Name: isolation and cryopreservation of neonatal rat cardiomyocytes
Uploaded: 2015-04-09T14:00:00.000+00:00
Duration: 7 min 5 s
Description: Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

The following protocol is designed for the isolation of cardiomyocytes from one litter of neonatal rat pups (10-14 pups). If litter size is significantly different, the procedure may have to be scaled to compensate. The pups should be around 48&#177;6 hr old. All procedures herein have been approved by the North Carolina State University Institutional Animal Care and Usage Committee (IACUC).
1. Cell Isolation Preparation
NOTE: Perform the following steps the day before isolation.
<ol>
	<li>Autoclave the following: large scissors, small scissors, small sharp forceps, large forceps. Use a gravity cycle for 60 min at a minimum of 121 &#176;C and a drying time of 30 min.</li>
	<li>Place 40 ml of 0.1% trypsin solution in refrigerator to thaw O/N. Refrigerate Hank&#8217;s Balanced Salts Solution (HBSS) if not already cold. Prepare a 20% fetal bovine serum (FBS) stock media solution.
	<ol>
		<li>In 400 ml of Iscove&#39;s Modified Dulbecco&#39;s media (IMDM) add 100 ml FBS, 5 ml L-glutamine (200 mM), 2.5 ml gentamicin (10 mg ml-1), and 0.9 ml 2-mercaptoethanol. Sterilize using vacuum filter. Store media at 4&#176;C for up to 3 weeks and this is scalable for smaller or larger volumes. Dilute 20% FBS stock media 1:1 with IMDM to produce 10% FBS NRCM media. Only make in small batches to prevent repeated heating and cooling of media.</li>
	</ol>
	</li>
</ol>
2. Cell Isolation Day 1
<ol>
	<li>Perform the following work in a culture hood to prevent contamination of the samples. For best timing start this work in the afternoon since there is an O/N incubation.</li>
	<li>Prepare workspace inside a sterile culture hood. Place bench pad in culture hood. Fill two 250 ml beakers with 70% ethanol and place surgical tools in them. Place small bucket of ice in hood. Ensure that it is large enough to hold several tubes and a 150 mm dish. UV sterilize the hood for 10 min. Place 40 ml 0.1% trypsin on ice.</li>
	<li>Retrieve neonatal rat pups from the mother. To keep the animals warm until use, place them in an insulated container such as an ice bucket wrapped in a bench pad to keep them warm.</li>
	<li>Fill a 150 mm culture dish with 25 ml HBSS and place on ice. Unwrap 2-3 gauze pads and place in work area. Holding pup by the skin on the back of the neck, spray it with 70% ethanol and place in hood. NOTE: To best maintain sterility, this is can be done by another person who then places the cleaned pup in the hood.</li>
	<li>Hold the pup by the skin on the back of the neck. Then using the large scissors, in one quick, strong cut euthanize by decapitation. Blot the blood with gauze. NOTE: Due to variances in IACUC requirements, it may be more appropriate to clean the pup with an ethanol wipe rather than spraying it. Be sure to check with the university IACUC as to their guidelines for this step.</li>
	<li>Continue cutting down anterior side of chest through the ribcage. Squeeze the skin on the neck to help open the ribcage until the heart is visible. Using the small scissors, remove the heart and place in the HBSS culture dish on ice. Repeat for the other pups.</li>
	<li>Remove any non-heart tissue from the samples and swirl the HBSS gently to wash the hearts. Transfer the heart tissue to another culture dish containing fresh HBSS and further wash the tissue. Cut the tissue further until the pieces are 1-3 mm3, and then transfer to the trypsin solution. Place trypsin solution on rocker in 4&#176;C refrigerator O/N.</li>
</ol>
Cell Isolation Day 2
NOTE: While aspirating in the following steps, use a pipette rather than a vacuum line. The tissue moves easily, and a vacuum line will clog or remove tissue still containing cells. It is best to use a pipette for aspiration instead. If tissue enters the pipette, pipette back out to remove it.
<ol>
	<li>Make three aliquots of 10% NRCM Media: one with 25 ml and two with 15 ml. Place one 25 ml tube of media in 37 &#176;C water bath. Add 4 ml HBSS to 5 different 15 ml conical tubes and place all on ice. Label each 15 ml tube #1-5.</li>
	<li>Add 40 ml HBSS to a 50 ml conical tube. Add 10 ml of HBSS to collagenase, pipette to suspend, and combine the solutions to create 50 ml of 1 mg ml-1 collagenase solution. Sterilize using 0.22 &#181;m vacuum filter, and store at RT.</li>
	<li>Retrieve heart tissue/trypsin tube. Ensure that trypsin is clear, and the tissue appears fluffy. Allow the tissue to settle to corner of tube and aspirate as much liquid as possible.</li>
	<li>Add 25 ml warm 10% FBS NRCM media to the tube and swirl by hand. Cap and rotate in 37 &#176;C water bath for 2 min. Aspirate liquid again. The tissue should float at this step; if so, aspirate from the bottom of the tube.</li>
	<li>Add 10 ml collagenase. Rotate in water bath for 2 min or until the solution is cloudy. Quickly aspirate the liquid. Add 10 ml fresh collagenase to tube containing the tissue and rotate in water bath for 2 min. Pipette to mix, transfer solution to tube #1, and place on ice. It may not be possible to remove all of the liquid. Repeat steps 3.2-3.6 for the other three 15 ml conical tubes. Transfer any remaining liquid to tube # 5.</li>
	<li>Centrifuge 15 ml tubes at 410 rcf for 8 min. While centrifuging, place 15 ml 10% FBS NRCM media tube in water bath. Place 40 &#181;m cell strainer on top of a 50 ml conical tube and wet with 2 ml cold HBSS.</li>
	<li>Aspirate supernatant from spun down cells. Add 6 ml cold HBSS to each tube and resuspend. Pipette cell suspensions through the strainer into 50 ml conical to remove any tissue. Rinse sides of each 15 ml tube with 3 ml cold HBSS and add to strainer. Centrifuge cells at 410 rcf for 10 min. Aspirate supernatant. Resuspend cell pellet in 10 ml 10% FBS NRCM media and transfer to T-75 flask. Rinse conical with 5ml media and add to flask.</li>
	<li>Incubate for 1 h at 37 &#176;C and 5% CO2. Place last 15 ml 10% FBS NRCM media tube into water bath. Transfer contents of T-75 to a new T-175 flask, rinse T-75 with 15 ml media and add to T-175. Incubate for 1 hr.</li>
</ol>
4. Cryopreservation
<ol>
	<li>Place freezing media on ice. Transfer cell media from T-175 to 50 ml conical tube, and collect 10 &#181;l&#160;of cell suspension for cell counting using Trypan blue. Centrifuge at 410 rcf for 5 min. While centrifuging, count cells using a cell counter.</li>
	<li>Aspirate culture media, and resuspend cells in freezing media at a concentration of 2-4 million cells per ml. Place 1 ml of cell suspension in cryogenic vial, then place vials a controlled cooling rate freezing container, and place in -80 &#176;C freezer O/N. Within 1-2 days, transfer vials in liquid nitrogen.</li>
</ol>
5. Thawing Cells
<ol>
	<li>Coat dish in fibronectin by dissolving 250 &#181;l&#160;of fibronectin (stock concentration 1 mg ml-1) into 9.75 ml water (final concentration 0.025 mg ml-1). Add enough solution to cover culture plate and incubate at 37oC for at least 30 min.
	<ol>
		<li>In four 15 ml&#160;conical tubes, make the DMSO/media mixtures as follows by adding DMSO to 20% Media. Add (5%) 9.5 ml media with 500 &#181;l&#160;DMSO. Add (2.5%) 9.75 ml media with 250 &#181;l&#160;DMSO. Add 10 ml&#160;media (make two of these).</li>
	</ol>
	</li>
	<li>Heat the aforementioned media mixtures in 37 &#176;C water bath. Remove cells from liquid nitrogen and place on dry ice. When media mixtures are warm, thaw cells in 37 &#176;C water bath until just thawed. Do not leave cells in water bath for too long as this may cause damage to the cells.</li>
	<li>Transfer contents of cryogenic vial to 5% DMSO/media tube. Centrifuge at 410 rcf for 8 min. Aspirate supernatant leaving cell pellet. Add contents of 2.5% DMSO/media tube to cell pellet and resuspend. Centrifuge at 410 rcf for 8 min. Aspirate supernatant leaving cell pellet.</li>
	<li>Add contents of one 0% DMSO/media tube to cell pellet and resuspend. Centrifuge at 410 rcf for 8 min. Aspirate supernatant leaving cell pellet.</li>
	<li>Add contents of second 0% DMSO/media to cell pellet and resuspend. Take small sample for cell counting. Centrifuge at 410 rcf for 8 min. While centrifuging count cells in a haemocytometer using Trypan Blue to label dead cells. Record cell amount, as well as viability.</li>
	<li>Aspirate supernatant leaving cell pellet. Resuspend in 10% NRCM media (optional: add 100 &#181;M bromodeoxyuridine (BrdU)) and plate cells on fibronectin-coated dishes.</li>
</ol>
6. Cell Culture
<ol>
	<li>Plate cells at about 100k cells per cm2. Adjust the plating density based on the intended use of the culture. Prepare 2% NRCM media by diluting 20% Media in IMDM in a 1:10 ratio. The following is an outline of a typical culture.</li>
	<li>On day 1, rinse dead cells away with warm IMDM, add 10% NRCM (optional 100 &#181;M BrdU). On day 2, rinse dead cells away with warm IMDM, add 10% NRCM. Day 3&#8212;add 2% NRCM. On day 4 add 2% NRCM. On day 5 ensure that the cells are confluent and beating. Maintain cells in culture for around one week. At that point fibroblasts may begin to significantly outgrow the NRCMs.</li>
</ol>

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The authors have nothing to disclose.

This protocol allows for the NRCMs to be isolated, cryopreserved, and thawed. Thawing the cells is a crucial portion of the procedure. A series of DMSO dilutions is used to slowly remove DMSO from the cells14. It is important that the extraction of DMSO be performed quickly as the cells are particularly sensitive to dying immediately after thawing. If more or less cells are required for thawing, the volumes of the DMSO solutions can be scaled as needed.
One challenge to NRCM isolation and culture is the rapid proliferation of fibroblasts. This protocol uses preplating which has been shown to reduce the number of fibroblasts15. Preplating works by using uncoated flasks to which the fibroblasts attach much faster than cardiomyocytes. The sequence of media changes can be altered to reduce fibroblast growth. Fibroblasts will outgrow NRCMs in 10% NRCM media, but fibroblast growth is significantly slower in 2% NRCM media. There are other methods such as Percoll gradients to remove fibroblast prior to plating16,17 or the addition of BrdU to slow fibroblast growth3. Excessive fibroblast growth can be hard to detect using phase microscopy, but is easily shown using NRCM specific staining such as &#945;-SA (Figure 3). If there remains excessive fibroblast growth, the amount of FBS in the media can be decreased to 2% FBS on day 2. If necessary, plating can be done in 5% FBS media, though this may result in increased cell death.
This procedure is limited and no longer beneficial if using cell cultures that require large amounts of cells18,19. In cases where the amount of cells needed approaches the half amount of cells possible to harvest from a litter, this procedure will require more animals since approximately half of the cells are lost due to cryopreservation. The benefits of cryopreservation are greater when using fewer cells for assays such as immunostaining.
This protocol illustrates the methods for the isolation, cryopreservation, and subsequent of NRCMs. There are few alternatives, such as buying frozen cells from vendors, to using this method but buying cells may be cost prohibitive to some labs. Our methods only require materials and reagents commonly used in cell culture. In addition, utilizing these methods can greatly increase the efficiency and flexibility of lab work while reducing the use of lab animals. The methods described produce viable cultures that can be used for subsequent in vitro studies.

Cell culture of cardiomyocytes is a critical tool in modern cardiac research. Neonatal rat cardiomyocytes (NRCMs) are commonly used since the isolation and culture is easier than that of adult rat cardiomyocytes1. The NRCM method still has several limitations including a long isolation procedure and limited cell proliferation in the dish. There are numerous protocols for the isolation of NRCMs with most generally requiring 4-48 hr of work2&#8211;6. In addition, the cells are frequently isolated from 1 to 2-day old rat pups2,4&#8211;7; the timing of the birth can be unpredictable and conflict with other work in the lab. The isolations can be inefficient and wasteful if only a small amount of cells are needed for experiments. Most efforts on improving the workflow focus on reducing the isolation time, yet this does not solve the problems of timing the birth of the pups.
As alternatives, many labs utilize cardiomyocytes derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, the reprogramming and/or differentiation process can be very time consuming and costly as well. There can be other problems when using these cells as in vitro myocyte models. Both ESC- and iPSC- derived cardiomyocytes have been shown to exhibit differences in electrophysiology from primary cardiomyocytes8&#8211;10.
Dissociated NRCMs are capable of being stored for several days using refrigeration11, yet this does allow for long term storage. Liquid nitrogen is typically used to preserve cells for a greater period of time, but requires a cryoprotectant such as dimethyl sulfoxide (DMSO). Previous research has shown that the ideal concentration between 5-10% DMSO in the freezing media allows for cryopreservation of NRCMs, yet even then the viability remains low12. Although DMSO helps protect the cells during freezing, it can be toxic to cells at concentrations above 1.5%13. Previous studies have shown that slowly removing DMSO from the cells, may improve cell viability14.
We sought to improve the efficiency of NRCM cell-based assays by cryopreserving the cells following isolation. This allows for the cells to be thawed and used when necessary, reducing the frequency of isolations and consumption of animals. Using this method, we show that it is possible to cryopreserve NRCMs and thaw them for use at a later time. After thawing the cells maintain an acceptable viability and produce NRCM cultures that are positive for &#945;-sarcomeric actinin (&#945;-SA) and contract spontaneously.

<table><tbody><tr><td>IMDM (+25 mM HEPES +L-glutamine)</td><td>Gibco</td><td>12440-046</td><td>With added 25 mM HEPES and L-glutamine</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>FBS</td><td>Hyclone</td><td>SH30070.03</td><td></td></tr><tr><td>Gentamicin</td><td>Gibco</td><td>15710-064</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco</td><td>21985-023</td><td></td></tr><tr><td>HBSS (+Ca +Mg)</td><td>Corning</td><td>21-020-CV</td><td>With added calcium and magnesium, pH 7.1-7.4</td></tr><tr><td>Trypsin 0.25%</td><td>Gibco</td><td>25300-056</td><td></td></tr><tr><td>Trypsin 0.05%</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cryostor CS5</td><td>BioLife Solutions</td><td>205102</td><td>Freezing media</td></tr><tr><td>Cryogenic Vial</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>Collagenase</td><td>Sigma</td><td>C1889-50MG</td><td></td></tr><tr><td>40 &amp;mu;m Cell Strainer</td><td>Greiner Bio-One</td><td>542040</td><td></td></tr><tr><td>Sterilizing Vacuum Filter (0.22&amp;mu;m)</td><td>Corning</td><td>431118</td><td></td></tr><tr><td>50 ml Conical</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>15 ml Conical</td><td>Corning</td><td>430790</td><td></td></tr><tr><td>trypan blue</td><td>Cellgro</td><td>25-900-CI</td><td></td></tr><tr><td>Mr Frosty Freezing Container</td><td>ThermoScientific</td><td>5100-0001</td><td></td></tr><tr><td>Millicell EZ&amp;nbsp;SLIDES</td><td>Millipore</td><td>PEZGS0416</td><td></td></tr><tr><td>&amp;alpha;-sarcomeric actinin antibody</td><td>Sigma</td><td>A7811</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>356008</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>BD Biosciences</td><td>51-7581KZ</td><td></td></tr></tbody></table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Following the isolation of the neonatal rat cardiomyocytes, the cells are frozen down to liquid nitrogen, and can be stored for at least several months. Typically upon thawing, the viability determined by Trypan Blue analysis will be between 40-60%. Though this is lower than other cell types, with proper seeding and culture the cells will proliferate (Figure 1). In order to verify contractility, the cells can be imaged using a phase contrast microscopy. The cells should begin to spontaneously contract within about three days (Figure 2). For immunocytochemistry (ICC) assays, cells were plated in 4-well culture slides with 200,000 cells per well. In order to show that the cells in culture are NRCMs the cells were labeled with &#945;-SA and imaged using fluorescence microscopy. There are numerous &#945;-SA positive cells indicating NRCMs in the culture (Figure 4). Other experiments have been performed including culturing NRCMs with human cardiac stem cell-derived exosomes to test regenerative properties of exosomes (Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/52726/52726fig1.jpg" /> 
Figure 1. NRCM Proliferation In Vitro. Fluorescence microscopy image of NRCM with staining for &#945;-SA (green), Ki67 (red), and DAPI (blue) for both (A) freshly isolated NRCMs and (B) cryopreserved then thawed NRCMs. Arrows indicates proliferating cells (ki67-positive). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52726/52726fig2.jpg" /> 
Figure 2. Contraction of Cryopreserved and Thawed NRCM In Vitro. NRCMs spontaneously contracting in vitro. Unlike freshly isolated NRCMs, it may take a few days before contractility is recovered. Video were recorded five days after thawing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/Figure2.m4v" target="_blank">Please click here to view this video.</a>
<img alt="Figure 3" src="/files/ftp_upload/52726/52726fig3.jpg" /> 
Figure 3. Contraction of Freshly Isolated NRCM In Vitro. Freshly isolated NRCMs spontaneously contracting in vitro. These cells will begin to exhibit contractility much sooner. Video were recorded 3 days after isolation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/figure_3_rgb.m4v" target="_blank">Please click here to view this video.</a>
<img alt="Figure 4" src="/files/ftp_upload/52726/52726fig4.jpg" /> 
Figure 4. Purity of NRCM Culture. Fluorescence microscope images of both (A) freshly isolated NRCMs in culture and (B) cryopreserved and thawed NRCMs after 5 days in culture. NRCMs show positive staining for &#945;-SA (green) whereas fibroblasts can be seen where nuclei (blue) are not surrounded by &#945;-SA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52726/52726fig5.jpg" /> 
Figure 5. Use of Thawed NRCM for Cell-Based Assay. Confocal microscopy image of NRCMs, highlighted by &#945;-SA (green), incubated with DiI-labeled exosomes (red) from cardiac stem cells. Cell nuclei labeled with DAPI (blue). In order to create an oxidative stress model, NRCMs were treated with 100 &#181;M hydrogen peroxide for 18 hr, prior to being incubated with exosomes for 4 hr to reverse the oxidative stress. Arrows indicate regions of high exosome concentrations in some cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52726/52726fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K Views.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Video: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Neuroscience

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 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 ...

Biology

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 ...

A Novel Minimally Invasive Slow Injection Method for Reducing Tissue Injury

A Novel Cell Injection Method with Minimum Invasion

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient amount of suspension solution to allow the cells to enter the tissue. The volume of the suspension solution affects the tissue, and this can cause major invasive injury as a result of the cell injection. This paper reports on a novel cell injection method, called slow injection, that aims to avoid this injury. However, pushing out the cells from the needle tip requires a sufficiently high injection speed according to Newton's law of shear force. To solve the above contradiction, a non-Newtonian fluid, such as gelatin solution, was used as the cell suspension solution in this work. Gelatins solution have temperature sensitivity, as their form changes from gel to sol at approximately 20 &#176;C. Therefore, to maintain the cell suspension solution in the gel form, the syringe was kept cooled in this protocol; however, once the solution was injected into the body, the body temperature converted it to a sol. The interstitial tissue fluid flow can absorb excess solution. In this work, the slow injection technique allowed cardiomyocyte balls to enter the host myocardium and engraft without surrounding fibrosis. This study employed a slow injection method to inject purified and ball-formed neonatal rat cardiomyocytes into a remote area of myocardial infarction in the adult rat heart. At 2 months following the injection, the hearts of the transplanted groups showed significantly improved contractile function. Furthermore, histological analyses of the slow-injected hearts revealed seamless connections between the host and graft cardiomyocytes via intercalated disks containing gap junction connections. This method could contribute to next-generation cell therapies, particularly in cardiac regenerative medicine.

Author Spotlight: A Novel Cell Injection Method with Minimum Invasion  

This method eliminates any major invasion during cell injections caused by the cell suspension solution.

Directly injecting cells into tissues is a necessary process in cell administration and/or replacement therapy. The cell injection requires a sufficient ...

Bioengineering

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

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 ...

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

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

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

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

Isolation, 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. ...

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

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