This project was funded by the UCSF Program for Breakthrough Biomedical Research (funded in part by the Sandler Foundation), the NIH Pathway to Independence Award (R00HL114738), and the Edward Mallinckrodt Jr. Foundation. JJ was supported by a postdoctoral fellowship from the NIH (T32HL007731). The authors are solely responsible for the contents of this work, which does not necessarily represent the official views of the NIH.

All procedures outlined here have been approved by the Institutional Animal Use and Care Committee at the University of California, San Francisco.
NOTE: Briefly, after extracting the heart from the mouse thorax, coronary retrograde perfusion is used to efficiently digest the extracellular matrix with collagenase and protease XIV. The ventricles are then isolated, mechanically dissociated and filtered into a single cell suspension. Gravity sedimentation is performed to isolate ...

The overall health of the isolated cardiomyocytes depends on several important aspects of this protocol. First, the time from heart extraction to perfusion is critical and should be performed in 5 min&#160;or less. Removal of calcium helps to dissociate cell-cell interactions, but can negatively impact cell health long-term.29-32 Thus, we find it sufficient to remove calcium during the initial few minutes of perfusion by EGTA (ethylene glycol tetraacetic acid) chelation,22 but quickly restore calciu...

Cultured cardiomyocytes are frequently used to monitor cell behavior in a well-controlled environment in vitro. For example, morphological, electrical, biochemical, or mechanical cell properties can be studied on engineered substrates,1,2 in defined media, and in response to small molecule drugs, peptides, gene regulation,3 or electrical stimulation.4 The cellular content can also be controlled using defined co-cultures.5 These in vitro experiments are useful in large drug or genetic screens and complement in vivo methods for various types of investigations involving cardiomyocyte biology.
Long-term culture enables experimental avenues that require extended periods of time to achieve phenotypic change. A timely example is that of adult mammalian cardiomyocyte proliferation, where dedifferentiation, cell cycle re-entry, and cell division is typically studied over several days to weeks.6,7 Here, the extended culture time facilitates genetic manipulation,7,8 functional dedifferentiation (e.g., sarcomeric disassembly)9 and potentially transcriptional dedifferentiation.6 Subsequent cell cycle re-entry and cell division requires even longer culture periods to observe, especially if multiple rounds of division are the experimental goal. The importance of the cardiomyocyte cell-cycle is central to several recent key scientific works in heart regeneration, where the dedifferentiation and proliferation of pre-existing cardiomyocytes has been shown responsible for heart regeneration in zebrafish and neonatal mice.10-12 Thus, the possibility to stimulate dedifferentiation and cell cycle re-entry in mammalian adult cardiomyocytes remains a key question in human heart regeneration13-15
In vitro experiments studying the cell cycle of mammalian cardiomyocytes have predominantly used rat sources, due to their relative ease of long-term culture compared to mouse models.16 However, murine systems offer a rich resource of well-characterized transgenic tools and disease models that are useful in both in vitro and in vivo protocols. For example, Cre-based lineage tracing has enabled the identification of pre-existing cardiomyocytes as a source of regenerating myocardium in the neonatal mouse heart in vivo.12 In vitro studies of lineage-traced neonatal mouse cardiomyocytes have enabled the examination of interactions with stromal cells through co-culture with fibroblasts.5 However, due to its challenges,17 few reports exist of the isolation and long-term culture of adult mouse cardiomyocytes.18,19
The isolation of viable adult mouse cardiomyocytes for short-term culture alone is known to be a challenging task. This protocol provides step-by-step instructions on how to achieve viable cardiomyocytes from adult mice that can be used for both short-term as well as long-term investigations. Cardiomyocytes isolated using this protocol can be efficiently transduced with adenoviral vectors20,21 and cultured for weeks. These methods provide a powerful system to study cardiomyocyte biology in vitro.
The methods described herein are based on several elements from previous works using variations of the Langendorff retrograde perfusion.18,22 Although several protocols have been published on the isolation of adult mouse cardiomyocytes for short-term culture and study,23-25 the advantage of this protocol is the ability to culture the isolated cardiomyocytes long-term. This will be useful in the study of cellular processes involving ectopic gene expression and requiring extended periods of time, such as in cellular reprogramming strategies.

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			<td height="21" style="height:21px;width:295px;">Equipment</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td style="width:161px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated water jacket</td>
			<td>Radnoti</td>
			<td>158831</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Circulating heated water bath, Isotemp</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:99px;">3013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Laboratory pump</td>
			<td style="width:148px;">Watson-Marlow</td>
			<td style="width:99px;">323</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Hemostats</td>
			<td style="width:148px;">Exelta</td>
			<td style="width:99px;">63042-090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tissue forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #7 curved forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91197-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Dumont #5 fine forceps</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small dissection scissors</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">470128034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Extra fine bonn scissors</td>
			<td>FST</td>
			<td style="width:99px;">14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fine spring scissors</td>
			<td style="width:148px;">FST</td>
			<td style="width:99px;">91500-09</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Name</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td>Comments</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Materials</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">NaCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">S9888</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">KCl</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25837</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O</td>
			<td style="width:148px;">Fisher</td>
			<td style="width:99px;">S25414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">86329</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butane dione monoxime (BDM)</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">B0753</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:148px;">Fisher&#160;</td>
			<td style="width:99px;">BP310100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">G-7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Insulin</td>
			<td style="width:148px;">Novo Nordisk</td>
			<td style="width:99px;">393153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EGTA</td>
			<td style="width:148px;">Amresco</td>
			<td style="width:99px;">0732-288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Protease, type XIV</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">P5147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Collagenase II</td>
			<td style="width:148px;">Worthington</td>
			<td style="width:99px;">LS004176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">MEM</td>
			<td style="width:148px;">Corning</td>
			<td style="width:99px;">15-010-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">FBS, heat inactivated</td>
			<td style="width:148px;">JRS</td>
			<td style="width:99px;">43613</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Primocin</td>
			<td style="width:148px;">Invitrogen</td>
			<td style="width:99px;">NC9141851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Ethyl Carbamate</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">AAA44804-18</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">215 micron mesh</td>
			<td style="width:148px;">Component supply</td>
			<td style="width:99px;">U-CMN-215-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G blunt ended needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305183</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">20 G beveled needle</td>
			<td style="width:148px;">Becton Dickinson</td>
			<td style="width:99px;">305176</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Lab tape</td>
			<td style="width:148px;">VWR</td>
			<td style="width:99px;">89097-990</td>
			<td></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:295px;">Surgical tape</td>
			<td style="width:148px;">3M</td>
			<td style="width:99px;">1527-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Silk suture, 7-0</td>
			<td style="width:148px;">Teleflex</td>
			<td style="width:99px;">15B051000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mouse anti-alpha-actinin antibody</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">A7811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Alexa Fluor 488 goat anti-mouse IgG1 antibody</td>
			<td style="width:148px;">Thermo Fisher</td>
			<td style="width:99px;">A21121</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

A wild type adult ICR (CD1) mouse heart typically yields 500,000 to 1 million cardiomyocytes from a successful isolation. Immediately after isolation, the cells maintain a mostly rod-shaped appearance (Figure 3A) with intact sarcomeres and can be used for functional studies involving cardiomyocyte contractility. A high percentage of rod-shaped cardiomyocytes (above 90%) is an indication of effective perfusion and digestion. Viable cardiomyocytes will be large (~ 100 - 200...

Watch this Scientific Journal Video about Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes at JoVE.com

Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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 ...

The overall goal of this procedure is to isolate viable primary adult mouse cardiomyocytes for culture and adenoviral vector transduction. This method will answer some the key question in the field of cardiovascular research. In particular, we can adjust what prevents adult mammalian cardiomyocytes from cell cycle entry and completion.The main advantage of this technique is that a high yield of viable adult mouse cardiomyocytes can be obtained for long term culture. Visual demonstration of this method is critical, as the heart extraction and cannulation procedures are difficult, but must be performed quickly and efficiently. To extract the heart, begin by treating an adult mouse with five international units of heparin per gram of body weight by intraperitoneal injection.Wait 20 minutes to allow the heparin to circulate to the heart. Then, after administering anesthesia, confirm a lack of response to toe pinch and corneal reflexes and tape the animal's limbs to a surgery platform. Sterilize the incision area, with liberal application of 70 percent ethynol, and then pat the hair dry with a lab wipe.Next, use forceps to lift the skin just below the sternum and make a lateral incision through the skin and peritoneum. Then, using hemostats, gently lift the rib cage via the sternum and use small dissection scissors to extend the incision in a V shape toward the axially. Cut through the first rib and puncture the diaphragm on each side, keeping the rib cage lifted with the hemostats.Using small Iris scissors, completely transect the diaphragm. Then use the hemostats to retract the ribcage rostrally and quickly but carefully extend the incision through the ribcage toward the axially on both sides. Retract the middle section of the ribcage fully, laying down the attached hemostats to hold the tissue in place, and remove the pericardium with fine forceps.Next, use curved forceps to gently lift the heart and transect the attached veins and arteries. Wash the heart in ice cold perfusion buffer, then transfer the heart onto a small piece of 215 micron mesh. Add ice cold perfusion buffer to slow myocardial contractions, and place the dish under a dissection microscope.Using fine iris scissors, trim any access non-cardiac tissue. Then transect the aorta near the brachiocephalic artery, taking care not to let bubbles or tissue debris enter the opening. To cannulate the heart, fill the Petri dish with ice cold perfusion buffer until the solution rises above the opening of the 18 gauge cannula.Then, keeping the tip of the cannula and aorta below the surface of the buffer, sheath the aorta onto the cannula until the needle tip is just distal to the aortic valve. Slide a pre-tied 7-0 silk suture down the shaft of the cannulation needle until the suture is just above the needle tip and tighten the knot with fine forceps. Place a second suture just above the first.Then slowly depress the syringe until 0.5 milliliters of perfusion buffer has been injected through the coronary vasculature. After removing the syringe, attach the cannula to the perfusion outlet port. At the end of the perfusion, use forceps to remove the heart from the cannula, and place the heart in an empty Petri dish.Using fine Iris scissors, quickly trim the extra ventricular tissue and wash the ventricles in a new Petri dish containing perfusion buffer. After washing, transfer the ventricles into a dry Petri dish and place in a sterile hood. Collect 7.5 milliliters of digestion buffer from the heat jacket of the perfusion system, and add 2.8 microliters of one molar calcium chloride.Mix the solution by inversion. And then add two to three milliliters of the calcium chloride supplemented digestion buffer to the ventricles. Using fine forceps, quickly tritcherate the ventricular tissue until most of the pieces are smaller than one cubic millimeter, and add the remaining calcium chloride supplemented digestion buffer to the dissociated tissues.Mix the fragments by gentle agitation. And incubate them at 37 degrees Celsius and five percent carbon dioxide with continued agitation every two minutes until a sufficient number of cardiomyocytes have been released. Then return the dish to the laminar flow hood and use a transfer pipette to gently dissociate the tissue further.Wearing sterile gloves, fold the 215 micron mesh into a cone and hold it over a 50 milliliter conical tube. Pipette the tissue slurry onto the mesh, and allow the filtrate to drain into the tube. Followed by a wash with 7.5 milliliters of pre-warmed stop buffer.Transfer the cell suspension to a 15 milliliter conical and allow the cells to settle by gravity for 15 minutes. Then use a new transfer pipette to carefully remove the supernatant until only 50 to 100 microliters of solution remains above the cells. Add 10 milliliters of pre-warmed equilibrated culture medium to the cells, and mix by gentle inversion.Finally, after another 15 minute room temperature incubation, use a new transfer pipette to remove the supernatant from the settled cells, until only 50 to 100 microliters of medium remain. Immediately after isolation, the cardiomyocytes maintain a mostly rod-shaped appearance with intact sarcomeres and a sharp outer membrane under bright field illumination. Immunostaining for cardiomyocyte-specific sarcomeric markers such as alpha actinin reveals a distinct sarcomeric banding pattern.After several days in culture, the cardiomyocytes assume a rounded morphology. Within one to two weeks, the live cardiomyocytes attach to the substrate and begin spreading. Transduction with adenovirus, as observed in these GFP positive cardiomyocytes, can be used to achieve a strong gene expression within 48 to 72 hours.Once mastered, this technique can be completed in four hours if it is performed properly. While attempting this procedure, it is important to remember to achieve a secure cannulation that is free of air bubbles in order to efficiently profuse the coronary vasculature. Following this procedure, other methods such as immunostaining or live imaging can be performed in order to answer questions such as why are adult mammalian cardiomyocytes resistant to cell division?Don't forget that working with adenovirus can be extremely hazardous, so appropriate precautions must be followed, such as working in a class two biosafety cabinet.

isolation-culture-and-transduction-of-adult-mouse-cardiomyocytes

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Biology

26.2K ビュー.  University of California, San Francisco. The overall goal of this procedure is to isolate viable primary adult mouse cardiomyocytes for culture and adenoviral vector transduction. This method will answer some the key question in the field of cardiovascular research. In particular, we can adjust what prevents adult mammalian cardiomyocytes from cell cycle entry and completion.The main advantage of this technique is that a high yield of viable adult mouse cardiomyocytes can be obtained for long term culture. Visual demonstratio...

ビデオ: Isolation, Culture and Transduction of Adult Mouse Cardiomyocytes

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.

isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes

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

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

analyzing oxygen consumption rate in primary cultured mouse neonatal cardiomyocytes using an extracellular flux analyzer

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the...

mesoscopic optical imaging of whole mouse heart

Mesoscopic Optical Imaging of Whole Mouse Heart

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet...

creating a structurally realistic finite element geometric model of a cardiomyocyte to study the role of cellular architecture in cardiomyocyte systems biology

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and...

simultaneous isolation and culture of atrial myocytes, ventricular myocytes, and non-myocytes from an adult mouse heart

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

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

isolation and culture of neonatal mouse cardiomyocytes

Isolation and Culture of Neonatal Mouse Cardiomyocytes

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...

isolation and culture of adult mouse cardiomyocytes for cell signaling and in vitro cardiac hypertrophy

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...

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

isolation and cultivation of adult rat cardiomyocytes

Isolation and Cultivation of Adult Rat 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...

isolation, culture, and functional characterization of adult mouse cardiomyoctyes

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...

isolation and physiological analysis of mouse cardiomyocytes

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

single cell transcriptional profiling of adult mouse cardiomyocytes

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Single cell expression profiling allows the detailed gene expression analysis of individual cells.  We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific...

isolation and culture of primary mouse keratinocytes from neonatal and adult mouse skin

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

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...

isolation and culture of primary oral keratinocytes from the adult mouse palate

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also...

isolation and culture of adult neural stem cells from the mouse subcallosal zone

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

isolation and culture of dental epithelial stem cells from the adult mouse incisor

Isolation and Culture of Dental Epithelial Stem Cells from the Adult 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...

simultaneous isolation of high quality cardiomyocytes, endothelial cells, and fibroblasts from an adult rat heart

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

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

isolation and cryopreservation of neonatal rat cardiomyocytes

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...

isolation and culture of adult zebrafish brain-derived neurospheres

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish...

live cell imaging of primary rat neonatal cardiomyocytes following adenoviral and lentiviral transduction using confocal spinning disk microscopy

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

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...

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 ...

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

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.

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 and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

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 ...

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.

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 and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...