Name: Pancreatic Islet Isolation and Purification from Lewis Rats Using Enzymatic Digestion and Density-Gradient Separation (Video) | JoVE Journal
Uploaded: 2026-04-03T12:53:00.000+00:00
Duration: 7 min 44 s
Description: Learn pancreatic islet isolation from Lewis rats using enzymatic digestion and density-gradient separation. This standardized protocol maximizes yield, purity, and viability for diabetes research and transplantation studies.

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This study was supported by the National Institutes of Health (NIH) R01DK133610-01 (A.G., N.K.) and NIH NIDDK R01DK132104 (A.G.), Breakthrough T1D SRA-2021-1078-S-B (AG) and SRA-2022-1224-S-B (A.G., N.K.), and the Vivian L. Smith Foundation.

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<li>Kenyon, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+survival+and+function+of+intrahepatic+islet+allografts+in+rhesus+monkeys+treated+with+humanized+anti-CD154%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Long-term survival and function of intrahepatic islet allografts in rhesus monkeys treated with humanized anti-CD154.</a> Proc Natl Acad Sci U S A. 96 (14), 8132-8137 (1999).</li>
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<li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adjustment+of+digestion+enzyme+composition+improves+islet+isolation+outcome+from+marginal+grade+human+donor+pancreata%5BTitle%5D)+AND+%22Cell+Tissue+Bank%22%5BJournal%5D)">Adjustment of digestion enzyme composition improves islet isolation outcome from marginal grade human donor pancreata.</a> Cell Tissue Bank. 8, 187-194 (2007).</li>
<li>McCall, M. D., Maciver, A. H., Pawlick, R., Edgar, R., Shapiro, A. M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Histopaque+provides+optimal+mouse+islet+purification+kinetics%3A+comparison+study+with+Ficoll%2C+iodixanol+and+dextran%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">Histopaque provides optimal mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran.</a> Islets. 3 (4), 144-149 (2011).</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/pubmed?term=((Automated+method+for+isolation+of+human+pancreatic+islets%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Automated method for isolation of human pancreatic islets.</a> Diabetes. 37 (4), 413-420 (1988).</li>
<li>BenchChem Technical Support Team. An in-depth technical guide to cell separation using iodixanol. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22BenchChem%22">BenchChem</a>. , (2026).</li>
<li>Dellê, H., Saito, M. H., Yoshimoto, P. M., Noronha, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+iodixanol+for+the+purification+of+rat+pancreatic+islets%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">The use of iodixanol for the purification of rat pancreatic islets.</a> Transplant Proc. 39 (2), 467-469 (2007).</li>
<li>Noguchi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Iodixanol-controlled+density+gradient+during+islet+purification+improves+recovery+rate+in+human+islet+isolation%5BTitle%5D)+AND+%22Transplantation%22%5BJournal%5D)">Iodixanol-controlled density gradient during islet purification improves recovery rate in human islet isolation.</a> Transplantation. 87 (11), 1629-1635 (2009).</li>
<li>Min, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Superiority+of+Visipaque+%28iodixanol%29-controlled+density+gradient+over+Ficoll-400+in+adult+porcine+islet+purification%5BTitle%5D)+AND+%22Transplant+Proc%22%5BJournal%5D)">Superiority of Visipaque (iodixanol)-controlled density gradient over Ficoll-400 in adult porcine islet purification.</a> Transplant Proc. 42 (5), 1825-1829 (2010).</li>
<li>Paez-Mayorga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neovascularized+implantable+cell+homing+encapsulation+platform+with+tunable+local+immunosuppressant+delivery+for+allogeneic+cell+transplantation%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Neovascularized implantable cell homing encapsulation platform with tunable local immunosuppressant delivery for allogeneic cell transplantation.</a> Biomaterials. 257, 120232(2020).</li>
<li>Paez-Mayorga, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Implantable+niche+with+local+immunosuppression+for+islet+allotransplantation+achieves+type+1+diabetes+reversal+in+rats%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Implantable niche with local immunosuppression for islet allotransplantation achieves type 1 diabetes reversal in rats.</a> Nat Commun. 13, 7951(2022).</li>
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</ol>

A.G. is a co-founder and chief scientific advisor of Continuity Biosciences, LLC. A.G., J.P-M., and C.Y.X.C. are inventors of intellectual properties licensed by continuity Biosciences, LLC. All other authors declare no competing interests.

This protocol outlines a high-throughput approach for isolating and purifying pancreatic islets from Lewis rats. The success of the method depends on several critical steps that collectively preserve islet integrity and function. Among these are the precise and complete intraductal perfusion of the pancreas, the careful cleaning of the perfused pancreas, and the exact enzymatic digestion time. Consistent whole organ perfusion is crucial to achieve uniform distension of the pancreas and ensure even distribution of the collagenase-thermolysin-DNase blend, which acts respectively to digest the extracellular matrix, enhance enzyme penetration, and reduce DNA-mediated viscosity21,22. Under-perfusion can result in incomplete tissue digestion and loss of a significant portion of islets, as the under-digested tissue is filtered out with the sieve. Similarly, inadequate removal of adipose or vascular tissue before digestion can introduce non-endocrine impurities that lower purity. Finally, prolonged digestion results in islet desegregation from excessive extracellular matrix breakage, while suboptimal digestion yields undigested tissue resistant to mechanical digestion via spinal needle. These steps represent the most frequent technical pitfalls and should be carefully monitored during training and optimization.
While this method was designed specifically for Lewis rats, it likely can be adapted to other strains or species through minor re-optimization of digestion time and enzyme ratio23,24,25,26. Some laboratories may choose to adjust the ratio of collagenase, thermolysin, and DNase I in the Working Dissociation Medium (WDM) to optimize yield for their specific enzyme lot or tissue characteristics. To do so, laboratories may carefully follow the manufacturer&#39;s step-by-step guide for enzyme and tissue optimization and, in all instances, avoid adding components such as 0.1 M EDTA, cysteine, mercaptoethanol, protease inhibitors, serum, or albumin, as these may inhibit collagenase, thermolysin, and DNase 1 enzyme activity27. Although no definitive working time for the enzyme blend before dissociation has been published, prior data show that extended cold preservation of pancreases adversely affects islet yield and function28. Empirically, we have found that, in our protocol, successful isolations were consistently achieved when the total cold ischemia time, defined as the interval between placing the first pancreas on ice for cleaning and transferring it to the 37 &#176;C water bath, was kept under 1.5 h. Further optimization of this parameter may be warranted depending on the end-user application.
Despite its robustness, this protocol has several practical and technical limitations. First, the enzyme blend (collagenase, thermolysin, and DNase I) and density gradients are essential for efficient tissue dissociation and islet isolation, yet impose significant cost. Our selection of enzyme combination was informed by the structural characteristics of the pancreatic extracellular matrix and the need to balance effective tissue dissociation with the preservation of islet integrity. Each enzymatic component facilitates a distinct role: collagenase targets fibrillar and basement-membrane collagens to liberate islets; thermolysin, a neutral protease, cleaves non-collagenous matrix proteins and cell-cell adhesion molecules, reducing the required collagenase exposure; and DNase I minimizes viscosity caused by DNA released from lysed cells29,30,31. Although lower-cost formulations of digestive enzymes are commercially available, their purity is variable and may compromise enzymatic performance13,23. Given that collagenase is isolated from bacterial cultures, additional purification steps reduce endotoxin contamination but substantially increase cost13,23.
For collagenase, we recommend preparations with an activity of approximately 2,000 to 4,000 W&#252;nsch units per bottle, which supports efficient dissociation at moderate dosing while avoiding prolonged digestion. Notably, the blend should include a balanced ratio of collagenase class I to class II, corresponding to a collagenase II/total collagenase fraction of approximately 0.3-0.5. This ratio provides strong collagenolytic activity while limiting excessive degradation of non-collagenous extracellular matrix. Additionally, the collagenase mixture should be complemented by a moderate level of neutral protease activity (roughly 150,000-250,000 units) and DNase, for reasons already commented.
From a quality control standpoint, each lot should include documentation of low endotoxin burden (&#8804;50 EU/mg protein), high enzyme purity (&#8805;85% by HPLC), and verified lot-to-lot activity, as enzyme purity and composition can vary considerably by manufacturer. Finally, variations in lot composition may affect digestion consistency and islet yield, necessitating the performance of lot-specific internal validation. Such validation can be achieved by following the standardized digestion conditions described in this protocol and subsequently assessing digestion efficiency and islet morphology. Indicators of suboptimal enzyme performance include incomplete tissue dissociation, islets with irregular or rough borders, or the need for excessive mechanical shear to achieve separation, in which case titration of the digestion time may be necessary.
Similar considerations apply to density gradients. We selected a polysucrose-based gradient, a non-ionic, highly hydrophilic sucrose-epichlorohydrin copolymer that forms stable gradients up to 1.2 g/mL and has a long track record in islet isolation32,33. Iodixanol is a well-validated alternative, and studies in human islets show that iodixanol-based gradients can improve islet recovery while maintaining viability and function34,35,36,37. However, rodent data are inconclusive, with some studies reporting superior outcomes using iodixanol and others demonstrating better viability and function with polysucrose-based gradients32,35. Critically, the use of commercially manufactured, quality-controlled gradient solutions is recommended to enhance reproducibility across laboratories. Polysucrose-based gradients are widely available at optimal densities for islet isolation, whereas iodixanol gradients are typically prepared by in-house dilution, introducing additional variability. A second limitation is that surgical harvesting requires a high level of technical proficiency, particularly for intraductal perfusion through the Sphincter of Oddi and rapid pancreas excision. Even minor delays or misalignment can result in tissue damage, leakage, uneven perfusion, or prolonged warm ischemia, which may compromise islet yield and viability. To mitigate this limitation, the protocol includes detailed procedural descriptions and original graphical aids with color-coded anatomical landmarks to facilitate understanding. New operators are encouraged to become comfortable with the perfusion technique prior to performing full isolations, and practice on carcasses is advised before attempting live harvests.
Third, for maximum efficiency, this protocol requires coordinated teamwork. During pancreas harvesting and enzymatic digestion, three trained operators are typically required: one performing sterile surgery, one cleaning the pancreas under sterile conditions, and one circulating and euthanizing animals under non-sterile conditions. Following digestion, downstream purification steps can generally be performed by fewer operators once proficiency is achieved. While this coordination represents an upfront logistical demand, it enables the isolation of many islets from multiple animals within a relatively short timeframe, improving overall throughput compared to less coordinated workflows. While this protocol is technically demanding and resource-intensive, these limitations are common to all islet isolation methods. By emphasizing standardized digestion conditions, structured training, and coordinated execution, this high-throughput approach can reduce time and labor per isolation and yield consistent, reproducible results when performed by a trained team.
Compared with conventional rodent islet isolation protocols, this method offers substantial advantages in throughput, reproducibility, and consistency. Traditional single-organ digestions rely heavily on manual hand-picking and often yield variable purity13. In contrast, our protocol utilizes pooled digestion and density-gradient purification, substantially reducing preparation time and inter-sample variability while maintaining high islet viability and functional responsiveness. The isolated islets were comprehensively characterized for morphology (dithizone staining), viability (live/dead assay), and function (glucose-stimulated insulin secretion), confirming the robustness of the method. Collectively, this standardized workflow enables the generation of plentiful, clean, and functional islet preparations suitable for downstream applications, including encapsulation, transplantation, and microphysiological modeling38,39,40. Importantly, Lewis rat islets isolated with this technique have reversed diabetes in preclinical models, underscoring the translational relevance of this reproducible and scalable approach41.

Pancreatic islet transplantation represents a viable strategy for restoring endogenous insulin production in individuals with type 1 diabetes (T1D)1. Beyond T1D, islet biology is increasingly relevant to type 2 diabetes (T2D), a condition characterized by progressive &#946;-cell dysfunction and loss, as well as to patients undergoing partial or total pancreatectomy for chronic pancreatitis, pancreatic tumors, or traumatic injury. In these contexts, access to high-quality islets is essential for advancing cellular therapies, modeling &#946;-cell failure, studying islet-immune and islet-stromal interactions, and developing emerging regenerative approaches2. Consequently, robust and reproducible islet isolation procedures are critical to support both mechanistic research and translational development across a wide range of pancreatic endocrine disorders.
Although islets constitute less than 2% of the pancreatic mass, they exert a disproportionately significant role in glucose homeostasis. Their intrinsic vulnerability to hypoxia, enzymatic over-digestion, oxidative stress, and mechanical shear makes them particularly sensitive to isolation-related injury3,4,5. Even subtle deviations in temperature, oxygenation, or tissue handling can impair &#946;-cell stimulus-secretion coupling or trigger apoptosis, owing to limited antioxidant defenses and high metabolic demand6,7. As a result, most conventional protocols emphasize rapid downstream use, typically within 24-48 h of harvesting in both clinical and experimental settings8,9,10,11,12. While this &#34;immediate-use&#34; framework minimizes deterioration, it restricts experimental flexibility, hampers longitudinal studies, and complicates efforts to accumulate sufficient islet numbers for large in vitro assays or in vivo transplantation experiments.
Among rodent models, Lewis rats are widely utilized for islet research due to their inbred genetic background, stable immune profile, and reproducible physiological characteristics14. Strain-dependent differences in islet yield, responsiveness, and resilience are well documented. De Groot et al. demonstrated that Lewis rat islets exhibit superior glucose-stimulated insulin secretion compared with other strains, underscoring their suitability for functional, metabolic, and transplantation studies15. Additional work has confirmed their reliability for immunological and allo-/xenotransplantation research14,15,16. However, across laboratories, variability in islet yield, purity, and function remains a significant barrier. Much of this variability arises from inconsistencies in enzyme blend, intraductal perfusion techniques, pancreas handling, digestion timing, and purification strategy17.
To address these limitations, we developed a reproducible, step-by-step protocol for surgical harvesting, enzymatic digestion, and density-gradient purification of pancreatic islets from Lewis rats. This workflow is optimized to maximize yield, minimize fragmentation, and preserve functional viability. Importantly, it establishes clear benchmarks, including islet equivalents (IEQ), viability over time, purity, and glucose-stimulated insulin secretion, that enable investigators to reliably distinguish successful from suboptimal isolations18,19,20. By improving both yield and longevity, the protocol enhances experimental flexibility and may reduce the number of donor animals required, supporting more ethical and scalable experimental design.
Distinct from many traditional approaches, the present method demonstrates that Lewis rat islets can be maintained in culture for up to 7 days with preserved viability and function, expanding their utility for extended in vitro studies. The protocol further incorporates a streamlined, pooled-processing strategy capable of handling up to 30 pancreases in a single batch, enabling uniform digestion and reducing inter-sample variability. With endocrine purity reaching 98%, the procedure eliminates the need for labor-intensive hand-picking and reduces opportunities for microbial contamination. Compared with single-step or bulk digestion methods that frequently produce impure or mechanically compromised preparations13, this high-throughput workflow reliably yields large numbers of clean, transplantation-quality islets suitable for in vitro assays, metabolic profiling, and in vivo transplantation studies.

<table><tbody><tr><td>21 G x 2 in. BD PrecisionGlide Needle</td><td>BD</td><td>305129</td><td></td></tr><tr><td>500 &amp;micro;m Brass Frame and 304 Stainless Steel Screen, 3&quot; Diameter x 1&quot; Deep (Sieve number 35)</td><td>McMaster-Carr</td><td>34735K525</td><td></td></tr><tr><td>Air-Tite Sterile Syringes, Luer lock</td><td>Fisher Scientific</td><td>14-817-178</td><td></td></tr><tr><td>Biogel PI UltraTouch M</td><td>M&amp;ouml;lnlycke</td><td>42665</td><td></td></tr><tr><td>Bonn Scissors; Straight; Sharp Points; 15 mm Blade Length; 3 1/2&quot; Overall Length</td><td>Roboz</td><td>RS-5840</td><td></td></tr><tr><td>Braintree Scientific GERMINATOR 500 DRY STERIL</td><td>Fisher Scientific</td><td>NC9956482</td><td></td></tr><tr><td>Cell-IQ Series 5.8 cu.ft. CO2 Laboratory Incubator</td><td>PHCBI</td><td>MCO-170AICUVHL-PA</td><td></td></tr><tr><td>Corning 100 mm Not TC-treated Culture Dish</td><td>Corning</td><td>430591</td><td></td></tr><tr><td>Corning Fetal Bovine Serum, 500 mL, Regular, USDA Safety Tested (Heat Inactivated)</td><td>Corning</td><td>35011CV</td><td></td></tr><tr><td>Corning Islet Gradient 1.069 (density 1.069 g/cm&lt;sup&gt;3&lt;/sup&gt;)</td><td>Corning</td><td>99-815-CIS</td><td>Requires special request from company</td></tr><tr><td>Corning Islet Gradient 1.096 (density 1.096 g/cm&lt;sup&gt;3&lt;/sup&gt;)</td><td>Corning</td><td>99-691-CIS</td><td>Requires special request from company</td></tr><tr><td>Corning Islet Gradient 1.108 (density 1.108 g/cm&lt;sup&gt;3&lt;/sup&gt;)</td><td>Corning</td><td>99-692-CIS</td><td>Requires special request from company</td></tr><tr><td>Covidien Tendersorb Underpads</td><td>Fisher Scientific</td><td>22-225-102</td><td></td></tr><tr><td>DNase I Grade II</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>Falcon&amp;nbsp;50 mL High Clarity Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>Fisherbrand Aluminum Foil, Standard-Gauge Roll</td><td>Fisher Scientific</td><td>01-213-100</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Nonwoven Gauze Sponges</td><td>Fisher Scientific</td><td>22-028-559</td><td></td></tr><tr><td>Fisherbrand Reusable Glass Heavy-Duty Low-Form Beakers</td><td>Fisher Scientific</td><td>FB101600</td><td></td></tr><tr><td>Fluorescence Mircoscope&amp;nbsp; BZ-X810</td><td>Keyence</td><td>BZ-X810</td><td></td></tr><tr><td>Glucose Solution</td><td>Gibco</td><td>A2494001</td><td></td></tr><tr><td>Halsey Micro Needle Holder</td><td>Fine Science Tools</td><td>12500-12</td><td></td></tr><tr><td>HBSS, calcium, magnesium, no phenol red</td><td>Gibco</td><td>14025092</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15630080</td><td></td></tr><tr><td>Hemostatic Forceps Classic Crile 5-1/2 Inch Length</td><td>McKesson</td><td>1231217</td><td></td></tr><tr><td>Hudson (Ewald) Forceps</td><td>Roboz</td><td>RS-5238</td><td></td></tr><tr><td>Hypodermic Needle Monoject 1-1/2 Inch Length 16 Gauge Regular Wall Without Safety</td><td>McKesson</td><td>43481</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25030081</td><td></td></tr><tr><td>Liberase T-Flex, Research Grade</td><td>Roche</td><td>5989132001</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td><td>Invitrogen</td><td>&amp;nbsp;L3224</td><td></td></tr><tr><td>MATLAB software package</td><td>MathWorks</td><td>&lt;a href=&quot;https://www.mathworks.com/pricing-licensing.html?prodcode=ML&quot;&gt;https://www.mathworks.com/pricing-licensing.html?prodcode=ML&lt;/a&gt;</td><td></td></tr><tr><td>NE-4000 Two Channel Programmable Syringe Pump</td><td>SyringePump</td><td>NE-4000-US</td><td></td></tr><tr><td>NEEDLE, HYPO STR DISP 27GX1/2&quot;(100/BX) EXLINT</td><td>McKesson</td><td>408877</td><td></td></tr><tr><td>Nunc Cell Culture/Petri Dishes</td><td>Thermo-Scientific</td><td>174926</td><td></td></tr><tr><td>Olympus CKX41 Inverted Phase Contrast Microscope</td><td>Olympus</td><td>CKX41-B</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010023</td><td></td></tr><tr><td>Penicillin&amp;sbquo; Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>RPMI 1640, no glucose</td><td>Gibco</td><td>11879020</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Sodium Pyruvate (100 mM stock)</td><td>Gibco</td><td>11360070</td><td></td></tr><tr><td>Sorval ST 16R</td><td>Thermo-Scientific</td><td>75004381</td><td></td></tr><tr><td>Stainless steel 304 syringe needle</td><td>Milipore Sigma</td><td>Z117013-1EA</td><td></td></tr><tr><td>SURGICAL DRAPE, DISPOSABLE, 36 IN X 100 YD</td><td>Covetrus</td><td>10408</td><td></td></tr><tr><td>Surgical Scissors - ToughCut</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr><tr><td>Thumb Dressing Forceps 4.5&quot; Serrated Delicate 1.3 mm Tip Width</td><td>Roboz</td><td>RS-8120</td><td></td></tr><tr><td>Thermo Scientific Nunc 200mL Centrifuge Tube</td><td>Thermo-Scientific</td><td>14-962-17</td><td></td></tr><tr><td>Thermo Scientific Nunc Centrifuge Bottle Adapters and Cushions</td><td>Thermo-Scientific</td><td>12-565-283B</td><td></td></tr><tr><td>TSDUB15 15L Dubnoff Shaking Water Bath</td><td>Fisher Scientific</td><td>300610298</td><td></td></tr><tr><td>TX-400 4 x 400 mL Swinging Bucket Rotor</td><td>Thermo-Scientific</td><td>75003181</td><td></td></tr><tr><td>Tygon S-50-HL</td><td>Tygon</td><td>AAXoooo3</td><td></td></tr><tr><td>Ultra Sensitive Rat Insulin ELISA kit</td><td>Crystal Chem</td><td>90060</td><td></td></tr></tbody></table>

pancreatic islet isolation and purification from lewis rats using enzymatic digestion and density-gradient separation

Pancreatic Islet Isolation and Purification from Lewis Rats Using Enzymatic Digestion and Density-Gradient Separation

Pancreatic islets are essential for both clinical islet transplantation and preclinical studies aimed at understanding the mechanisms and progression of diabetes. However, progress in these fields is often limited by the availability of high-quality islets, as emerging therapeutic strategies require larger, cleaner, and more standardized preparations. This article presents a high-throughput, standardized protocol for surgical harvesting, enzymatic digestion, and density-gradient purification of pancreatic islets from Lewis rats. The method combines intraductal pancreas perfusion with a defined enzyme blend, pooled digestion of multiple pancreases, and density-based separation to maximize yield while minimizing fragmentation and eliminating the need for routine hand-picking. Islet yield is quantified using two complementary approaches: a traditional aliquot-based counting method commonly used in clinical settings, and a semi-automated whole-preparation imaging-based method. In a representative isolation from 12 pancreases, manual counting yielded approximately 3,300 islet equivalents (IEQ) and 2,700 absolute islets per pancreas (~39,600 total IEQ), with a size distribution of 62% (50-100 &#181;m), 17% (100-150 &#181;m), 10% (150-200 &#181;m), 5% (200-250 &#181;m), and 6% (&#62;250 &#181;m). In comparison, whole-prep automated analysis reported 16,350 total IEQ (1,362 IEQ/pancreas), highlighting inherent variability in aliquot-based quantification. Preparations had a purity of 98% by dithizone staining, and islet viability was maintained at 98% in vitro on days 3 and 7 after isolation. Functional assessment by static glucose-stimulated insulin secretion demonstrated a 10-fold increase in insulin release following a 1-h stimulation with 16.7 mM glucose. Overall, this protocol provides a reproducible and efficient approach for generating high-purity, functionally intact islet preparations that can be used immediately without additional cleanup. Islets retain robust function in culture for up to seven days, enabling flexibility for large-scale in vitro assays as well as in vivo transplantation studies.

The following results represent a typical islet isolation performed using the described method, with twelve Lewis rat pancreases processed in parallel.
Yield and size distribution
Manual aliquot counting and IEQ conversion
Two independent 100 µL aliquots were obtained from the mixed islet suspension, as described in section "Islet Counting," Step 6.1, and counted by diameter bins (50-100 µm, 100-150 µm, 150-200 µm, 200-250 µm, 250-300 µm, 250-300 µm). Counts were converted to islet equivalents (IEQ; 150 µm reference) using standard equivalency tables and extrapolated to the total suspension volume (see Figure 4A). Across the test isolation (n = 12), yield was 3330 IEQ/pancreas with a median islet diameter in the 50-100 µm bin. Replicate aliquots agreed by 18.8% per bin (coefficient of variation), indicating moderate variation yet expected with the use of manual counting methods. Over the previous 14 islet isolations, this method produced an average of 1353 (SD 607) IEQ/pancreas.
Automated whole-plate quantification
To benchmark manual IEQ, brightfield images were acquired at D1 using a microscope and stitched to generate whole-dish composites (4× objective; 1/100 s exposure). Automated sizing and object quantification were performed using Fiji and coding with a predefined size threshold of 50-600 µm. Images were first preprocessed to reduce background variation and enhance contrast uniformity. Islet structures were identified based on intensity differences relative to surrounding tissue, generating binary object masks. Adjacent or partially touching islets were separated to avoid underestimation of object number. Objects outside the specified size range were excluded to remove debris and non-islet tissue. For each retained object, morphometric parameters including area and x- and y-axis diameters were extracted. Islets were then binned by diameter, and IEQ was calculated by multiplying the number of islets in each size bin by the corresponding conversion factor. Automated sizing yielded 98,832 total objects, with mean x- and y-axis diameters of 117 µm and 116 µm, respectively, and an estimated total of 16,350 IEQ, which strongly correlated with manual IEQ measured at D0 (Spearman r = 0.937, p = 0.0048; see Figure 4A-C).
Islet cell shedding
Whole-plate images were acquired 2 h post-purification and again at Day 1 (D1), using the same methodology in "Automated whole-plate quantification" to evaluate early islet stabilization ("shedding"). Object counts changed by 0.9% and IEQ by -6.11% from 2 h to D1 (see Figure 4D). Paired comparison of islet diameters between D0 and D1 showed a statistically significant median decrease of 2 µm (Wilcoxon signed-rank test, p = 0.0136, n = 2820). However, the small magnitude of this change is unlikely to be biologically or clinically meaningful.
<img alt="Islet size analysis; bar charts on manual vs. automatic distribution and Spearman correlation graph." class="xfigimg" src="/files/ftp_upload/70414/70414fig4.jpg" data-altupdatedat="1778558422000" data-alt="Figure 4"> 
Figure 4: Quantitative assessment of islet size distribution, correlation, and early shedding. Graphical summary of islet measurements obtained through manual and automated analyses. (A) Day 0 (D0) islet size distribution based on manual quantification across standardized islet-equivalent IEQ size bins. (B) Day 1 (D1) islet size distribution determined by automated image-based measurements across the same standardized IEQ size bins. (C) Correlation of counts between manual D0 and automated D1 quantification across IEQ size bins. (D) Box plot of the distribution of islet diameters between D0 and D1 for automated measurements of the same plate. <a href="https://www.jove.com/files/ftp_upload/70414/70414fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Purity (endocrine vs. exocrine content)
Dithizone (DTZ) staining
On D1, an aliquot of islets (n = 451) was stained with DTZ for 30 s at room temperature and immediately imaged using brightfield microscopy (4× objective; 1/20 s exposure). In Fiji, background pixels were removed such that the remaining pixel area corresponded to the total islet area. DTZ-negative exocrine tissue regions, identified by darker color contrast, were manually selected using a lasso tool, and the area of each region of interest was automatically recorded in pixels. Endocrine purity was calculated as the percentage of DTZ-positive pixels relative to total islet pixels, yielding a purity of 98.56 ± 0.38% (Figure 5A).
Viability and functional performance
Glucose-stimulated insulin secretion (GSIS)
At each time point (D1, D3, D7), 15 size-matched islets were hand-picked and placed into individual wells of a 24-well plate, prepared in quadruplicate. The islets were incubated at 37 °C 5% CO2 in Krebs buffer under a low-high-low glucose stimulation protocol: first in low glucose (2.8 mM, 60 min), followed by high glucose (16.7 mM, 60 min, see Table 1). Effluent fractions were collected at the end of each incubation period, and insulin concentrations were quantified using a rat insulin ELISA assay. The stimulation indices were calculated as the ratio of insulin secretion during high to low glucose conditions. Stimulation indices were 9.37, 8.34, and 6.7, at days 1, 3, and 7, respectively, with no significant differences (Figure 5B).
Live/Dead staining
At D1, D3, and D7, 15 islets were hand-picked and stained with the Live/Dead viability kit according to the manufacturer's instructions. Briefly, islets were incubated in the Live/Dead staining solution for 30 min at 25 °C, then imaged under consistent microscope settings to ensure comparability across time points (4X objective; 1/30 s exposure, Texas Red and GFP filters). Viability was quantified using a Python script employing scikit-image for islet segmentation and fluorescence measurement. Islets were identified by combining the green and red fluorescence channels, applying Li's thresholding method to generate a binary mask, and using watershed segmentation to separate touching islets. Objects were filtered by area to exclude debris and artifacts. For each islet, viability was calculated as the percentage of live integrated density (green pixels) relative to total integrated density (green plus red pixels).The results showed viability of 68 ± 9% at D1, 98.2 ± 0.6% at D3, and 100% at D7. A positive control sample treated with 70% ethanol for 10 min exhibited 2.7 ± 7.2% viability (see Figure 5C-F).
<img alt="Cell morphology study; microscopy images; GSIS bar graph; fluorescent analysis D1-D7, PS assay results." class="xfigimg" src="/files/ftp_upload/70414/70414fig5.jpg" data-altupdatedat="1778558422000" data-alt="Figure 5"> 
Figure 5: Assessment of islet purity, viability, and functional responsiveness. Representative images and quantitative summaries of isolated islet characteristics over time. (A) Representative 10× brightfield image of islets stained with dithizone (DTZ), showing red-stained endocrine islets and unstained exocrine tissue. (B) Glucose-stimulated insulin secretion (GSIS) values of high-glucose/low-glucose (HG/LG) stimulation index for Days 1, 3, and 7. (C-E) Live/dead fluorescence images of islets at Day 1 (C), Day 3 (D), and Day 7 (E). (F) Live/dead fluorescence image of positive-control islets treated with ethanol. <a href="https://www.jove.com/files/ftp_upload/70414/70414fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(A) Washing Media</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>Amount (mL)</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>-</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>10%</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Pen-Strep (10,000 U/mL)</td>
			<td>1%</td>
			<td>5.5 mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>25 mM</td>
			<td>12.5 mL from 1M stock</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>2 mM</td>
			<td>5 mL from 200 mM stock</td>
		</tr>
		<tr>
			<td>(B) Islet Culture Media</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>Amount (mL or g)</td>
		</tr>
		<tr>
			<td>RPMI 1640, No glucose</td>
			<td>-</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>10%</td>
			<td>50 mL</td>
		</tr>
		<tr>
			<td>Pen-Strep (10,000 U/mL)</td>
			<td>1%</td>
			<td>5.5 mL</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>20 mM</td>
			<td>11 mL from 1M stock</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>1 mM</td>
			<td>5.5 mL from 100 mM stock</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>5.5 mM</td>
			<td>2.75 mL from 200 g/L stock</td>
		</tr>
		<tr>
			<td>(C) Penicillin-Streptomycin Rinse Solution</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>Amount (mL or g)</td>
		</tr>
		<tr>
			<td>Saline Solution, 0.9%</td>
			<td>-</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Penicillin‚ Streptomycin (10,000 U/mL)</td>
			<td>2%</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>(D) Quenching Buffer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>Amount (mL or g)</td>
		</tr>
		<tr>
			<td>HBSS </td>
			<td>-</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>20%</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>2.50%</td>
			<td>2.5 mL</td>
		</tr>
		<tr>
			<td>(E) Krebs Buffer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Final concentration</td>
			<td>Amount (mL or g)</td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>-</td>
			<td>975 mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>115 mM</td>
			<td>6.72 g</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>24 mM</td>
			<td>2.02 g</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5 mM</td>
			<td>0.3728 g</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>25 mM</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
			<td>0.0952 g</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.10%</td>
			<td>1 g</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>2.5 mM</td>
			<td>0.2775 g</td>
		</tr>
	</tbody>
</table>
Table 1: Solution list. Composition and preparation details for all solutions used throughout the islet isolation and purification protocol. Each subsection lists components, final concentrations, preparation volumes, and catalog numbers. (A) Washing Media, used for rinsing and washing tissue during digestion and purification. (B) Islet Culture Media, used for post-isolation islet maintenance and viability. (C) Penicillin-Streptomycin Rinse Solution, used to sterilize and rinse harvested pancreases. (D) Quenching Buffer, used to terminate enzymatic digestion and preserve cell viability. (E) Krebs Buffer, used during glucose-stimulated insulin secretion (GSIS) assays.
<table border="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number of Pancreases</td>
			<td>Collagenase (15.38 mg/mL)</td>
			<td>Thermolysin         (1 mg/mL)</td>
			<td>DNAse               (100 mg/mL)</td>
			<td>Working buffer</td>
			<td>600 mL Beakers to use</td>
			<td>Volume/Beaker</td>
		</tr>
		<tr>
			<td>30</td>
			<td>4.5 mL</td>
			<td>2.25 mL</td>
			<td>450 µL</td>
			<td>450 mL</td>
			<td>5</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>24</td>
			<td>3.6 mL</td>
			<td>1.8 mL</td>
			<td>360 µL</td>
			<td>360 mL</td>
			<td>4</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>20</td>
			<td>3 mL</td>
			<td>1.5 mL</td>
			<td>300 µL</td>
			<td>300 mL</td>
			<td>3</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>16</td>
			<td>2.4 mL</td>
			<td>1.2 mL</td>
			<td>240 µL</td>
			<td>240 mL</td>
			<td>2</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>12</td>
			<td>1.8 mL</td>
			<td>0.9 mL</td>
			<td>180 µL</td>
			<td>180 mL</td>
			<td>2</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>10</td>
			<td>1.5 mL</td>
			<td>0.75 mL</td>
			<td>150 µL</td>
			<td>150 mL</td>
			<td>2</td>
			<td>25 mL</td>
		</tr>
		<tr>
			<td>Final conc.</td>
			<td>0.15 mg/mL</td>
			<td>5 mg/mL</td>
			<td>0.1 mg/mL</td>
			<td colspan="3"></td>
		</tr>
	</tbody>
</table>
Table 2: Digestive enzyme solution by pancreas quantity. Volumes of collagenase, thermolysin, and DNase I required to prepare the Working Dissociation Medium (WDM) for varying numbers of rat pancreases. Final concentrations of each enzyme are listed at the bottom of the table.
<table border="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number of Pancreases</td>
			<td>Number 50 mL tubes</td>
			<td>Volume/ syringe</td>
			<td>Number of 1.096 syringes</td>
			<td>Number of 1.068 syringes</td>
		</tr>
		<tr>
			<td>30</td>
			<td>10 tubes</td>
			<td>50 mL</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>24</td>
			<td>8 tubes</td>
			<td>40 mL</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>20</td>
			<td>6 tubes</td>
			<td>30 mL</td>
			<td>2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>16</td>
			<td>4 tubes</td>
			<td>40 mL</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>12</td>
			<td>4 tubes</td>
			<td>40 mL</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>10</td>
			<td>2 tubes</td>
			<td>20 mL</td>
			<td>1</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 3: Gradient loading by pancreas quantity. Number of 50 mL conical tubes and corresponding gradient volumes required for islet purification based on the number of pancreases processed.
<table border="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Islet Diameter     Range (µm)</td>
			<td>Islet Particle     Number (AI)</td>
			<td>IEQ Conversion Factor</td>
			<td>IEQ per Range</td>
		</tr>
		<tr>
			<td>50-100</td>
			<td>a</td>
			<td>* 0.167</td>
			<td>a * 0.167</td>
		</tr>
		<tr>
			<td>101-150</td>
			<td>b</td>
			<td>* 0.648</td>
			<td>b * 0.648</td>
		</tr>
		<tr>
			<td>151-200</td>
			<td>c</td>
			<td>* 1.685</td>
			<td>c * 1.685</td>
		</tr>
		<tr>
			<td>201-250</td>
			<td>d</td>
			<td>* 3.500</td>
			<td>d * 3.500</td>
		</tr>
		<tr>
			<td>251-300</td>
			<td>e</td>
			<td>* 6.315</td>
			<td>e * 6.315</td>
		</tr>
		<tr>
			<td>301-350</td>
			<td>f</td>
			<td>* 10.352</td>
			<td>f * 10.352</td>
		</tr>
		<tr>
			<td>&gt; 350</td>
			<td>g</td>
			<td>* 15.833</td>
			<td>g * 15.833</td>
		</tr>
	</tbody>
</table>
Table 4: Islet equivalence conversion. Conversion of islet diameter ranges to standardized islet equivalents (IEQ). The table lists the observed islet diameter bins, the number of islet particles per range, the corresponding IEQ conversion factor for each size range, and the resulting IEQ contribution per range.

This protocol describes a standardized method for surgical harvesting, enzymatic digestion, and density-gradient purification of Lewis rat pancreatic islets, enabling high-yield, high-purity preparations for direct use in transplantation or in vitro studies.

Pancreatic islets are essential for both clinical islet transplantation and preclinical studies aimed at understanding the mechanisms and progression of ...

Our research focuses on developing a standardized high throughput Lewis Rat eyelet isolation protocol to optimize yield, purity, viability, and functional longevity. Existing methods yield variable impure short-lived eyelets. This protocol standardizes digestion, purification and pooling to improve consistency and longevity.To begin, place a euthanized rat under a microscope. Using skin scissors, make a V-shaped incision starting from the pubic synthesis toward each lateral rib cage to access the abdominal cavity. Using flat forceps, retract the small and large intestines to the left side of the animal to clear the abdominal cavity workspace.Then use the flat forceps and curved hemostats to unfurl the upper duodenum. Ensure the common bile duct and hepatic portal vein are within the field of view. Now use the curved hemostats to retract the liver in the cephalon direction.Clamp the common bile duct and hepatic vein one to two millimeters from the liver. Under the microscope view, with flat forceps and flat hemostats, gently mobilize the contents within the duodenum to clear the sphincter of audi. Then gently clamp the duodenum approximately five millimeters from each side of the sphincter of audi to isolate the surrounding region.Ensure minimal compression to preserve tissue integrity. Carefully insert the needle of a syringe containing sterile WDM through the duodenal wall. Pass the needle through the sphincter body and advance it into the main pancreatic duct for perfusion.While maintaining constant pressure, perfuse seven to 10 milliliters of WDM into the pancreas at a rate of about 500 microliters per second until the pancreas is fully distended. Now, remove both flat hemostats from the duodenum. Then gently dissect and free the pancreas from surrounding structures starting from the spleen, stomach, large intestine, duodenum, and mesentary.Gently lift the perfused pancreas with flat forceps and use fine dissecting scissors to sever any remaining connective tissue attachments. Immediately transfer the excised pancreas into a Petri dish containing ice cold rinse solution and proceed to harvest the next pancreas while the excised pancreas is being cleaned. To clean the pancreas's use a pair of fine curved forceps and fine dissecting scissors to carefully remove lymph nodes, adipose tissue, mesentary, and large vasculature from the perfused pancreas if present.Transfer the cleaned pancreas into a sterile 600 milliliter beaker containing 25 milliliters of cold WDM. Cover the beaker with sterile aluminum foil to maintain cleanliness during transfer from the surgical workspace to the cell culture workspace. Transfer the beaker into an orbital shaker water bath set at 130 revolutions per minute for 10 minutes.At the five minute mark, manually swirl the beaker clockwise and then counterclockwise to aid tissue dissociation. At the 10 minute mark, rapidly transfer the beaker and submerge it in ice to slow enzyme activity. Then add an equal volume of quenching buffer to the solution in the beaker to stop the digestion.Using a 50 milliliter syringe fitted with a 14 gauge spinal needle, gently aspirate and expel the tissue suspension repeatedly to dissociate large tissue fragments. Transfer the suspension into 200 milliliter conical tubes. After centrifuging the suspension, carefully decant the supernatant and resuspend the pellet in 100 milliliters of washing media.Now filter the suspension through a sterile 500 micrometer sieve placed over a sterile 600 milliliter beaker to remove large undigested tissue. Transfer the filtrate into new 200 milliliter conical tubes before centrifuging again. For eyelet purification, use a pre wedded 10 milliliter serological pipette to resuspend each tissue pellet in 15 milliliters of gradient solution per two milliliters of pellet volume.Mix gently until the suspension is uniform. Carefully dispense 15 milliliters of the cell suspension into the bottom of each 50 milliliter conical tube avoiding contact with the tube walls to ensure clean gradients. Then use a syringe pump to dispense 10 milliliters of each gradient at a rate of five milliliters per minute while positioning the 16 gauge needle tip against the inner wall of the tube to prevent mixing.After centrifuging the tubes, identify the eyelet layer located at the interfaces between the gradient layers. Using a wedded past pipette, collect the eyelet layer, transfer the collected eyelets into a pre chilled 200 milliliter conical tube containing five milliliters of washing media. Bring up the volume to 200 milliliters with washing media and perform the following washing and centrifugation steps.Then evenly plate the eyelets into non tissue culture treated 100 millimeter Petri dishes using one plate for every five pancreas'processed. Across the test isolation, yield was 3, 330 eyelet equivalents per pancreas with a median eyelet diameter in the 50 to 100 micrometer bin. Automated sizing yielded 98, 832 total objects, which strongly correlated with manual eyelid equivalents measured at day zero.The endocrine purity was about 98.56%based on dithozone staining at day one. Stimulation indices were 9.37 at day one, 8.34 at day three and 6.7 at day seven with no significant differences between time points. This method enables the study of eyelet transplantation, beta cell function, diabetes reversal, eyelet encapsulation, and eyelet immune or microenvironment interactions.This protocol can be applicable to preclinical transplantation, diabetes modeling, encapsulation studies, and invitro functional assays requiring high purity Lewis Rat eyelets.

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JoVE Journal

Biology

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part II: purification and culture of human islets) using a modified automated method.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes.  Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

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.

Isolation of Human Islets from Partially Pancreatectomized Patients

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. This protocol explains a method for isolating pancreatic islets by first perfusing collagenase via Ampulla of Vater to digest the exocrine pancreatic tissue and a simplified density gradient centrifugation to purify the islets from mice.

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet β-cell replication and to develop methods for stimulating β-cell regeneration. Herein a high-content screening method to identify and assess the β-cell replication-promoting activity of small molecules is presented.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250–350 high quality and fully functional islets per mouse.

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

This study developed a simplified method to efficiently extract functional primary islets and acinar cells from the mouse pancreas, offering a valuable tool for studying intercellular communication in the pathogenesis of Acute Pancreatitis-Induced Diabetes.

A Simple and Rapid Method for Simultaneous Isolation of Primary Islets and Primary Pancreatic Acinar Cells from Mice

Learn pancreatic islet isolation from Lewis rats using enzymatic digestion and density-gradient separation. This standardized protocol maximizes yield, purity, and viability for diabetes research and transplantation studies.

Pancreatic Islet Isolation and Purification from Lewis Rats Using Enzymatic Digestion and Density-Gradient Separation

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