Name: isolation of human islets from partially pancreatectomized patients
Uploaded: 2011-07-30T13:00:00
Duration: 11 min 10 s
Description: Watch this Scientific Journal Video about Isolation of Human Islets from Partially Pancreatectomized Patients at JoVE.com

We want to thank our many colleagues who provided help, advice and critical input at various stages of the project. Production of this video article was supported with funds from the German Ministry for Education and Research (BMBF) to the German Centre for Diabetes Research (DZD, <a href="http://www.dzd-ev.de">http://www.dzd-ev.de</a>), IMIDIA (<a href="http://www.imidia.org">http://www.imidia.org</a>) and the University Hospital Carl Gustav Carus at the University of Technology Dresden. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n&deg; 115005.

1. Pancreatic tissue collection in the operating room
<ol>
	<li>The surgeon performs a partial resection of the pancreas.</li>
	<li>After placing the pancreatic specimen in a box on ice, deliver it immediately to the pathologist.</li>
</ol>
2. Tissue selection for islet isolation
<ol>
	<li>The pathologist selects tissue that appears soft and healthy, retaining the damaged tissue for diagnostic purposes. Fibrotic tissue and specimens offering less than 2 g of usable tissue are excluded from islet isolation.</li>
	<li>Immerse the pancreatic tissue in Euro Collins Solution and deliver on ice to the laboratory.</li>
</ol>
3. Isolation of human islets
<ol>
	<li>Weigh the pancreatic tissue, then place it into a 10 cm dish.</li>
	<li>Put 150 ml RPMI media in a 500 ml flask.</li>
	<li>In a 250 ml flask, prepare the digestive enzyme solution by combining 130 ml RPMI media with 100 mg/ml DNase and 20 ml of 5mg/ml Liberase RI. Draw 10 ml of this solution into a syringe to inject the pancreatic tissue.</li>
	<li>Inject the digestive enzyme solution into the pancreatic tissue, aiming to distend it homogeneously. If this is prevented by fibrosis, the digestion will likely fail. Next, mince the tissue into ~4 mm3 pieces on ice.</li>
</ol>
4. Human pancreas digestion circuit
<ol>
	<li>Assemble the digestion circuit as shown in Figure 1. The flow direction in the Ricordi chamber is in at the bottom and out at the top of the chamber.</li>
	<li>Add the remaining digestive enzyme solution to the 500 ml flask up to a total volume of 300 ml and insert a thermometer.</li>
	<li>Transfer the pancreatic tissue into the chamber, insert the mesh (pore diameter: 600 &mu;m) and three Silicon Nitride Marbles (diameter: 15 mm), close the chamber and start the pump with the flow set at 140 ml/min.</li>
	<li>When the temperature of the circuit reaches 37 &deg;C, start timing and periodically take samples to determine when to stop the digestion. Staining with dithizone (2 mg/ml) allows microscopic visualization of islets within the digested tissue4.</li>
	<li>Once the islets are separated from the surrounding acinar tissue, quickly stop the digestion by placing the heating coil on ice and adding 200 ml of cold wash media (900 ml RPMI 1640 with 5.5 mM glucose and 10% FBS) to the circuit.</li>
	<li>Collect the islet solution in 250 ml conical tubes as it comes out of the sample tube. Continue until the circuit is empty.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/2962/2962fig1.jpg" /> 
Figure 1. The human pancreas digestion circuit The human pancreas digestion circuit includes a Ricordi chamber containing mesh (pore diameter: 600 &mu;m) and three silicon nitride marbles (diameter: 15 mm). Outflow from the chamber is driven to the tissue collection flask by the peristaltic pump (140 ml/min). Arrows indicate the flow direction. An optimal temperature for enzymatic digestion is maintained by immersing the heating coil in a 37 &deg;C water bath.
5. Washing post-digestion
<ol>
	<li>Centrifuge the 250 ml conical tubes containing the islet solution at 1000 rpm, 4 &deg;C for 5 min.</li>
	<li>Discard the supernatant, resuspend the islet pellet in wash media (900 ml RPMI 1640 with 5.5 mM glucose and 10% FBS) and distribute it in equal fractions to 50 ml conical tubes. (Optional: Distribute into additional conical tubes to improve washing.) Centrifuge at 1000 rpm, 4 &deg;C for 5 min.</li>
	<li>Discard supernatant and gently loosen pellets by manual shaking.</li>
</ol>
6. Purification on a Ficoll gradient
<ol>
	<li>Resuspend the pellet with Ficoll media at a density of 1.125 g/ml, pH 7.4 and slowly overlay 10 ml aliquots of Ficoll media with densities of 1.080, 1.060 and 1.037 g/ml.</li>
	<li>Centrifuge the Ficoll gradients at 2400 rpm, 4&deg;C for 20 min.</li>
</ol>
7. Islet Collection and Culture
<ol>
	<li>After centrifugation, different tissue components can be distinguished by their partitioning in the gradient.</li>
	<li>Discard the upper layer consisting of fat and connective tissue.</li>
	<li>Carefully harvest the first layer of islet aggregates at the 1.037-1.060 g/ml interphase. This layer contains the most pure isolated islets. Collect fractions separately for efficient isolation.</li>
	<li>Collect the second, less pure fraction of islet aggregates at the 1.060-1.080 g/ml interphase.</li>
	<li>Dilute the Ficoll gradient by adding wash media (900 ml RPMI 1640 with 5.5 mM glucose and 10% FBS) to each conical tube up to a total volume of 50 ml. Centrifuge at 1000 rpm, 4&deg;C for 5 min.</li>
	<li>Discard the supernatant by suction. Use care as the pellet might be loose due to the Ficoll gradient.</li>
	<li>Wash the pellets with wash media (900ml RPMI 1640 with 5.5 mM glucose and 10% FBS) and centrifuge at 1000 rpm, 4&deg;C for 5 min. Repeat using islet culture media</li>
	<li>Resuspend the islets in culture media and place them in the incubator with 5% CO2 at 37 &deg;C for 24-48 hours before further processing.</li>
</ol>

<ol>
	<li>Kahn, S. E., Zraika, S., Utzschneider, K. M., Hull, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta+cell+lesion+in+type+2+diabetes:+there+has+to+be+a+primary+functional+abnormality.">The beta cell lesion in type 2 diabetes: there has to be a primary functional abnormality.</a> Diabetologia. 52, 1003-1012 (2009).</li><li>Deng, S., Vatamaniuk, M., Huang, X., Doliba, N., Lian, M. M., Frank, A., Velidedeoglu, E., Desai, N. M., Koeberlein, B., Wolf, B., Barker, C. F., Naji, A., Matschinsky, F. M., Markmann, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+abnormalities+in+the+islets+isolated+from+type+2+diabetic+subjects.">Structural and functional abnormalities in the islets isolated from type 2 diabetic subjects.</a> Diabetes. 53, 624-632 (2004).</li><li>Ricordi, C., Lacy, P. E., Finke, E. H., Olack, B. J., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+method+for+isolation+of+human+pancreatic+islets.">Automated method for isolation of human pancreatic islets.</a> Diabetes. 37, 413-420 (1988).</li><li>Latif, Z. A., Noel, J., Alejandro, 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+simple+method+of+staining+fresh+and+cultured+islets.">A simple method of staining fresh and cultured islets.</a> Transplantation. 45, 827-830 (1988).</li><li>Ehehalt, F., Knoch, K., Erdmann, K., Krautz, C., J&#228;ger, M., Steffen, A., Wegbrod, C., Meisterfeld, R., Kersting, S., Bergert, H., Kuhlisch, E., Bornstein, S., Bonifacio, E., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+insulin+turnover+in+islets+from+type+2+diabetic+patients.">Impaired insulin turnover in islets from type 2 diabetic patients.</a> Islets. 2, 30-36 (2010).</li><li>Hardt, P. D., Brendel, M. D., Kloer, H. U., Bretzel, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+pancreatic+diabetes+(type+3c+diabetes)+underdiagnosed+and+misdiagnosed?.">Is pancreatic diabetes (type 3c diabetes) underdiagnosed and misdiagnosed?.</a> Diabetes Care. 31, S165-S169 (2008).</li><li>Meisterfeld, R., Ehehalt, F., Saeger, H. D., Solimena, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+disorders+and+diabetes+mellitus.">Pancreatic disorders and diabetes mellitus.</a> Exp Clin Endocrinol Diabetes. 116, S7-S12 (2008).</li><li>Verge, C. F., Gianani, R., Kawasaki, E., Yu, L., Pietropaolo, M., Jackson, R. A., Chase, H. P., Eisenbarth, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+type+I+diabetes+in+first-degree+relatives+using+a+combination+of+insulin,+GAD,+and+ICA512bdc/IA-2+autoantibodies.">Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies.</a> Diabetes. 45, 926-933 (1996).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabet Med. 15, 539-553 (1998).</li><li>Marselli, L., Thorne, J., Ahn, Y. B., Omer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+of+Purified+{beta}-Cell+Tissue+Obtained+from+Human+Pancreas+with+Laser+Capture+Microdissection.">Gene Expression of Purified {beta}-Cell Tissue Obtained from Human Pancreas with Laser Capture Microdissection.</a> Journal of Clin. Endocrinol. Metab. 93, 1046-1053 (2008).</li></ol>

No conflicts of interest declared.

Using our protocol, human islets can be isolated from pancreatic tissue collected from a partial pancreatectomy. The success of this protocol relies on the care taken during a few critical points. To preserve beta-cell viability, it is essential that the specimen be transported rapidly on ice to the laboratory. Additionally, the duration of tissue digestion must be optimized empirically according to the grade of tissue fibrosis and the enzymatic activity of the media. This is also true for the degree of mechanical force applied manually to the Ricordi chamber. Thus to obtain a good islet yield, the protocol is best performed by a dedicated scientist or technical assistant. Initial failure is the norm unless the team is already experienced in islet isolation.
There are key differences between our islet isolation protocol and the standard human islet isolation method: 1) Although we use pancreatic tissue subjected to several hours of ischemia during the surgical procedure, it is immediately processed on site. This is in contrast to pancreata explanted from brain-dead donors for pancreas/islet transplantation, which remain ischemic for several hours during allocation and delivery to the islet isolation facility. 2) We inject collagenase directly into the pancreatic tissue, whereas the standard protocol is to infuse it into the pancreatic duct. 3) We separate the islets using a discontinuous Ficoll gradient instead of collecting the islets from a continuous Ficoll gradient using a COBE cell processor.
In cases where the surgical specimen is too fibrotic or scarce for isolating an adequate number of islets, tissue can still be retrieved by laser capture microdissection (LCM)10. This allows gene expression data to be recovered from virtually every specimen, even if the islet isolation fails or the yield is very low. Unfortunately, LCM does not produce living cells for functional studies and their amount is typically insufficient for proteomic analysis. Thus, using LCM in parallel with our collagenase digestion protocol to retrieve the islets may be the most effective way to process surgical specimens.
When collecting tissue for islet isolation from partial pancreatectomies, it is important to carefully examine the patient&#x2019;s clinical history and metabolic state. A partially pancreatectomized patient could be affected by type 3c diabetes, i.e. diabetes secondary to the pancreatic disorder leading to surgery6. Among the 43 participating patients who underwent this surgery in our department in 2010, 32 were non-diabetic, 5 were affected by type 2 diabetes and 6 had type 3c diabetes. These data agree with previous studies pointing to impaired glucose metabolism and diabetes in a sizeable fraction of patients suffering from pancreatic cancer or chronic pancreatitis6. We considered diabetes to be of primary origin if it was diagnosed at least one year prior to the onset of symptoms leading to pancreatic surgery7. The levels of antibodies against islet autoantigens should also be measured to evaluate a potential autoimmune origin of the diabetes8. Because a patient undergoing pancreatectomy could suffer from undiagnosed diabetes or be glucose intolerant, all non-diabetic patients should be given an oral glucose tolerance test prior to the pancreatectomy9.

<table border="1">
 <tbody>
 <tr>
 <th>
 Name</th>
 <th>
 Company</th>
 <th>
 Catalogue number</th>
 </tr>
 <tr>
 <th>
 Equipment</th>
 <th>
 </th>
 <th>
 </th>
 </tr>
 <tr>
 <td>1 ml serological pipette (CELLSTAR)</td>
 <td>greiner bio-one</td>
 <td>604181</td>
 </tr>
 <tr>
 <td>10 ml serological pipette (CELLSTAR)</td>
 <td>greiner bio-one</td>
 <td>607180</td>
 </tr>
 <tr>
 <td>25 ml serological pipette (CELLSTAR)</td>
 <td>greiner bio-one</td>
 <td>760180</td>
 </tr>
 <tr>
 <td>10 ml Syringe</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>300912</td>
 </tr>
 <tr>
 <td>50 ml Syringe</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>300865</td>
 </tr>
 <tr>
 <td>5 ml Syringe</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>309050</td>
 </tr>
 <tr>
 <td>18 G 1,1/2 Needle Microlance 3</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>304622</td>
 </tr>
 <tr>
 <td>27 Gx1/2 Needle</td>
 <td>Braun</td>
 <td>4658300</td>
 </tr>
 <tr>
 <td>27 Gx3/4 Needle Microlance 3</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>302200</td>
 </tr>
 <tr>
 <td>3 cm cell culture dish 2x2 mm grid</td>
 <td>Nunc</td>
 <td>174926</td>
 </tr>
 <tr>
 <td>20 cm Tissue culture dish</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>6 cm Easy grip petri dish</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>351016</td>
 </tr>
 <tr>
 <td>150 ml storage bottle</td>
 <td>Corning Incorporated</td>
 <td>431175</td>
 </tr>
 <tr>
 <td>250 ml Erlenmeyer flasks</td>
 <td>Schott Duran</td>
 <td>21 217 36</td>
 </tr>
 <tr>
 <td>500 ml Erlenmeyer flasks</td>
 <td>Schott Duran</td>
 <td>21 217 44</td>
 </tr>
 <tr>
 <td>250 ml Screw Cap Conical Bottom Centrifuge Tube</td>
 <td>Corning Incorporated</td>
 <td>430776</td>
 </tr>
 <tr>
 <td>50 ml Falcon conical tube</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>352070</td>
 </tr>
 <tr>
 <td>260 ml Easyflask Non-treated</td>
 <td>Nunc</td>
 <td>156800</td>
 </tr>
 <tr>
 <td>Millex GV Filter Unit Duropore (0,22 &mu;m)</td>
 <td>Millipore</td>
 <td>SLGV033RB</td>
 </tr>
 <tr>
 <td>Bottle Top Vacuum Filter (PES 70 mm Diameter Membran, 0,22 &mu;m pore size, 45 mm neck size, 500 mL volume)</td>
 <td>Corning Incorporated</td>
 <td>431118</td>
 </tr>
 <tr>
 <td>Densitometer (Densito 30PX) LWE37463</td>
 <td>Mettler Toledo</td>
 <td>51324450</td>
 </tr>
 <tr>
 <td>Fast PES Filter Unit 0,2 &mu;m</td>
 <td>Nalgene</td>
 <td>569-0020</td>
 </tr>
 <tr>
 <td>Heating Coil</td>
 <td>BioRep Technologies Inc</td>
 <td>HC-02</td>
 </tr>
 <tr>
 <td>Multifuge 4 KS-R</td>
 <td>Heraeus</td>
 <td>75015680</td>
 </tr>
 <tr>
 <td>Pump Typ PD5206</td>
 <td>Heidolph</td>
 <td>523-52060-00-2</td>
 </tr>
 <tr>
 <td>Ricordi chamber (metal mesh with 600 &mu;m pore diameter)</td>
 <td>BioRep Technologies Inc.</td>
 <td>Model 50-U-01, Serial 0503-001</td>
 </tr>
 <tr>
 <td>Preparation Stand</td>
 <td>BioRep Technologies Inc.</td>
 <td>50-PS-01</td>
 </tr>
 <tr>
 <td>O-Rings</td>
 <td>BioRep Technologies Inc.</td>
 <td>OR-50-U-01</td>
 </tr>
 <tr>
 <td>Silicon Nitride Marbles (15 mm set of 9)</td>
 <td>BioRep Technologies Inc.</td>
 <td>SN-01</td>
 </tr>
 <tr>
 <td>Silicon Tubing</td>
 <td>Tygon</td>
 <td>R 3603 8,0X4,0 mm</td>
 </tr>
 <tr>
 <td>Surgical forceps</td>
 <td>Braun</td>
 <td>BD168R</td>
 </tr>
 <tr>
 <td>Surgical scissors (Aesculap)</td>
 <td>Braun</td>
 <td>BC273R</td>
 </tr>
 <tr>
 <td>Water bath OLS 200</td>
 <td>Grant</td>
 <td>OLS 200</td>
 </tr>
 <tr>
 <th>
 Reagents</th>
 <th>
 </th>
 <th>
 </th>
 </tr>
 <tr>
 <td>Collagenase -&gt; Liberase RI (100 mg)</td>
 <td>Roche</td>
 <td>11815032001</td>
 </tr>
 <tr>
 <td>D(+)-Glucose</td>
 <td>Merck</td>
 <td>1.08337.1000</td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Merck</td>
 <td>1.16743.1000</td>
 </tr>
 <tr>
 <td>Diphenylthiocarbazone (DTZ)</td>
 <td>Sigma Aldrich</td>
 <td>D5130</td>
 </tr>
 <tr>
 <td>DNase I (100 mg)</td>
 <td>Roche</td>
 <td>10104159001</td>
 </tr>
 <tr>
 <td>Dulbeccos 1xPBS</td>
 <td>PAA Laboratories</td>
 <td>H15-002</td>
 </tr>
 <tr>
 <td>Electrolytsolution E154</td>
 <td>Serumwerk</td>
 <td>00509</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>PAA Laboratories</td>
 <td>A15-101</td>
 </tr>
 <tr>
 <td>Ficoll 400</td>
 <td>Sigma Aldrich</td>
 <td>F9378</td>
 </tr>
 <tr>
 <td>Foetal Bovine Serum (FBS)</td>
 <td>Gibco</td>
 <td>10270-106</td>
 </tr>
 <tr>
 <td>2-(4-(2-Hydroxyethyl)- 1-piperazinyl)-ethansulfons&permil;ure (HEPES)</td>
 <td>Roth</td>
 <td>9105.3</td>
 </tr>
 <tr>
 <td>NaOH, 5 M</td>
 <td>clinical pharmacy, hospital</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Penicillin/Streptomycin (100x)</td>
 <td>PAA Laboratories</td>
 <td>P11-010</td>
 </tr>
 <tr>
 <td>Pipettaid (EXPRESS)</td>
 <td>BD (Becton, Dickinson and Company)</td>
 <td>357591</td>
 </tr>
 <tr>
 <td>Potassiumchlorid</td>
 <td>Fluka</td>
 <td>60132</td>
 </tr>
 <tr>
 <td>Potassiumdihydrogenphospate anhydrous</td>
 <td>JT Baker</td>
 <td>0241</td>
 </tr>
 <tr>
 <td>Potassiummonohydrogenphosphate</td>
 <td>JT Baker</td>
 <td>3246-01</td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 [-]D-Glucose [+]L-Glutamine</td>
 <td>Gibco</td>
 <td>11879-020</td>
 </tr>
 <tr>
 <td>RPMI Medium 1640 [+]L-Glutamine</td>
 <td>PAA Laboratories</td>
 <td>E15-840</td>
 </tr>
 <tr>
 <td>Sodiumhydrogencarbonat</td>
 <td>Merck</td>
 <td>1.06329.0500</td>
 </tr>
 </tbody>
</table>
 
Media and Solutions
Dithizone Solution
<ul>
 <li>Dissolve 100 mg DTZ in 10 ml DMSO and dilute in 40 ml PBS</li>
 <li>Sterile filter the solution (0.22 &mu;m)</li>
</ul>
Enzyme Solution
<ul>
 <li>Dissolve 100 mg Liberase RI in 20 ml RPMI 1640 media (without supplements)</li>
 <li>Dissolve 100 mg DNase in 1 ml RPMI 1640 media (without supplements)</li>
 <li>Sterile filter the solution (0.22 &mu;m) and keep the enzyme on ice</li>
</ul>
Wash media (1000ml/5.5mM Glucose)
<ul>
 <li>Dissolve 100 ml FBS (heat inactivated)</li>
 <li>Add 450 ml RPMI 1640 (no glucose)</li>
 <li>Add 450 ml RPMI 1640 (11 mM glucose)</li>
 <li>Sterile filter the solution (0.22 &mu;m)</li>
</ul>
Islet culture media (500ml/5,5mM Glucose)
<ul>
 <li>Dissolve 10 ml 1M HEPES (pH 7.4)</li>
 <li>Add 50 ml FBS</li>
 <li>Add 5 ml penicillin/streptomycin (100x)</li>
 <li>Add 2.75 ml 1 M Glucose Solution</li>
 <li>Add 432.25 ml RPMI 1640 (no glucose)</li>
 <li>Sterile filter the solution (0.22 &mu;m)</li>
</ul>
Euro Collins Solution
<ul>
 <li>4,083 g potassium hydrogenphosphate</li>
 <li>70,0 g glucose</li>
 <li>2,237 g potassium chloride</li>
 <li>14,80 g potassium monohydrogenphosphate</li>
 <li>1,680 g sodium hydrogencarbonate</li>
 <li>40 ml electrolyte solution (E 154)</li>
 <li>pH 7.4</li>
 <li>Add distilled water up to 2000 ml</li>
 <li>Sterile filter the solution (0.22 &mu;m)</li>
</ul>
Ficoll Stock Solution
<ul>
 <li>Add 1000 ml of Euro Collins Solution to 500 g Ficoll</li>
 <li>Add 8.94 g HEPES</li>
 <li>Stir overnight until Ficoll is solved</li>
 <li>pH 7.4</li>
 <li>Measure the density</li>
</ul>
Ficoll Gradients
<ul>
 <li>Calculate the amount of FBS, Euro Collins Solution and Ficoll Stock Solution according to the density</li>
 <li>Combine the 3 solutions and measure the density with a densitometer</li>
 <li>Add Euro Collins Solution or Ficoll Stock Solution until the density is adjusted</li>
 <li>Sterile filter the solution (0.22 &mu;m) and store at 4&deg;C</li>
</ul>

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.

Our protocol yielded an average of ~500 islets per gram of pancreatic tissue, although this greatly varied between preparations due largely to differences in fibrosis and collagenase activity. We achieved &gt; 90% islet purity by first staining with Dithizone during tissue processing, then handpicking them 24 hours after isolation. To determine the quality of the purified islets, we tested for 25 mM glucose-stimulated insulin secretion under static conditions for 2 hours. We found insulin secretion comparable to that of islets obtained from ECIT islet isolation and transplantation centers. As islets isolated from partially pancreatectomized pancreata are not meant for islet transplantation, assessment of the total IEQ was not considered to be a critical factor.
Importantly, our method enabled us to isolate islets for functional studies from 25 partial pancreatectomized patients between 2005 and 20085. These islets were analyzed for glucose-stimulated insulin secretion and specific protein expression by western blotting and immunohistochemistry. Islets isolated from partially pancreatecomized pancreata could also be used to compare gene and protein expression profiles, ultrastructural appearance and functional responses of non-diabetic and diabetic islets. Because there are more patients undergoing partial pancreatectomy than there are organ donors, our protocol could allow enough samples and data to be collected for robust statistical analysis.

Watch this Scientific Journal Video about Isolation of Human Islets from Partially Pancreatectomized Patients at JoVE.com

Isolation of Human Islets from Partially Pancreatectomized Patients

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 ...

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Research

JoVE Journal

Medicine

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Dynamic Visual Tests to Identify and Quantify Visual Damage and Repair Following Demyelination in Optic Neuritis Patients

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to assess changes in myelination at the central nervous system (CNS). However, standard measurements, including routine visual tests and MRI scans, are not sensitive enough for this purpose. We present two visual tests addressing dynamic monocular and binocular functions which may closely associate with the extent of myelination along visual pathways. These include Object From Motion (OFM) extraction and Time-constrained stereo protocols. In the OFM test, an array of dots compose an object, by moving the dots within the image rightward while moving the dots outside the image leftward or vice versa. The dot pattern generates a camouflaged object that cannot be detected when the dots are stationary or moving as a whole. Importantly, object recognition is critically dependent on motion perception. In the Time-constrained Stereo protocol, spatially disparate images are presented for a limited length of time, challenging binocular 3-dimensional integration in time. Both tests are appropriate for clinical usage and provide a simple, yet powerful, way to identify and quantify processes of demyelination and remyelination along visual pathways. These protocols may be efficient to diagnose and follow optic neuritis and multiple sclerosis patients.
In the diagnostic process, these protocols may reveal visual deficits that cannot be identified via current standard visual measurements. Moreover, these protocols sensitively identify the basis of the currently unexplained continued visual complaints of patients following recovery of visual acuity. In the longitudinal follow up course, the protocols can be used as a sensitive marker of demyelinating and remyelinating processes along time. These protocols may therefore be used to evaluate the efficacy of current and evolving therapeutic strategies, targeting myelination of the CNS.

Object From Motion (OFM) and Time-constrained stereo protocols are sensitive tools to identify monocular and binocular dynamic visual function deficits, which are uniquely affected in optic neuritis patients. Furthermore, these tests may be used as quantitative noninvasive tools to assess the extent of myelination along visual pathways.

In order to follow optic neuritis patients and evaluate the effectiveness of their treatment, a handy, accurate and quantifiable tool is required to ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

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 ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

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 ...

Full-root Aortic Valve Replacement by Stentless Aortic Xenografts in Patients with Small Aortic Roots

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial valve might not meet the functional needs. The implantation of a too-small stented pericardial valve, leading to an effective orifice area indexed to a body surface area less than 0.85 cm2/m2, is regarded as prosthesis-patient mismatch (PPM). A PPM negatively affects the regression of left ventricular hypertrophy and thus the normalization of left ventricular function and the alleviation of symptoms. Persistent left ventricular hypertrophy is associated with an increased risk of arrhythmias and sudden cardiac death. In the case of predictable PPM, there are three options: 1) accept the PPM resulting from the implantation of a stented pericardial valve when comorbidities of the patient forbid the more technically demanding operative technique of implanting a larger prosthesis, 2) enlarge the aortic root to accommodate a larger stented valve substitute, or 3) implant a stentless biological valve or a homograft. Compared to classical aortic valve replacement with stented pericardial valves, the full-root implantation of stentless aortic xenografts offers the possibility of implanting a 3-4 mm larger valve in a given patient, thus allowing significant reduction in transvalvular gradients. However, a number of cardiac surgeons are reluctant to transform a classical aortic valve replacement with stented pericardial valves into the more technically challenging full-root implantation of stentless aortic xenografts. Given the potential hemodynamic advantages of stentless aortic xenografts, we have adopted full-root implantation to avoid PPM in patients with small aortic roots necessitating an aortic valve replacement. Here, we describe in detail a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses. Limitations of this technique and alternative options are discussed.

Full-root aortic valve replacement by stentless aortic xenograft is a viable option in patients with small aortic roots. We describe, a technique for the full-root implantation of stentless aortic xenografts, with emphasis on the management of the proximal suture line and coronary anastomoses, and discuss its limitations and alternative options.

In patients with small aortic roots who need an aortic valve replacement with biological valve substitutes, the implantation of the stented pericardial ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...