1. Cardiomyocyte Isolation
Materials (Figure 1)
Microdissecting forceps 
Tissue forceps 
Delicate hemostatic forceps 
Hemostatic forceps 
Microdissecting, serrated, curved forceps 
Operating scissors, straight 
Operating scissors, curved 
15 ml Falcon tubes (5) 
60 mm Petri dish 
Phosphate Buffered Saline (PBS) 
Nylon Mesh - 400 &#956;m pore size 
Small funnel 
Wax coated, braided silk 4-0, 19 mm, 7-10 cm long 
Non-hypodermic needle, blunt end, 24 G or commercial animal-feeding needle (24 x 1 in, W/1-1/4) 
Heparin sodium 
Ketamine/xylazine mixture 
Pasteur Pipettes 
OptiVision Dissecting Goggles 
IonOptix MMSYS system
Note: If culturing cells, see section 2 on cell culture before beginning this protocol.
Note: All mice were cared for in a barrier facility and sacrificed according to approved IACUC regulations, practices, and procedures. 
Note: Prior to beginning the procedure, the entire system should be preheated to ensure that all the elements of the Langendorff system (Figure 2A, Figure 3) are warmed to 37 &#176;C to allow for proper and complete collagenase activity.
<ol>
	<li>Inject adult mice (~12 weeks) with 150 USP units heparin sodium into the abdominal space.</li>
	<li>Anesthetize with an injection of a ketamine/xylazine mixture at a weight-dependent dose (Table 1).
	<ol>
		<li>80:12 mg/kg ketamine:xylazine recipe: 2.6 ml ketamine (100 mg/ml) + 0.4 ml xylazine (100 mg/ml) + 37 ml PBS. Final: ketamine = 6.5 mg/ml; xylazine = 1 mg/ml</li>
	</ol>
	</li>
	<li>Secure the sedated mouse to the operating pad, excise the heart, and place into a dish of room temperature PBS or 0.9% saline (Figure 1J). Physiologic saline allows the intact heart to contract for better extrusion of blood in the chamber and easy identification of the aorta prior to step 1.6. Mice were checked to ensure that they are deeply anesthetized via loss of hind limb toe pinch reflex and slowing of respiratory rate prior to onset of thoracotomy.</li>
	<li>Use OptiVision dissecting goggles (Figure 1A) to identify and expose the aorta. Removing the tissue around the aorta, although it does facilitate the visualization of the vessel, is not necessary for the optimization of the cell isolation if the aortic root is clearly visible.</li>
	<li>Prime the pump with perfusion buffer (Table 2). Temperature should be held at 37 &#176;C for the length of the procedure using a controlled, circulating water bath (Figure 2D).</li>
	<li>Mount the heart onto the Langendorff Apparatus (Figure 2A). Using two micro-dissecting forceps (Figures 1D-E), prime the aorta around the non-hypodermic, blunt end needle (24 G) with silicone tube at the distal tip. Alternatively a commercial animal-feeding needle (24 x 1 in, W/1-1/4) can be used. The silicone tube on the 24 G needle or the animal feeding needle will allow for securing the suture over the groove. Place the aorta on the needle as a sock. (Figure 2C).
	<ol>
		<li>Note: It is important to be sure that the end of the needle rests in the ascending aorta, ideally in the aortic root distal to the right innominate, but that it does not extend through the aortic valve into the left ventricle, as this will severely limit the ability of the buffers to effectively perfuse through the heart via the coronary arteries.</li>
	</ol>
	</li>
	<li>Secure the heart to the needle by tying a small length of suturing silk (7-10 cm long) (Figure 1K) around the top of the aorta, ensuring that the tie is at the level of the ascending aorta, below the right innominate artery, but is above the end of the needle.</li>
	<li>Lower the heart into the interior of the conical glass (Figure 2B) to help maintain a suitable ambient temperature.</li>
	<li>Perfuse the heart with perfusion buffer for 5 min at a rate of 1 ml/min and clamp the glass chamber outflow to allow the perfusate to collect in the conical glass and envelope the heart. The schematic for digesting the heart is shown in Figure 3.
	<ol>
		<li>At this point you should see the volume of the heart increase. Additionally, blood in cardiac tissue will be replaced by perfusate, causing tissue pallor.</li>
	</ol>
	</li>
	<li>Release the outflow clamp to drain the perfusate. Stop the pump to move the tubing from the perfusion buffer container to the enzyme buffer container (Figure 2E). Restart the pump to begin to perfuse the heart with enzyme buffer (Table 2) at 1 ml/min. Clamp the outflow again to allow the enzyme buffer to envelope the heart.
	<ol>
		<li>Here, as the enzyme is perfusing the heart and digesting the connective tissue, the heart will become paler and the structural integrity will diminish.</li>
		<li>To determine when to cut the ventricles from the digested heart, lift the heart out of the conical glass, gently squeeze the heart with forceps and collect a few drops of perfusate in a small Petri dish and check to see whether or not single cells are dropping.</li>
		<li>For the novice it is helpful to connect the perfusion line with a manometer. When the heart is getting digested the pressure will rapidly decline, as the connective tissue does not hold the resistance. The manometer is also useful for the novice to ensure that the position of the needle is above the aortic valve and not in the ventricle. Low pressure will indicate that the needle is in the ventricular chamber and high pressure indicated that the needle touches the valve.</li>
	</ol>
	</li>
	<li>Cut the ventricles from the heart into a dish full of transfer buffer (A) (Table 2) when the ventricular pressure begins to drop dramatically or when you are able to see single cells in the perfusate. This usually takes ~7-10 min.</li>
	<li>Again using micro-dissecting forceps (Figure 1C), mince the ventricles into small pieces to dissociate. A successful digestion will leave almost no solid chunks of tissue after mincing, with most of the tissue becoming amorphous upon dissociation.
	<ol>
		<li>To further dissociate the tissue, pipette the solution in/out using a plastic Pasteur pipette (Figure 4) from which the tip had been cut at an ~45&#176; angle. This modification serves to increase the inner diametric space of the pipette tip thus decreasing the amount of shear stress on the cells as the tissue is triturated.</li>
		<li>Before dissecting the tissue with the forceps it is possible to separate the individual chambers and separately dissociate atrial, left ventricular or right ventricular cells.</li>
	</ol>
	</li>
	<li>Secure the squared mesh (Figure 1M) to a small funnel (Figure 1L) using clamps and place this filtering apparatus into a 15ml Falcon tube (Figure 1I).</li>
	<li>Use the modified Pasteur pipette to remove the cell solution from the dish and filter the solution into the Falcon tube.</li>
	<li>Allow the live cells to settle to the bottom of the tube (live cell sedimentation should take 2-4 min).</li>
	<li>Aliquot 2.5ml of each of the four calcium solutions (Table 3) into 4 separate 15ml falcon tubes.</li>
	<li>Again using a standard Pasteur pipette, carefully remove the cell pellet from the bottom of the tube and transfer the cells to the first of the calcium solutions.</li>
	<li>Allow the cells to settle to the bottom of the tube and repeat step 1.17 in the subsequent calcium solutions until the cells are in the last solution.</li>
	<li>Do not let the cells settle in the fourth calcium solution. Cap the tube and turn it on its side.</li>
</ol>
2. Cell Culture
<ol>
	<li>Before the isolation, plate 1 ug/ml natural mouse laminin onto glass coverslips and incubate for at least 1 hr at 37 &#176;C and 2% CO2. Also allow your plating and culture media (Table 4) to warm in the incubator at 37 &#176;C and 2% CO2. This allows the dissolved CO2 in the media to equilibrate.
	<ol>
		<li>Do not remove the top from the media. Simply loosen the top to avoid contamination.</li>
	</ol>
	</li>
	<li>Allow the isolated cells to pellet at the bottom of the Falcon tube.</li>
	<li>Remove the pellet using a standard plastic Pasteur pipette and resuspend in the appropriate amount of plating media.</li>
	<li>Plate the cells on the laminin-coated coverslips in the appropriate sized dish and incubate in 37 &#176;C and 2% CO2 for at least 30-60 min to allow the cells to adhere to the coverslips.</li>
	<li>**If transfecting with adenovirus, dilute the virus in an appropriate amount of plating media and replace the plating media in the dish with the virus-containing media. Let the virus incubate on the cells for 2 hr in 37 &#176;C and 2% CO2.**</li>
	<li>Remove the plating media from the dish and replace with culture media.</li>
	<li>Carefully change the culture media each day so as not to disturb the cells on the coverslips.</li>
</ol>
Using this procedure, cells have been successfully cultured for up to 72 hr. Images of cultured and GFP transfected cells can be found in Figure 5.
3. MMSYS System
<ol>
	<li>Power the MMSYS system ensuring that the arc lamp is initiated first.</li>
	<li>Connect the tubes from the pump to the appropriate inlet and outlets of the MMSYS chamber (Figure 6).</li>
	<li>Prime the system with calcium buffer (B) (Table 2).</li>
	<li>Place an appropriately sized glass coverslip into the chamber and fasten (Figure 6E). Most MMSYS chambers use either 22 mm or 25 mm square coverslips. Consult your MMSYS user manual to determine the appropriate coverslip for your chamber.</li>
	<li>Transfer 500 &#956;l of the cell solution to a small Eppendorf tube.</li>
	<li>Add 0.5 &#956;l Fura2-AM (stock solution of 1 &#956;g/&#956;l) to the small tube of cells and allow them to incubate in the dark at RT for 5-7 min. This allows the Fura2-AM to &#34;load&#34; into the cells through rapid passive diffusion through the cell membrane.
	<ol>
		<li>Let the Fura-2 loaded cells pellet to the bottom of the tube, and wash the excess Fura with 500 &#956;l of calcium buffer B.</li>
	</ol>
	</li>
	<li>Turn off the room lights.</li>
	<li>Using a standard Pasteur pipette, drop 1-2 drops (depending of the cell concentration) of the &#34;loaded&#34; cell solution into the center of the chamber (Figure 6C) and allow the cells to settle onto the coverslip for 5 min.
	<ol>
		<li>The density of the cells in the chamber should be such that single non-overlapping cells can be easily viewed in the MMSYS data acquisition platform.</li>
	</ol>
	</li>
	<li>Carefully begin to flow calcium buffer (B) through the chamber.</li>
	<li>Increase the chamber temperature to 37 &#176;C.
	<ol>
		<li>IMPORTANT: Do not turn on the heating apparatus until the flow has been initiated. This can severely damage the feedback thermocoupler (Figure 6A).</li>
	</ol>
	</li>
	<li>Make sure that the chamber is connected to the MyoPacer via the connection wires (Figure 6B) and begin to pace the cells 5 Hz at 1.5x the pacing threshold for the cells.</li>
	<li>Choose a cell that is beating at the correct frequency and move it into the framing aperture.</li>
	<li>Adjust the camera and framing aperture dimensions so that the entire cell is in the center of the window, directed horizontally, with the sarcomeres apparent.
	<ol>
		<li>For cell length adjust the red and green (right and left) photodiode indicators to the edge of the cell. Adjust the contrast to optimize the black/white contrast at the edges of the cell. Refer to the Ionoptix manual for further details.</li>
	</ol>
	</li>
	<li>Place the box in an area of the cell containing well-defined sarcomeres and adjust the focus to optimize the peak of the power spectrum (red). Also adjust the brightness of the microscope to optimize the blue smoothing window and black contrast information.</li>
	<li>Adjust the green threshold bars to capture as much of the red power spectrum (peak) and as little background as possible.</li>
	<li>Begin recording.</li>
	<li>After enough data has been recorded, click &#34;Pause&#34; to temporarily stop recording and, ensuring that neither the focus nor dimensions of the viewing window are altered, move the microscope&#39;s stage to an area of the coverslip in which no cells or cellular material can be seen. Length of recording varies depending on the investigator&#39;s experimental protocol.
	<ol>
		<li>Note: Clicking &#34;Stop&#34; will terminate the recording and no background will be able to be recorded for that cell.</li>
	</ol>
	</li>
	<li>Click &#34;Resume&#34; to record a background measurement.</li>
	<li>After enough background has been recorded click &#34;Stop&#34; to terminate the recording.</li>
	<li>Click &#34;File &#62;&#62; Save&#34; to save all the traces for that cell in a single .zpt file.</li>
	<li>Repeat steps 3.12 - 3.20 until enough cells have been recorded.</li>
	<li>Consult the IonOptix-MMSYS User Manual 12 for instructions on analysis of the recorded data. Characteristic recorded data of wild-type cardiomyocytes are shown in in Figure 7.</li>
</ol>
4. Patch Clamp
Patch clamping of different cell types have been well described previously in this journal25-28. We therefore focus on some critical parameters for successful patch clamping of adult cardiomyocytes isolated using our protocol described.
<ol>
	<li>Using a pipette puller and borosilicate glass (with filament: O.D. 1.5 mm, I.D. 0.86 mm - 10 cm length) pull a pipette that exhibits ~2.0-3.0 M&#937; when filled with pipette solution (Table 5).</li>
	<li>Fire-polish the pipette tip gently using a Narishige or other vertical fire polisher. The resistance of the patch pipette should be 2-4 MO after fire polishing.</li>
	<li>Turn on the computer, amplifier (Axopatch 200B), A-D converter (Digidata 1440A, Axon Instruments) and Micromanipulator (MP-285). For whole cell patch clamping, we start with standard parameters as previously described.</li>
	<li>For cultured adult CMs, remove them from the incubator and gently wash the coverslip on which the CMs are plated with PBS at room temperature.</li>
	<li>Gently remove the coverslip and place it in the perfusion chamber on the inverted microscope (Nikon TE 2000).
	<ol>
		<li>Ensure that the inlet for the perfusion system and outlet (for suction) are just above the surface of the coverslip.</li>
	</ol>
	</li>
	<li>Start perfusing with the appropriate extra-cellular solution (Table 5).</li>
	<li>For freshly dissociated adult CMs, place a collagen-coated coverslip in the perfusion system as above.
	<ol>
		<li>Since the cells will be in solution, gently replace the solution with extracellular buffer.</li>
	</ol>
	</li>
	<li>Using the modified Pasteur pipette (Figure 4), gently resuspend the cells in the extracellular buffer, take an aliquot and place it on the coverslip.</li>
	<li>Let the cells attach for 10-15 min before starting the perfusion as in 4.4.</li>
	<li>Back-fill the whole cell patch pipette with intra-cellular buffer using a Microfil (World Precision Instruments, 28 G) needle. Ensure that there are no air bubbles in the tip of the pipette; gently tap to allow the air bubbles to float to the surface of the solution.</li>
	<li>Attach the filled patch pipette to the pipette-holder, ensuring that there is contact between the microelectrode and the internal solution.
	<ol>
		<li>We use silver chloride electrodes, but take care of &#39;chloriding&#39; the silver wires at least once a week by immersing overnight in household bleach.</li>
	</ol>
	</li>
	<li>Gently lower the pipette into the bath, ensuring that the bath electrode is immersed at all times in the bath/extra-cellular solution. Offset any liquid junction potentials at this time. Large junction potentials may be a sign of deteriorating silver chloride on the electrodes.</li>
	<li>Healthy cylindrical adult CMs are identified in the microscope and centered in the field of vision. As previously described, a combination of moving the micromanipulator and adjusting the focus allows for close proximity of the patch pipette and the surface of the CM.
	<ol>
		<li>Once they are in the same focal plane, we use a combination of visualization of the cell and test pulse (available in the Axopatch 200B).</li>
		<li>Once the resistance of the pipette decreases in half (suggesting that the pipette is in contact with the surface of the cell), and the cell continues to look cylindrical, we establish gigaohm seal using gentle suction.</li>
		<li>For CMs, we prefer mouth suction to establish negative pressure. If a gigaseal is successfully obtained, we again use gentle rhythmic mouth suction to &#39;break-in&#39; to the cell to establish whole cell patch mode.</li>
	</ol>
	</li>
	<li>Typical resting potentials of healthy CMs are in the order of -85 to -90 mV. Once a gigaseal is obtained, measure the resting membrane potential using current clamp with I=0 pA.
	<ol>
		<li>If the resting membrane potential is depolarized this can indicate a damaged cell or a poor seal.</li>
	</ol>
	</li>
	<li>Because the CMs are large cells with a lot of surface area, careful attention needs to be paid to capacitance compensation, especially when rapidly activating currents such as the sodium channel needs to be studied. If adequate capacitance compensation cannot be obtained using the built in compensation settings on the pulse generator, prepulses at sub-threshold voltage and opposite polarity may have to be applied and the resultant current subtracted from the recorded signal. Such a protocol is easily programmed in Axopatch.</li>
	<li>Once whole cell patch mode has been established, wait 15 min to allow for equilibration and to ensure stability of the seal prior to beginning voltage or current clamp protocols. We use standard protocols that have been described previously in this and other journals and will not be elaborated upon.</li>
</ol>

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M., Siegmund, B., Schwartz, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longevity+of+adult+ventricular+rat+heart+muscle+cells+in+serum-free+primary+culture.">Longevity of adult ventricular rat heart muscle cells in serum-free primary culture.</a> J. Mol. Cell. Cardiol. 23, 161-173 (1991).</li><li>Guatimosim, S., Guatimosim, C., Song, L. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Molecular Biology. 689, 205-214 (2010).</li><li>Yang, J., Delaloye, K., Lee, U. S., Cui, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+and+Perfusion+Techniques+for+Studying+Ion+Channels+Expressed+in+Xenopus+oocytes.">Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes.</a> J. Vis. Exp. (47), e2269 (2011).</li><li>Brown, A. L., Johnson, B. E., Goodman, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recording+of+Ion+Channels+Expressed+in+Xenopus+Oocytes.">Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes.</a> J. Vis. Exp. (20), e936 (2008).</li><li>Arman, A. C., Sampath, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+Clamp+Recordings+from+Mouse+Retinal+Neurons+in+a+Dark-adapted+Slice+Preparation.">Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation.</a> J. Vis. Exp. (43), e2107 (2010).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+patch+clamp+recordings+from+motor-neuron+and+target+skeletal+muscle+in+zebrafish.">Paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish.</a> J. Vis. Exp. (45), e2351 (2010).</li><li>Maier, S., Westenbroek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+unexpected+role+for+brain-type+sodium+channels+in+coupling+of+cell+surface+depolarization+to+contraction+in+the+heart.">An unexpected role for brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart.</a> Proc. Natl. Acad. Sci. U.S.A. 99 (6), 4073-4078 (2002).</li><li>Shroff, S. G., Saner, D. R., Lal, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+micromechanical+properties+of+cultured+rat+atrial+myocytes+measured+by+atomic+force+microscopy.">Dynamic micromechanical properties of cultured rat atrial myocytes measured by atomic force microscopy.</a> Am. J. Physiol. 269, 286-292 (1995).</li><li>Domke, J., Parak, W. J., George, M., Gaub, H. E., Radmacher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+mechanical+pulse+of+single+cardiomyocytes+with+the+atomic+force+microscope.">Mapping the mechanical pulse of single cardiomyocytes with the atomic force microscope.</a> European Biophysics Journal. 28, 179-186 (1999).</li><li>Smith, B. A., Tolloczko, B., Martin, J. G., Gr&#252;tter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+Viscoelastic+Behavior+of+Cultured+Airway+Smooth+Muscle+Cells+with+Atomic+Force+Microscopy:+Stiffening+Induced+by+Contractile+Agonist.">Probing the Viscoelastic Behavior of Cultured Airway Smooth Muscle Cells with Atomic Force Microscopy: Stiffening Induced by Contractile Agonist.</a> Biophysical Journal. 88, 2994-3007 (2005).</li></ol>

The authors declare that they have no competing financial interests.

In this report, we have described the techniques necessary for successful isolation and culture of adult CMs from the mouse heart. Our technique allows for subsequent study of CM function and excitability using the methods described above. The critical parameter for studying functionality of adult CMs is the health and quality of the isolated CMs. As described above, our techniques allow for a high yield of functional cells that are amenable to manipulation of gene expression using adenoviral/lentiviral infections in ...

Murine models of cardiovascular disease have served as effective tools for elucidating fundamental disease mechanisms1,2 as well as for identifying potential therapeutic targets1,3. In particular, the use of both murine models of acquired heart disease (such as pressure-overload)4,5 and transgenic mouse models have advanced our understanding of heart disease6-8. The use of cell culture techniques to study signaling cascades3,9,10 and alterations in individual proteins that underlie cellular excitability and excitation-contraction coupling in the heart11-13 at the level of the single cell have complemented the in vivo mouse models. However, the lack of adequate cell lines that reflect adult CM structure and function has been a significant limitation. Investigators have sought to overcome this by studying individual proteins, such as ion channels, in heterologous expression systems14, and while these studies have provided us with useful information in terms of ion channel biophysics or protein trafficking, inadequate representation of the native microenvironment of CMs is a significant limitation. Secondly, since most of these heterologous cells do not have a mature contractile apparatus, it has not been possible to study contractile function and the complex interplay between cellular excitability and contraction. For this reason, researchers have turned to primary cardiac cell cultures for many of their in vitro functional studies. Finally, isolated cardiomyocyte studies allow assessment of contractile function without the confounding factors of multicellular preparation including the effect of scar or fibrosis and fiber orientation.
Primary neonatal rat ventricular cardiomyocytes (NRVMs) are relatively easy to culture, can be infected with adenoviruses and lentiviruses to manipulate gene expression15, and have therefore been used successfully1, but have limitations of their own. Although they provide a physiologic microenvironment1 and have been the workhorse of the signaling field, substantial differences between the morphology and subcellular organization of NRVMs and adult cardiomyoctyes make them an inadequate model for the investigation of ionic fluxes and excitation-contraction coupling in the adult heart. Most notably NRVMs lack a definitive t-tubular subsystem4. Since Ca2+ flux and dynamics are critically dependent on mature t-tubular and sarcoplasmic reticulum (SR) structure6, Ca2+ dynamics and functional studies of the cardiac contractility in NRVMs are not an accurate reflection of these critical processes in adult cardiomyocytes. Further, some components of signaling pathways differ between neonatal and adult mice9, thereby providing another limitation for studying disease processes and their impact on cellular excitability and contractility in NRVMs. Finally, the distribution of the contractile machinery leads to multidirectional and non-uniform cell shortening limiting the accuracy of the contractile measurements.
The use of isolated adult cardiomyocytes provides therefore a more accurate in vitro modeling system. The extraordinary growth of knowledge made possible by the genetic manipulation of mice underlines the significance of obtaining functional isolated cardiomyocytes from mice. In fact, the characterization of adult CMs isolated from mouse models has shed light on many biological and pathological events. Isolated CMs from transgenic mouse models have allowed for studies of the gain or loss of function of proteins on the contractile properties of single cells2,16, and viability in disease models such as ischemia/reperfusion17,18, thereby complementing information gained from in vivo studies on these mice. Use of isolated adult CMs from murine models of acquired heart disease3,19,20 (such as transverse aortic constriction-induced pressure overload, that mimics hypertension or aortic valve stenosis) or exercise5,21 (for modeling physiological hypertrophy) allows for examination of the interaction of signaling cascades implicated in these processes with cellular excitability and excitation-contraction coupling at the level of the single cell. Furthermore, the ability to manipulate gene expression using adenoviral-driven gene expression in adult CMs affords us the opportunity to dissect the components of complex signaling pathways. 
From an electrophysiological perspective, whole-cell voltage and current clamp experiments on isolated adult CMs have been critical in elucidating the nature of ionic fluxes at baseline and in various disease states. Because of the complex structure of the cellular membrane and the differential protein scaffolding structures between adult CMs and NRVMs or heterologous cell lines, the ability to patch adult cells gives a much better representation of the effects of certain membrane proteins, structural proteins, and ion channel interacting partners on the electrophysiological components of the adult heart. 
Despite such prominent advantages in studying adult murine cardiomyocytes, isolating and culturing adult mice cardiomyocytes has been challenging, urging the need for a systematic and accurate description of the methodology to isolate viable mouse cardiomyocytes and to maintain them in culture to allow further genetic manipulation using viral vectors. Previous studies have used either acutely isolated mouse adult CMs or cultured rat adult CMs. The latter are easier to culture than adult mouse CMs, and most experiments manipulating gene expression in vitro have used rat adult CMs. Few studies have successfully altered and investigated functional gene expression in mouse adult CMs, presenting a large limitation in the scope of experiments. Therefore, here we present in detail such methodologies, modified from previous investigations, for the isolation7,8,22, culture3,10,15,23, adenoviral infection11-13,15, and functional analysis of adult mouse ventricular cardiomyoctyes. This isolation protocol results in Ca2+-tolerant, excitable cardiomyocytes that we have successfully cultured for up to 72 hr and transiently transfected with adenovirus. The functionality of these isolated cells can be assessed using the MMSYS imaging system14,24 and patch clamp, which will also be discussed.

<table border="1">
<tbody>
 <tr>
 <td>Name of 			Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Sodium Chloride</td>
 <td>Sigma</td>
 <td>S7653</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium Chloride</td>
 <td>Sigma</td>
 <td>P9333</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium Chloride</td>
 <td>Sigma</td>
 <td>M8266</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>Sigma</td>
 <td>H3375</td>
 <td></td>
 </tr>
 <tr>
 <td>Sodium Phosphate 			Monobasic</td>
 <td>Sigma</td>
 <td>S8282</td>
 <td></td>
 </tr>
 <tr>
 <td>D-glucose minimum</td>
 <td>Sigma</td>
 <td>G8270</td>
 <td></td>
 </tr>
 <tr>
 <td>Taurine</td>
 <td>Sigma</td>
 <td>T0625</td>
 <td></td>
 </tr>
 <tr>
 <td>2,3-Butanedione 			monoxime</td>
 <td>Sigma</td>
 <td>B0752</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase B</td>
 <td>Roche Applied 			Science</td>
 <td>11088807001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche Applied 			Science</td>
 <td>11088858001 </td>
 <td></td>
 </tr>
 <tr>
 <td>Protease 			XIV from Streptomyces griseus</td>
 <td>Sigma</td>
 <td>P5147</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin from Bovine 			Serum</td>
 <td>Sigma</td>
 <td>A2153</td>
 <td></td>
 </tr>
 <tr>
 <td>Calcium Chloride</td>
 <td>Sigma</td>
 <td>C8106</td>
 <td></td>
 </tr>
 <tr>
 <td>Minimum Essential 			Media</td>
 <td>Sigma</td>
 <td>51411C</td>
 <td></td>
 </tr>
 <tr>
 <td>Albumin solution 			from bovine serum</td>
 <td>Sigma</td>
 <td>A8412</td>
 <td></td>
 </tr>
 <tr>
 <td>L-glutamine</td>
 <td>Sigma</td>
 <td>G3126</td>
 <td></td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Sigma</td>
 <td>P4333</td>
 <td></td>
 </tr>
 <tr>
 <td>Insulin-transferrin-sodium 			selenite media supplement</td>
 <td>Sigma</td>
 <td>I1884</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Chloride</td>
 <td>Sigma</td>
 <td><a href="http://www.sigmaaldrich.com/catalog/product/sial/289329?lang=en&amp;region=US" target="_blank">289329</a></td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Sigma</td>
 <td>G3291</td>
 <td></td>
 </tr>
 <tr>
 <td>Adenosine 			5'-triphosphate magnesium salt</td>
 <td>Sigma</td>
 <td>A9187</td>
 <td></td>
 </tr>
 <tr>
 <td>Ethylene 			glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic 			acid</td>
 <td>Sigma</td>
 <td>E3889</td>
 <td></td>
 </tr>
 <tr>
 <td>Cesium Hydroxide 			Solution</td>
 <td>Sigma</td>
 <td>232041</td>
 <td></td>
 </tr>
 <tr>
 <td>Tetraethylammonium 			hydroxide solution</td>
 <td>Sigma</td>
 <td>86643</td>
 <td></td>
 </tr>
 <tr>
 <td>OptiVisor optical 			glass binocular 			visor</td>
 <td>Dohegan Optical 			Company Inc.</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Tissue 			forceps, 5.5", 1x2 teeth</td>
 <td>Roboz Scientific</td>
 <td>RS-8164</td>
 <td></td>
 </tr>
 <tr>
 <td>Moloney 			forceps - 4.5" (11.5 cm) long slight curve, serrated </td>
 <td>Roboz Scientific</td>
 <td>RS-8254</td>
 <td></td>
 </tr>
 <tr>
 <td>Dumont 			#3 Forceps, Dumostar, tip size 0.17 x 0.10mm</td>
 <td>Roboz Scientific</td>
 <td>RS-4966</td>
 <td></td>
 </tr>
 <tr>
 <td>Packer 			Mosquito Forceps 5" Straight Flat</td>
 <td>Roboz Scientific</td>
 <td>RS-7114</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro 			Dissecting Scissors 4.5" Curved Sharp/Sharp </td>
 <td>Roboz Scientific</td>
 <td>RS-5917</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Dissecting Scissors 3.5" Straight Sharp/Sharp20mm</td>
 <td>Roboz Scientific</td>
 <td>RS-5907</td>
 <td></td>
 </tr>
 </tbody>
</table>

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of heart disease. In particular, the ability to study ion homeostasis, ion channel function, cellular excitability and excitation-contraction coupling and their alterations in diseased conditions and by disease-causing mutations have led to significant insights into cardiac diseases. Furthermore, the lack of an adequate immortalized cell line to mimic adult CMs, and the limitations of neonatal CMs (which lack many of the structural and functional biomechanics characteristic of adult CMs) in culture have hampered our understanding of the complex interplay between signaling pathways, ion channels and contractile properties in the adult heart strengthening the importance of studying adult isolated cardiomyocytes. Here, we present methods for the isolation, culture, manipulation of gene expression by adenoviral-expressed proteins, and subsequent functional analysis of cardiomyocytes from the adult mouse. The use of these techniques will help to develop mechanistic insight into signaling pathways that regulate cellular excitability, Ca2+ dynamics and contractility and provide a much more physiologically relevant characterization of cardiovascular disease.

The isolation of adult cardiomyoctyes results in rod-shaped, striated, and quiescent (not spontaneously beating) cells (Figure 5A). Dead cells will look rounded and no striations will be present. Quiescent cells can be cultured and transfected with adenovirus to manipulate gene expression (Figures 5B and 5C). After 24 hr of culture, the morphology of the live cells does not change, they are still Ca2+-tolerant, and they can be paced by field stimulation. With ...

Watch this Scientific Journal Video about Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes at JoVE.com

Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Here we describe the isolation of adult mouse cardiomyoctyes using a Langendorff perfusion system. The resulting cells are Ca2+-tolerant, electrically quiescent and can be cultured and transfected with adeno- or lentiviruses to manipulate gene expression. Their functionality can also be analyzed using the MMSYS system and patch clamp techniques.

The use of primary cardiomyocytes (CMs) in culture has provided a powerful complement to murine models of heart disease in advancing our understanding of ...

isolation-culture-functional-characterization-adult-mouse

Research

JoVE Journal

Biology

24.3K ビュー.  Beth Israel Deaconess Medical Center, Harvard Medical School. ここでは、ランゲンドルフ灌流システムを使用して、成体マウスcardiomyoctyesの単離を記載している。得られた細胞は、電気的に休止状態、のCa 2 +·トレラントであり、培養およびアデノ又は遺伝子発現を操作するためのレンチウイルスでトランスフェクトすることができる。それらの機能は、MMSYSシステムおよびパッチクランプ技術を用いて分析すること?...

ビデオ: Isolation, Culture, and Functional Characterization of Adult Mouse Cardiomyoctyes

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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 ...

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). ...

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 ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...