Name: isolation and cryopreservation of neonatal rat cardiomyocytes
Uploaded: 2015-04-09T14:00:00
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.

निम्नलिखित प्रोटोकॉल नवजात चूहे पिल्ले (10-14 पिल्ले) के एक कूड़े से cardiomyocytes के अलगाव के लिए बनाया गया है। कूड़े का आकार काफी अलग है, तो प्रक्रिया क्षतिपूर्ति करने के लिए बढ़ाया जाना पड़ सकता है। पिल्ले चारो?...

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NRCMs, पृथक cryopreserved, और thawed किया जा करने के लिए इस प्रोटोकॉल की अनुमति देता है। कोशिकाओं विगलन प्रक्रिया का एक महत्वपूर्ण हिस्सा है। DMSO के dilutions की एक श्रृंखला धीरे-धीरे 14 कोशिकाओं से DMSO के दूर करने के लिए प्रय?...

Cardiomyocytes की सेल संस्कृति आधुनिक हृदय अनुसंधान के क्षेत्र में एक महत्वपूर्ण उपकरण है। नवजात चूहे cardiomyocytes (NRCMs) आमतौर पर अलगाव के बाद से इस्तेमाल किया जाता है और संस्कृति वयस्क चूहे cardiomyocytes एक की तुलना में आसान है। NRCM पद्धति अभी भी डिश में एक लंबे समय के अलगाव की प्रक्रिया और सीमित सेल प्रसार सहित कई सीमाएं हैं। सबसे आम तौर पर काम 2-6 से 4-48 घंटे की आवश्यकता के साथ NRCMs के अलगाव के लिए कई प्रोटोकॉल रहे हैं। इसके अलावा, कोशिकाओं अक्सर 2 दिन पुराने चूहे पिल्ले 2,4-7 1 से अलग कर रहे हैं; जन्म के समय प्रयोगशाला में अन्य काम के साथ अप्रत्याशित और संघर्ष हो सकता है। कोशिकाओं का केवल एक छोटी राशि के प्रयोगों के लिए आवश्यक हैं अगर isolations अक्षम और बेकार हो सकता है। अलगाव के समय को कम करने पर कार्यप्रवाह ध्यान केंद्रित में सुधार लाने पर अधिकांश प्रयासों, अभी तक इस पिल्ले के जन्म के समय की समस्याओं का समाधान नहीं करता है। विकल्प के रूप में, कई प्रयोगशालाओं का उपयोगभ्रूण स्टेम कोशिकाओं (ESCs) या प्रेरित स्टेम कोशिकाओं (IPSCs) से व्युत्पन्न cardiomyocytes। हालांकि, reprogramming और / या भेदभाव की प्रक्रिया बहुत समय लगता है और महंगा के रूप में अच्छी तरह से हो सकता है। इन विट्रो myocyte मॉडल के रूप में इन कोशिकाओं का उपयोग करते समय अन्य समस्या हो सकती है। दोनों ESC- और iPSC- व्युत्पन्न cardiomyocytes प्राथमिक cardiomyocytes 8-10 से इलेक्ट्रोफिजियोलॉजी में मतभेद प्रदर्शन करने के लिए दिखाया गया है। अलग NRCMs प्रशीतन 11 का उपयोग कर कई दिनों के लिए भंडारित किया जा रहा करने में सक्षम हैं, अभी तक इस लंबी अवधि के भंडारण के लिए अनुमति नहीं है। तरल नाइट्रोजन आम तौर पर समय का एक बड़ा अवधि के लिए कोशिकाओं की रक्षा करने के लिए प्रयोग किया जाता है, लेकिन डाइमिथाइल sulfoxide (DMSO) के रूप में एक cryoprotectant ऐसे आवश्यकता है। पिछले अनुसंधान ठंड मीडिया में 5-10% DMSO के बीच आदर्श एकाग्रता NRCMs की cryopreservation के लिए अनुमति देता है कि दिखाया गया है, अभी तक फिर भी व्यवहार्यता कम 12 बनी हुई है। DMSO के मुक्त दौरान कोशिकाओं की रक्षा में मदद करता हैZing, यह 1.5% 13 से ऊपर सांद्रता में कोशिकाओं को विषाक्त हो सकता है। पिछले अध्ययनों से धीरे धीरे कोशिकाओं से DMSO के हटाने, सेल व्यवहार्यता 14 में सुधार हो सकता है कि पता चला है। हम अलगाव निम्नलिखित cryopreserving कोशिकाओं द्वारा NRCM सेल आधारित assays की दक्षता में सुधार करने की मांग की। यह कोशिकाओं thawed और आवश्यक, isolations और जानवरों की खपत की आवृत्ति को कम करने के लिए जब इस्तेमाल किया जा करने के लिए अनुमति देता है। इस विधि का उपयोग करना, हम यह NRCMs cryopreserve और बाद में उपयोग के लिए उन्हें पिघलना करने के लिए संभव है कि पता चलता है। विगलन के बाद कोशिकाओं को एक स्वीकार्य व्यवहार्यता बनाए रखने और अनायास α-sarcomeric actinin (α-एसए) और अनुबंध के लिए सकारात्मक रहे हैं कि NRCM संस्कृतियों का उत्पादन।

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM 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&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL 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&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-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.

नवजात चूहे cardiomyocytes के अलगाव के बाद, कोशिकाओं तरल नाइट्रोजन के लिए नीचे जमे हुए हैं, और कम से कम कई महीनों के लिए भंडारित किया जा सकता है। आमतौर पर विगलन पर, Trypan ब्लू विश्लेषण द्वारा निर्धारित व्यवहार्यता 40-60% ...

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

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes (Video) | JoVE

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.

अलगाव और नवजात चूहे cardiomyocytes की cryopreservation

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

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Developmental Biology

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

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चूहा हेड Branchiomeric मांसपेशियों से अलगाव और उपग्रह कोशिकाओं की विशेषता

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5 (Video) | JoVE

Gata4, Mef2c और Tbx5 का इष्टतम अनुपात व्यक्त एक Polycistronic निर्माण का उपयोग प्रेरित cardiomyocytes की बेहतर पीढ़ी

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

Isolation of Murine Embryonic Hemogenic Endothelial Cells (Video) | JoVE

Murine भ्रूण Hemogenic endothelial कोशिकाओं के अलगाव

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas (Video) | JoVE

नवजात माउस अग्न्याशय से कार्यात्मक आइलेट्स की प्रभावी अलगाव

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes (Video) | JoVE

प्रसव के बाद cardiomyocytes में कोशिका चक्र बदलाव के दृश्य और Nucleation का निर्धारण

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization (Video) | JoVE

बॉटलनोज़ डॉल्फिन (Tursiops truncatus) शुक्राणु: Collection, Cryopreservation, और Heterologous इन विट्रो निषेचन

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation (Video) | JoVE

इलेक्ट्रिकल उत्तेजना का उपयोग करने वाले बायोवायर में मानव स्टेम कोशिका-व्युत्पन्न कार्डिय्योमायसाइट्स का परिपक्वता

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification (Video) | JoVE

अलगाव और प्राथमिक चूहा वाल्व मध्यवर्ती कोशिकाओं के लक्षण वर्णन: एक नया मॉडल महाधमनी वाल्व कड़ा अध्ययन करने के लिए

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation (Video) | JoVE

हैंडलिंग और हार्मोनल परिपक्वता और शुक्राणु Cryopreservation के लिए पुरुष यूरोपीय ईल (एंगुइला एंगुइला) का उपचार

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...