We like to acknowledge Marcelo Toro for the assistance with flow cytometry. This study was supported by Swedish Heart- and Lung Foundation, EU Commission FP7 "CardioCell", Swedish Research Council, AFA insurances and ALF. O.B. was supported by Deutsche Forschungsgemeinschaft.

1. Isolation of the Cardiac Nuclei
<ol>
 <li>Coat ultracentrifuge tubes (Beckman Centrifuge Tubes #363664) with 10 ml of 1%BSA/PBS coating solution. Cap the tubes and let them rotate for 30 min in a tube rotator. Remove the coating solution and let the centrifuge tubes air dry (one tube per mouse heart is required for the analysis of single mouse hearts, alternately up to 5 mouse hearts or 1 g of heart tissue from a different species (e.g. human) can be processed in one tube).</li>
 <li>All following steps should be performed on ice. Dissect the left ventricle from fresh or snap-frozen mouse heart with a scalpel. Note, this protocol is optimized for mouse heart but can also be adapted to rat or human heart. Alternatively, use up to 1 g of heart tissue from a different species.</li>
 <li>Trim the specimen into small cubicles with a scalpel.</li>
 <li>Transfer the tissue pieces into a 50 ml Falcon tube filled with 15 ml of lysis buffer.</li>
 <li>Homogenize the heart tissue with a T-25 Ultra-Turrax probe homogenizer (IKA) at 24,000 rpm for 10 sec.</li>
 <li>Dilute the homogenate with equal volume of lysis buffer to 30 ml.</li>
 <li>Use a glass douncer (40 ml) to further homogenize the tissue and free the nuclei. Perform eight strokes with a large clearance pestle.</li>
 <li>Pass the crude nuclei isolate through a 100 &mu;m and 70 &mu;m nylon mesh cell strainer (BD Biosciences), consecutively.</li>
 <li>Spin down the crude nuclei isolate in a refrigerated centrifuge (4 &deg;C) at 700 x g for 10 min.</li>
 <li>Remove the supernatant carefully by inverting the tubes and wipe the inside of the tube with paper towel. Be careful not to disturb the nuclei pellet.</li>
 <li>Dissolve the crude nuclei isolate in 5 ml of sucrose buffer by pipetting the solution several times up and down. Add a further 25 ml of sucrose buffer to the dissolved pellet.</li>
 <li>Add 10 ml of freshly prepared sucrose buffer to the coated ultracentrifuge tube (see step 1.1).</li>
 <li>Carefully overlay the added 10 ml of sucrose buffer with the resuspended nuclei pellet dissolved in sucrose buffer from step 1.9.</li>
 <li>Balance the centrifuge tubes before placing them into a JS13.1 free swinging rotor and place the rotor into a high-speed centrifuge (Beckman Avanti S-25).</li>
 <li>Spin the nuclei sample at 13,000 x g at 4 &deg;C for 60 min.</li>
 <li>When the spin has completed, remove the tubes carefully from the rotor and discard the supernatant by inverting the tubes and wiping the remaining debris from the inside of the tubes with paper towel.</li>
 <li>Dissolve the nuclei pellet in 1 ml of nuclei storage buffer (NSB plus buffer). Note: NSB plus contains 1.5 mM spermine as a DNA stabilizer.</li>
 <li>Proceed with step 2.1, immunostaining of cardiomyocyte nuclei.</li>
</ol>
2. Immunostaining for Flow Cytometry
<ol>
 <li>Prepare the negative control for immunostaining. Take an aliquot of 20 &mu;l out of the nuclei sample and add 980 &mu;l of NSB plus buffer.</li>
 <li>Add anti-pericentriolar material 1 antibody (rabbit anti-PCM-1, Atlas Antibodies) to the nuclei sample in a dilution of 1:500 to immunolabel cardiomyocyte nuclei. Add the isotype antibody in the same dilution as the anti-PCM-1 antibody to the negative control, prepared in step 2.1.</li>
 <li>Incubate negative control and sample tube at 4 &deg;C overnight.</li>
 <li>Wash negative control and sample at least once with NSB plus buffer (spin down tubes in a refrigerated centrifuge (4 &deg;C) at 700 x g for 10 min. Discard supernatant and dissolve the nuclei pellet in 1 ml of NSB plus buffer).</li>
 <li>Add anti-rabbit fluorescent secondary antibody (FITC or APC) to negative control and sample tube in a dilution of 1:1000.</li>
 <li>Incubate negative control and sample tube at 4 &deg;C for 1 h.</li>
 <li>Wash negative control and sample at least once with NSB plus buffer (spin down tubes in a refrigerated centrifuge (4 &deg;C) at 700 x g for 10 min. Discard supernatant and dissolve the nuclei pellet in 1 ml of NSB plus buffer).</li>
 <li>Proceed with the flow cytometric analysis and sorting.</li>
</ol>
3. Flow Cytometry
<ol>
 <li>Coat nuclei collection tubes (Falcon 15 ml) with 1%BSA/PBS solution before you start flow cytometry sorting as described in step 1.1.</li>
 <li>Filter the sample and the negative control through a 30 &mu;m cell strainer and load first the negative control to the flow cytometer (BD Influx). Define the first and second gate to define nuclei and singlets (single nuclei), based on forward scatter (FSC), forward scatter pulse width (FS pulse width) and side scatter (SSC) (Fig. 2a and b). Adding a DNA stain (DRAQ5 (1:500)) to the sample can help to identify the nuclei population initially.</li>
 <li>Load the immunolabeled sample and define the third gate to isolate cardiomyocyte nuclei (PCM-1-positve) from non-cardiomyocyte nuclei (PCM-1-negative). Start the sorting (Fig. 2c and d).</li>
</ol>
Optional: In order to analyze the nuclear DNA content (ploidy) and to perform cell cycle analysis add an appropriate DNA stain to the nuclei (e.g. Hoechst 33342 or DRAQ5) (Fig. 2e).
<ol start="4">
 <li>After flow cytometry sorting, place the nuclei on ice and re-analyze to determine the sorting purity (Fig. 3a and b).</li>
 <li>Spin down the sorted nuclei in the collection tubes at 1500 x g in a refrigerated centrifuge for 15 min.</li>
 <li>Dissolve the nuclei pellet in a buffer compatible with the downstream application.</li>
</ol>
4. Representative Results
Nuclei morphology and integrity can be assessed by DNA stains and visualized by microscopy (Fig. 1). Successful PCM-1 labeling can be assessed by epifluorescence microscopy and by flow cytometry (Fig. 1 and Fig. 2c and d). PCM-1-positive and negative populations should be well separated from each other (Fig. 2c and d). In murine left ventricle about 30% of all nuclei should be cardiomyocyte nuclei (Fig. 2d). Sorting purity can be assessed by re-analyzing the sorted nuclei (Fig. 3a and b). Both nuclei populations should have a sorting purity exceeding 95%.
<img alt="Figure 1" src="/files/ftp_upload/4205/4205fig1.jpg" /> 
Figure 1. PCM-1 identifies cardiomyocyte nuclei. Cardiac nuclei (a) are stained with antibodies to PCM-1 (b) and to Nkx2.5 (c) in an adult mouse heart. (d) PCM-1-labeled nuclei are surrounded by cardiomyocyte cytoplasm (myosin heavy chain (MHC)) and express the transcription factor Nkx2.5, documenting the accurate identification of cardiomyocyte nuclei by PCM-1 staining (scale bars 20 &mu;m and 10 &mu;m (d, inset)). (e) Cardiac nuclei isolates visualized with the DNA stain DRAQ5. (f and g) Cardiomyocyte nuclei are labeled with antibodies against PCM-1 (scale bar 10 &mu;m). Note, the epinuclear staining pattern of PCM-1 in myocyte nuclei in tissue section and in isolated nuclei (arrows).
<img alt="Figure 2" src="/files/ftp_upload/4205/4205fig2.jpg" /> 
Figure 2. Flow cytometric sorting of cardiomyocyte nuclei. (a) Cardiac nuclei are identified by forward scatter (FSC) and side scatter (SSC). (b) A second gate identifies single nuclei by FSC and FS pulse width5. (c, d) Fluorescent gating allows the separation of cardiomyocyte nuclei (PCM-1-positive) and non-cardiomyocyte (PCM-1-negative) nuclei from heart tissue. (e) Mouse cardiomyocyte are mostly (&gt;80%) diploid (2n), only a small subset is tetraploid (4n)6. Note, human cardiomyocytes contain a higher frequency of polyploidy nuclei (&gt;2n)7,8.
<img alt="Figure 3" src="/files/ftp_upload/4205/4205fig3.jpg" /> 
Figure 3. Purity analysis of sorted cardiomyocyte and non-cardiomyocyte nuclei. Re-analysis of sorted non-cardiomyocyte (a) and cardiomyocyte nuclei (b). Both populations show a sorting purity exceeding 99%.

<ol>
	<li>Ang, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limitations+of+conventional+approaches+to+identify+myocyte+nuclei+in+histologic+sections+of+the+heart.">Limitations of conventional approaches to identify myocyte nuclei in histologic sections of the heart.</a> American journal of physiology. Cell physiology. , 298-1603 (2010).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardiomyocyte+nuclei+and+assessment+of+ploidy+for+the+analysis+of+cell+turnover.">Identification of cardiomyocyte nuclei and assessment of ploidy for the analysis of cell turnover.</a> Experimental cell research. 327, 188-194 (2011).</li><li>Bergmann, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Renewal+in+Humans.">Evidence for Cardiomyocyte Renewal in Humans.</a> Science. 324, 98-102 (1126).</li><li>Walsh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+cell+cycle+control+and+growth+estimation.">Cardiomyocyte cell cycle control and growth estimation.</a> Cardiovascular Research. , 1-31 (2010).</li><li>Spalding, K., Bhardwaj, R. D., Buchholz, B., Druid, H., Frisén, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrospective+birth+dating+of+cells+in+humans.">Retrospective birth dating of cells in humans.</a> Cell. 122, 133-143 (2005).</li><li>Adler, C. P., Friedburg, H., Herget, G. W., Neuburger, M., Schwalb, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+cardiomyocyte+DNA+content,+ploidy+level+and+nuclear+number+in+mammalian+hearts.">Variability of cardiomyocyte DNA content, ploidy level and nuclear number in mammalian hearts.</a> Virchows Arch. 429, 159-164 (1996).</li><li>Herget, G. W., Neuburger, M., Plagwitz, R., Adler, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+content,+ploidy+level+and+number+of+nuclei+in+the+human+heart+after+myocardial+infarction.">DNA content, ploidy level and number of nuclei in the human heart after myocardial infarction.</a> Cardiovascular Research. 36, 45-51 (1997).</li><li>Adler, C. P., Friedburg, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+DNA+content.+ploidy+level+and+cell+number+in+geriatric+hearts:+postmortem+examinations+of+human+myocardium+in+old+age.">Myocardial DNA content. ploidy level and cell number in geriatric hearts: postmortem examinations of human myocardium in old age.</a> Mol. Cell Cardiol. 18, 3953-39 (1986).</li><li>Dubois, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SIRPA+is+a+specific+cell-surface+marker+for+isolating+cardiomyocytes+derived+from+human+pluripotent+stem+cells.">SIRPA is a specific cell-surface marker for isolating cardiomyocytes derived from human pluripotent stem cells.</a> Nature. 29, 1011-1018 (2011).</li><li>Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature Methods. 7, 61-66 (2010).</li><li>Fransioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+the+c-kit-Positive+Cell+Response+to+Pathological+Challenge+in+the+Myocardium.">Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium.</a> Stem Cells. 26, 1315-1324 (2008).</li><li>Elliott, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NKX2-5(eGFP/w)+hESCs+for+isolation+of+human+cardiac+progenitors+and+cardiomyocytes.">NKX2-5(eGFP/w) hESCs for isolation of human cardiac progenitors and cardiomyocytes.</a> Nature Methods. 8, 1037-1040 (2011).</li><li>Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Cardiomyocyte+Repopulation+by+Extracardiac+Progenitors+in+Transplanted+Human+Hearts.">Evidence for Cardiomyocyte Repopulation by Extracardiac Progenitors in Transplanted Human Hearts.</a> Circulation Research. 90, 634-640 (2002).</li><li>Srsen, V., Fant, X., Heald, R., Rabouille, C., Merdes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosome+proteins+form+an+insoluble+perinuclear+matrix+during+muscle+cell+differentiation.">Centrosome proteins form an insoluble perinuclear matrix during muscle cell differentiation.</a> BMC cell biology. 10, 28 (2009).</li><li>Spoelgen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+flow+cytometry-based+technique+to+measure+adult+neurogenesis+in+the+brain.">A novel flow cytometry-based technique to measure adult neurogenesis in the brain.</a> Journal of neurochemistry. 119, 165-175 (2011).</li><li>Soonpaa, M. H., Kim, K. K., Pajak, L., Franklin, M., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiomyocyte+DNA+synthesis+and+binucleation+during+murine+development.">Cardiomyocyte DNA synthesis and binucleation during murine development.</a> The American journal of physiology. 271, H2183-H2189 (1996).</li><li>Olivetti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aging,+cardiac+hypertrophy+and+ischemic+cardiomyopathy+do+not+affect+the+proportion+of+mononucleated+and+multinucleated+myocytes+in+the+human+heart.">Aging, cardiac hypertrophy and ischemic cardiomyopathy do not affect the proportion of mononucleated and multinucleated myocytes in the human heart.</a> J Mol Cell Cardiol. 28, 1463-1477 (1996).</li><li>Okada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+sorting+of+neuronal+and+glial+nuclei+from+central+nervous+system+tissue.">Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue.</a> Journal of cellular physiology. 226, 552-558 (2011).</li><li>Matevossian, A., Akbarian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+Nuclei+Isolation+from+Human+Postmortem+Brain+Tissue.">Neuronal Nuclei Isolation from Human Postmortem Brain Tissue.</a> J. Vis. Exp. (20), e914 (2008).</li></ol>

No conflicts of interest declared.

Accurate identification of cardiomyocyte nuclei is crucial for the analysis of regenerative processes in the myocardium2,3. Conventional techniques to isolate cardiomyocytes from fresh tissue are mainly based on enzymatic digestion of extracellular matrix proteins and the subsequent purification from interstitial cells by low speed centrifugation. Further purification of living cardiomyocytes from embryonic stem cells (ESC) can be performed by immunolabeling with surface markers such as SIRPA9 or mi...

<table border="1">
 <tbody>
 <tr>
 <td>1. Lysis Buffer</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>0.32 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 8)</td>
 </tr>
 <tr>
 <td>5 mM CaCl2</td>
 </tr>
 <tr>
 <td>5 mM magnesium acetate</td>
 </tr>
 <tr>
 <td>2.0 mM EDTA</td>
 </tr>
 <tr>
 <td>0.5 mM EGTA</td>
 </tr>
 <tr>
 <td>1 mM DTT</td>
 </tr>
 </tbody>
</table>
 
<table border="1">
 <tbody>
 <tr>
 <td>2. Sucrose buffer</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>2.1 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 8)</td>
 </tr>
 <tr>
 <td>5 mM magnesium acetate</td>
 </tr>
 <tr>
 <td>1 mM DTT</td>
 </tr>
 </tbody>
</table>
 
<table border="1">
 <tbody>
 <tr>
 <td>3. Nuclei storage buffer (NSB plus)</td>
 </tr>
 <tr>
 <td>Name of the reagent</td>
 </tr>
 <tr>
 <td>0.44 M sucrose</td>
 </tr>
 <tr>
 <td>10 mM Tris-HCl (pH = 7.2)</td>
 </tr>
 <tr>
 <td>70 mM KCl</td>
 </tr>
 <tr>
 <td>10 mM MgCl2</td>
 </tr>
 <tr>
 <td>1.5 mM spermine</td>
 </tr>
 </tbody>
</table>

<table border="1">
 <tbody>
 <tr>
 <td>Reagents and Equipment</td>
 <td>Company</td>
 </tr>
 <tr>
 <td>Isotype rabbit IgG- ChIP Grade, #ab37415</td>
 <td>Abcam</td>
 </tr>
 <tr>
 <td>Rabbit anti-PCM-1 antibody, #HPA023374</td>
 <td>Atlas Antibodies</td>
 </tr>
 <tr>
 <td>Donkey sec. antibody, anti-rabbit Alexa 488 Fluor, #A-21206 or equivalent sec. fluorescent antibody</td>
 <td>Life Technologies</td>
 </tr>
 <tr>
 <td>DRAQ5</td>
 <td>Biostatus</td>
 </tr>
 <tr>
 <td>cell strainers 30 &mu;m, 70 &mu;m and 100 &mu;m</td>
 <td>BD Biosciences</td>
 </tr>
 <tr>
 <td>Glass douncer (40 ml) and pestle "L"</td>
 <td>VWR (Wheaton Industries Inc.)</td>
 </tr>
 <tr>
 <td>T-25 Ultra-Turrax Homogenizer</td>
 <td>IKA Germany</td>
 </tr>
 <tr>
 <td>Dispersing tool S25 N-18 G</td>
 <td>IKA Germany</td>
 </tr>
 <tr>
 <td>Beckman Avanti Centrifuge</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Falcon Tubes 15 ml and 50 ml</td>
 <td>VWR</td>
 </tr>
 <tr>
 <td>Beckman Centrifuge Tubes #363664</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>JS13.1 free swinging rotor</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Influx cytometer</td>
 <td>Beckman Coulter</td>
 </tr>
 <tr>
 <td>Tube Rotator</td>
 <td>VWR</td>
 </tr>
 </tbody>
</table>

isolation of cardiomyocyte nuclei from post-mortem tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Watch this Scientific Journal Video about Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue at JoVE.com

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

isolation-of-cardiomyocyte-nuclei-from-post-mortem-tissue

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16.9K Views.  University of Lund. Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

Video: Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Electromechanical Assessment of Optogenetically Modulated Cardiomyocyte Activity

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including the heart. While Channelrhodopsin-2 (ChR2) is a common tool to depolarize the membrane potential in cardiomyocytes (CM), potentially eliciting action potentials (AP), an effective tool for reliable silencing of CM activity has been missing. It has been suggested to use anion channelrhodopsins (ACR) for optogenetic inhibition. Here, we describe a protocol to assess the effects of activating the natural ACR GtACR1 from Guillardia theta in cultured rabbit CM. Primary readouts are electrophysiological patch-clamp recordings and optical tracking of CM contractions, both performed while applying different patterns of light stimulation. The protocol includes CM isolation from rabbit heart, seeding and culturing of the cells for up to 4 days, transduction via adenovirus coding for the light-gated chloride channel, preparation of patch-clamp and carbon fiber setups, data collection and analysis. Using the patch-clamp technique in whole-cell configuration allows one to record light-activated currents (in voltage-clamp mode, V-clamp) and AP (current-clamp mode, I-clamp) in real time. In addition to patch-clamp experiments, we conduct contractility measurements for functional assessment of CM activity without disturbing the intracellular milieu. To do so, cells are mechanically preloaded using carbon fibers and contractions are recorded by tracking changes in sarcomere length and carbon fiber distance. Data analysis includes assessment of AP duration from I-clamp recordings, peak currents from V-clamp recordings and force calculation from carbon fiber measurements. The described protocol can be applied to the testing of biophysical effects of different optogenetic actuators on CM activity, a prerequisite for the development of a mechanistic understanding of optogenetic experiments in cardiac tissue and whole hearts.

We present a protocol for evaluating the electromechanical effects of GtACR1 activation in rabbit cardiomyocytes. We provide detailed information on cell isolation, culturing and adenoviral transduction, and on functional experiments with the patch-clamp and carbon-fiber techniques.

Over the past two decades, optogenetic tools have been established as potent means to modulate cell-type specific activity in excitable tissues, including ...

Model of Ischemic Heart Disease and Video-Based Comparison of Cardiomyocyte Contraction Using hiPSC-Derived Cardiomyocytes

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with small-animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart, underscoring the need for clinically relevant models to study heart disease. Here, we present a protocol to model ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs) and to quantify the damage and functional impairment of the ischemic cardiomyocytes. Exposure to 2% oxygen without glucose and serum increases the percentage of injured cells, which is indicated by staining of the nucleus with propidium iodide, and decreases cellular viability. These conditions also decrease the contractility of hiPS-CMs as confirmed by displacement vector field analysis of microscopic video images. This protocol may furthermore provide a convenient method for personalized drug screening by facilitating the use of hiPS cells from individual patients. Therefore, this model of ischemic heart disease, based on iPS-CMs of human origin, can provide a useful platform for drug screening and further research on ischemic heart disease.

We present a model of ischemic heart disease using cardiomyocytes derived from human induced pluripotent stem cells, together with a method for quantitative evaluation of tissue damage caused by ischemia. This model can provide a useful platform for drug screening and further research on ischemic heart disease.

Ischemic heart disease is a significant cause of death worldwide. It has therefore been the subject of a tremendous amount of research, often with ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

Preparation of a Non-Cardiomyocyte Cell Suspension for Single-Cell RNA Sequencing from a Post-Myocardial Infarction Adult Mouse Heart

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more than half of the total cardiac cell population, and they contribute to adaptive compensations upon myocardial injury, including inflammatory responses, tissue repair, and scar formation. To study the post-MI cardiac microenvironment, single-cell RNA sequencing (scRNA-seq) is widely used to identify different cardiac cell types and intercellular communications. Among the procedures of scRNA-seq sample preparation, preparing the cell suspension is one of the most critical steps, because the cell viability can affect the quality of the scRNA-seq results. Therefore, we designed an experimental protocol for preparing a non-cardiomyocyte cell suspension from post-MI mouse hearts with an extra focus on improving the cell viability by choosing mild digestive enzymes, controlling the digestion time, and applying fluorescence-activated cell sorting (FACS). Finally, we isolated CD45+ cells from the non-cardiomyocyte cell suspension obtained through this protocol, and then we performed scRNA-seq.

Here, we describe a protocol to isolate a sufficient amount of single non-cardiomyocytes with high viability from a post-myocardial infarction (MI) mouse heart. This can be used for subsequent single-cell sequencing, flow cytometry analysis, and primary cell culture.

Myocardial infarction (MI) is one of the most common cardiovascular diseases, with increasing mortality worldwide. Non-cardiomyocytes account for more ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...