Rinse the cells on the coverslip in 12-well plate with 1 mL PBS three times and incubate the cells with donkey anti-rabbit Alexa Fluor-488 antibody (1:500) and nuclear staining solution TO-PRO (10 &#181;M) for 1 h at RT.</li>
		<li>Rinse the cells on the coverslip in 12-well plate with 1 mL PBS, aspirate the PBS, and repeat the step three times.</li>
		<li>Take the coverslip out of the 12-well plate and mount it on a glass slide with a drop of mounting medium. The cells are ready to be visualized under a fluorescent/confocal microscope. Use an excitation filter of 490 nm for vWF and 640 nm for nuclear staining.</li>
	</ol>
	</li>
</ol>

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Authors have nothing to declare.

In this article, a reproducible protocol for the isolation and culture of cardiac myocytes, endothelial cells, and fibroblasts is described. This protocol describes the simultaneous and high quality isolation of the major cell types of cardiac tissue, i.e. cardiomyocytes, endothelial cells, and fibroblasts as opposed to just one cell type. A critical step in the isolation procedure is the proper digestion of the cardiac tissue. If the digestion is incomplete, a large number of myocytes may still be obtained but ...

Rodent models have long been used as tools to broaden our understanding of cardiovascular physiology in health and disease.1 Although these animal models allow us to understand the pathophysiology of a disease at the organ level and to analyze the pharmacokinetics and pharmacodynamics of various pharmacological agents used to treat cardiovascular diseases, understanding of the molecular mechanisms of cardiovascular disease development and the contribution of a particular cell type requires the use of in vitro cell culture models. For this purpose different immortalized cell lines from the cardiovascular system have been developed;2,3 however, freshly isolated primary cells are physiologically and functionally more relevant to living tissues and organisms.
The heart is a versatile organ containing all major types of cells of the cardiovascular system, and the rat heart is still a commonly used model for the understanding of cardiovascular physiology. During the last few decades, different methods for the isolation of individual cell types from cardiac tissue have been described;4,5,6,7 however, these methods focus only on the isolation of one specific cell type resulting in the loss of other types of cells that can no longer be used for cellular analysis. Here, an optimized protocol is described that enables the simultaneous and high quality isolation of the major cell types of cardiac tissue, i.e. cardiomyocytes, endothelial cells, and fibroblasts. All of these cell types can be used in different experimental setups8,9,10 and for the analysis of cell-cell interactions from the same animal.

<table><tbody><tr><td>anti-vWF</td><td>Santa Cruz Biotech.</td><td>SC-14014</td><td></td></tr><tr><td>Calcium chloride</td><td>Merck</td><td>102378</td><td></td></tr><tr><td>Carnitin</td><td>Sigma-Aldrich</td><td>C0283</td><td></td></tr><tr><td>Collagenase type II</td><td>Worthington</td><td>LS004176</td><td></td></tr><tr><td>Creatin</td><td>Sigma-Aldrich</td><td>C0780</td><td></td></tr><tr><td>D-Glucose</td><td>Merck</td><td>108342</td><td></td></tr><tr><td>Dil-Ac-LDL</td><td>Thermo Scientific</td><td>L3484</td><td></td></tr><tr><td>EDTA Solution (0.2 M)</td><td>Biochrome AG</td><td>L2113</td><td></td></tr><tr><td>Embeding solution</td><td>Citiflour</td><td>AF1-25</td><td></td></tr><tr><td>Endothelial cell medium MV2</td><td>PromoCell</td><td>C-22022</td><td></td></tr><tr><td>Foetal calf serum (FCS)</td><td>Biochrome AG</td><td>S0615</td><td></td></tr><tr><td>Gentamicin</td><td>Serva Chemicals</td><td>47991</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H0887</td><td></td></tr><tr><td>Isoflurane</td><td>Abbott</td><td>TU 061219</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Roche/Sigma</td><td>11243217001</td><td></td></tr><tr><td>M199 medium</td><td>Thermo Scientific</td><td>11150059</td><td></td></tr><tr><td>M199 medium (Powder)</td><td>Biochrome AG</td><td>T061</td><td></td></tr><tr><td>Magnesium sulphate</td><td>Sigma-Aldrich</td><td>63138</td><td></td></tr><tr><td>Mouse anti-rat CD31 antibody (TLD-3A12)</td><td>Thermo Scientific</td><td>MA1-81051</td><td></td></tr><tr><td>NaCl solution (0.9%), Sterile</td><td>B. Braun</td><td>30820080</td><td></td></tr><tr><td>Pan mouse IgG beads (Dynabeads)</td><td>Thermo Scientific</td><td>11041</td><td></td></tr><tr><td>Paraformaldehyde (PFA) 4% Solution</td><td>Santa Cruz Biotech.</td><td>sc-281692</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td></td></tr><tr><td>Phosphate buffer saline (PBS) 1x&amp;nbsp;</td><td>PAN-Biotech</td><td>P04-36500</td><td></td></tr><tr><td>Plastic consumables</td><td>Greiner Bio-One</td><td></td><td></td></tr><tr><td>Potassium Chloride</td><td>Merck</td><td>4933</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>7873</td><td></td></tr><tr><td>Sodium Chloride</td><td>Merck</td><td>6404</td><td></td></tr><tr><td>Sodium Hydroxide Solution (2 N)</td><td>Merck</td><td>109136</td><td></td></tr><tr><td>Sterile filtration system</td><td>Thermo Scientific</td><td>5660020</td><td></td></tr><tr><td>Taurine</td><td>Sigma-Aldrich</td><td>T8691</td><td></td></tr><tr><td>TO-PRO</td><td>Thermo Scientific</td><td>T3605</td><td></td></tr><tr><td>Trypsin-EDTA Solution (10x)</td><td>Sigma-Aldrich</td><td>T4174</td><td></td></tr><tr><td>Water, Sterile</td><td>B. Braun</td><td></td><td></td></tr></tbody></table>

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.

The isolation procedure results in a yield of 70 - 80% viable, rod-shaped, striated cardiomyocytes (Figures 2A and 2C) that can be used for planned experiments. In our laboratory cardiomyocytes are routinely used for analysis of Ca2+ signaling. Figure 5A shows intracellular calcium [Ca2+]i oscillations in response to ischemia/reperfusion in cardiomyocytes loaded with Fura-AM (5 &#181;M). F...

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

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 ...

simultaneous-isolation-high-quality-cardiomyocytes-endothelial-cells

Research

JoVE Journal

Biology

13.3K Views.  University Hospital Giessen.  Verscheidene protocollen zijn ontwikkeld en beschreven voor de isolatie van verschillende hartcel typen uit een rat hart. Hier wordt een geoptimaliseerd protocol beschreven dat de isolatie van hoge kwaliteit grote hartcellen (cardiomyocyten, endotheelcellen en fibroblasten) van een enkel preparaat mogelijk maakt, waardoor de experimentele kosten worden verminderd. 

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

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.

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 ...

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 ...

Isolation of Endocardial and Coronary Endothelial Cells from the Ventricular Free Wall of the Rat Heart

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. Consequently, these two cell populations play unique roles in cardiac diseases. Current studies involving isolated endothelial cells investigate cell populations consisting of both EECs and CECs. This protocol outlines a method to independently isolate these two cell populations for cell-specific characterization. Following the collection of the left and right ventricular free wall, endothelial cells from the outer surface and inner surface are separately liberated using a digestion buffer solution. The sequential digestion of the outer surface and the inner endocardial layer retained separation of the two endothelial cell populations. The separate isolation of EECs and CECs is further verified through the identification of markers specific to each population. Based on previously published single cell RNA profiling in the mouse heart, the Npr3, Hapln1, and Cdh11 gene expression is unique to EECs; while Fabp4, Mgll, and Cd36 gene expression is unique to CECs. qPCR data revealed enriched expression of these characteristic markers in their respective samples, indicating successful EEC and CEC isolation, as well as maintenance of cell phenotype, enabling further cell-specific functional analysis.

We present a protocol for the isolation of endocardial and coronary endothelial cells from rat hearts through sequential tissue digestion in a digestion buffer, cell collection from recurrent centrifuge cycles, and cell purification using anti-rat CD31 microbeads.

It has been shown that endocardial endothelial cells (EECs) and coronary endothelial cells (CECs) differ in origin, development, markers, and functions. ...

Developmental Biology

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Medicine

Isolation of Atrial Cardiomyocytes from a Rat Model of Metabolic Syndrome-related Heart Failure with Preserved Ejection Fraction

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease.
We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling).
We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.

Here, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes from a rat model of metabolic syndrome-related heart failure with preserved ejection fraction. A manual regulation of intraluminal pressure of cardiac cavities is implemented to yield functionally intact myocytes suitable for excitation-contraction-coupling studies.

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of ...

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes&#8217; terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

We describe a reliable method for isolation of adult mouse cardiomyocytes. This protocol yields a consistent result for the culture of functional adult cardiomyocytes from a variety of genetically modified mice.

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and accessibility of in vitro culture enables fine control over biochemical analyses, live imaging, and electrophysiology. Long-term culture of cardiomyocytes offers access to additional experimental approaches that cannot be completed in short term cultures. For example, the in vitro investigation of dedifferentiation, cell cycle re-entry, and cell division has thus far largely been restricted to rat cardiomyocytes, which appear to be more robust in long-term culture. However, the availability of a rich toolset of transgenic mouse lines and well-developed disease models make mouse systems attractive for cardiac research. Although several reports exist of adult mouse cardiomyocyte isolation, few studies demonstrate their long-term culture. Presented here, is a step-by-step method for the isolation and long-term culture of adult mouse cardiomyocytes. First, retrograde Langendorff perfusion is used to efficiently digest the heart with proteases, followed by gravity sedimentation purification. After a period of dedifferentiation following isolation, the cells gradually attach to the culture and can be cultured for weeks. Adenovirus cell lysate is used to efficiently transduce the isolated cardiomyocytes. These methods provide a simple, yet powerful model system to study cardiac biology.

This protocol describes a step-by-step method for the reproducible isolation and long-term culture of adult mouse cardiomyocytes with high yield, purity, and viability.

Cultured cardiomyocytes can be used to study cardiomyocyte biology using techniques that are complementary to in vivo systems. For example, the purity and ...

A Simple and Effective Method to Consistently Isolate Mouse Cardiomyocytes

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and molecular functional experiments (e.g., contraction, electrophysiology, calcium cycling, etc.) on cardiomyocytes are the gold standard for establishing mechanism(s) of disease. The mouse is the species of choice for functional experiments and the described technique is specifically for the isolation of mouse cardiomyocytes. Previous methods requiring a Langendorff apparatus require high levels of training and precision for aortic cannulation, often resulting in ischemia. The field is shifting toward Langendorff-free isolation methods that are simple, are reproducible, and yield viable myocytes for physiological data acquisition and culture. These methods greatly diminish ischemia time compared to aortic cannulation and result in reliably obtained cardiomyocytes. Our adaptation to the Langendorff-free method includes an initial perfusion with ice-cold clearing solution, use of a stabilizing platform that ensures a steady needle during perfusion, and additional digestion steps to ensure reliably obtained cardiomyocytes for use in functional measurements and culture. This method is simple and quick to perform and requires little technical skill.

The gold standard in cardiology for cellular and molecular functional experiments are cardiomyocytes. This article describes adaptations to the non-Langendorff technique to isolate mouse cardiomyocytes.

The need for reproducible yet technically simple methods yielding high-quality cardiomyocytes is essential for research in cardiac biology. Cellular and ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

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

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