Name: observing islet function and islet-immune cell interactions in live pancreatic tissue slices
Uploaded: 2021-04-12T13:30:00.000+00:00
Duration: 5 min 51 s
Description: Watch this Scientific Journal Video about Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices at JoVE.com

This work was funded by NIH grants R01 DK123292, T32 DK108736, UC4 DK104194, UG3 DK122638, and P01 AI042288. This research was performed with the support of the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641), a collaborative type 1 diabetes research project sponsored by JDRF (nPOD: 5-SRA-2018-557-Q-R), and The Leona M. &#38; Harry B. Helmsley Charitable Trust (Grant #2018PG-T1D053). The content and views expressed are the responsibility of the authors and do not necessarily reflect the official view of nPOD. Organ Procurement Organizations (OPO) partnering with nPOD to provide research resources are listed at http://www.jdrfnpod.org/for-partners/npod-partners/. Thank you to Dr. Kevin Otto, University of Florida, for providing the vibratome used to generate mouse slices.&#160;

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The authors declare no competing interests.

The objective of this protocol is to explicate the generation of pancreas slices and the procedures needed to employ the slices in functional and immunological studies. There are many benefits to using live pancreatic slices. However, there are several critical steps that are essential for the tissue to remain viable and useful during the described experiment protocols. It is imperative to work quickly. The length of time between injecting the pancreas and generating the slices on the vibratome should be minimized to mai...

Involvement of the pancreas is pathognomonic to diseases such as pancreatitis, T1D, and T2D1,2,3. The study of function in isolated islets usually involves removal of the islets from their surrounding environment4. The live pancreatic tissue slice method was developed to allow for the study of pancreatic tissue while maintaining intact islet microenvironments and avoiding the use of stressful islet isolation procedures5,6,7. Pancreatic tissue slices from human donor tissue have been successfully used to study T1D and have demonstrated processes of beta cell loss and dysfunction in addition to immune cell infiltration8,9,10,11,12,13. The live pancreatic tissue slice method can be applied to both mouse and human pancreatic tissue5,6,8. Human pancreatic tissue slices from organ donor tissues are obtained through a collaboration with the Network for Pancreatic Organ Donors with Diabetes (nPOD). Mouse slices can be generated from a variety of different mouse strains.
This protocol will focus on non-obese diabetic-recombination activating gene-1-null (NOD.Rag1-/-) and T cell receptor transgenic (AI4) (NOD.Rag1-/-.AI4&#945;/&#946;) mouse strains. NOD.Rag1-/- mice are unable to develop T and B cells due to a disruption in the recombination-activating gene 1 (Rag1)14. NOD.Rag1-/-.AI4&#945;/&#946; mice are used as a model for accelerated type 1 diabetes because they produce a single T cell clone that targets an epitope of insulin, resulting in consistent islet infiltration and rapid disease development15. The protocol featured here describes procedures for functional and immunological studies using live human and mouse pancreatic slices through the application of confocal microscopy approaches. The techniques described herein include viability assessments, islet identification and location, cytosolic Ca2+ recordings, as well as staining and identification of immune cell populations.

<table><tbody><tr><td>#3 Style Scalpel Handle</td><td>Fisherbrand</td><td>12-000-163</td><td></td></tr><tr><td>1 M HEPES</td><td>Fisher Scientific</td><td>BP299-100</td><td>HEPES Buffer, 1M Solution</td></tr><tr><td>10 cm Untreated Culture Dish</td><td>Corning</td><td>430591</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe</td><td>BD</td><td>301029</td><td>BD Syringe with Luer-Lok Tips</td></tr><tr><td>27 G Needle</td><td>BD</td><td>BD 305109</td><td>BD General Use and PrecisionGlide Hypodermic Needles</td></tr><tr><td>35 mm coverglass-bottom Petri dish</td><td>Ibidi</td><td>81156</td><td>&amp;micro;-Dish 35 mm, high</td></tr><tr><td>50 mL syringe</td><td>BD</td><td>309653</td><td></td></tr><tr><td>8-well chambered coverglass</td><td>Ibidi</td><td>80826</td><td>&amp;micro;-Slide 8 Well</td></tr><tr><td>APC anti-mouse CD8a antibody</td><td>Biolegend</td><td>100712</td><td></td></tr><tr><td>BSA</td><td>Fisher Scientific</td><td>199898</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C5670</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma</td><td>C7902</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (dihydrate)</td></tr><tr><td>Compact Digital Rocker</td><td>Thermo Fisher Scientific</td><td>88880020</td><td></td></tr><tr><td>Confocal laser-scanning microscope</td><td>Leica</td><td>SP8</td><td>Pinhole&amp;thinsp;= 1.5-2 airy units; acquired with 10x/0.40 numerical aperture HC PL APO CS2 dry and 20x/0.75 numerical aperture HC PL APO CS2 dry objectives at 512&amp;thinsp;&amp;times;&amp;thinsp;512 pixel resolution</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma</td><td>G7021</td><td>C&lt;sub&gt;6&lt;/sub&gt;H&lt;sub&gt;12&lt;/sub&gt;O&lt;sub&gt;6&lt;/sub&gt;</td></tr><tr><td>ddiH2O</td><td></td><td></td><td></td></tr><tr><td>Dithizone</td><td>Sigma-Aldrich</td><td>D5130-10G</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td>Dimethyl sulfoxide</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>2805</td><td></td></tr><tr><td>Falcon 35 mm tissue culture dish</td><td>Corning</td><td>353001</td><td>Falcon Easy-Grip Tissue Culture Dishes</td></tr><tr><td>FBS</td><td>Gibco</td><td>10082147</td><td></td></tr><tr><td>Feather No. 10 Surgical Blade</td><td>Electron Microscopy Sciences</td><td>7204410</td><td></td></tr><tr><td>fluo-4-AM</td><td>Invitrogen</td><td>F14201</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Gel Control Super Glue</td><td>Loctite</td><td>45198</td><td></td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>11049-10</td><td></td></tr><tr><td>Hardened Fine Scissors</td><td>Fine Science Tools</td><td>14090-09</td><td></td></tr><tr><td>HBSS</td><td>Gibco</td><td>14025092</td><td>Hanks Balanced Salt Solution</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td>C&lt;sub&gt;8&lt;/sub&gt;H&lt;sub&gt;18&lt;/sub&gt;N&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;S</td></tr><tr><td>Ice bucket</td><td>Fisherbrand</td><td>03-395-150</td><td></td></tr><tr><td>Isoflurane</td><td>Patterson Veterinary</td><td>NDC 14043-704-05</td><td></td></tr><tr><td>Johns Hopkins Bulldog Clamp</td><td>Roboz Surgical Store</td><td>RS-7440</td><td>&amp;nbsp;Straight; 500-900 Grams Pressure; 1.5&quot; Length</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34705</td><td>Kimtech Science&amp;trade; Kimwipes&amp;trade; Delicate Task Wipers, 2-Ply</td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td>This kit contains the calcein-AM live cell dye.</td></tr><tr><td>Low glucose DMEM</td><td>Corning</td><td>10-014-CV</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma</td><td>M9272</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt; (hexahydrate)</td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma</td><td>M2773</td><td>MgSO&lt;sub&gt;4&lt;/sub&gt; (heptahydrate)</td></tr><tr><td>Magnetic Heated Platform</td><td>Warner Instruments</td><td>PM-1</td><td>Platform for imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Microwave</td><td>GE</td><td>JES1460DSWW</td><td></td></tr><tr><td>Nalgene Syringe Filter</td><td>Thermo Fisher Scientific</td><td>726-2520</td><td></td></tr><tr><td>No.4 Paintbrush</td><td>Michaels</td><td>10269140</td><td></td></tr><tr><td>Open Diamond Bath Imaging Chamber</td><td>Warner Instruments</td><td>RC-26</td><td>Imaging chamber for dynamic stimulation recordings</td></tr><tr><td>Oregon Green 488 BAPTA-1-AM</td><td>Invitrogen</td><td>O6807</td><td>cell-permeable Ca&lt;sup&gt;2+&lt;/sup&gt; indicator</td></tr><tr><td>Overnight imaging chamber</td><td>Okolab</td><td>H201-LG</td><td></td></tr><tr><td>PBS</td><td>Thermo Fisher Scientific</td><td>20012050</td><td>To make agarose for slice generation</td></tr><tr><td>PE-labeled insulin tetramer</td><td>Emory Tetramer Research Core</td><td>sequence YAIENYLEL</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma</td><td>P5405</td><td>KCl</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P5655</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Razor Blades</td><td>Electron Microscopy Sciences</td><td>71998</td><td>For Vibratome; Double Edge Stainless Steel, uncoated</td></tr><tr><td>RPMI 1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>SeaPlaque low melting-point agarose</td><td>Lonza</td><td>50101</td><td>To make agarose for slice generation</td></tr><tr><td>Slice anchor</td><td>Warner Instruments</td><td>64-1421</td><td></td></tr><tr><td>Slice anchor (dynamic imaging)</td><td>Warner Instruments</td><td>640253</td><td>Slice anchor for dynamic imaging chamber</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S5886</td><td>NaCl</td></tr><tr><td>Sodium phosphate monohydrate</td><td>Sigma</td><td>S9638</td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4 &lt;/sub&gt;(monohydrate)</td></tr><tr><td>Soybean Trypsin Inhibitor</td><td>Sigma</td><td>T6522-1G</td><td>Trypsin inhibitor from Glycine max (soybean)</td></tr><tr><td>Stage Adapter</td><td>Warner Instruments</td><td>SA-20MW-AL</td><td>To fit imaging chamber for dynamic stimulation recordings on the microscope stage</td></tr><tr><td>Stage-top incubator</td><td>Okolab</td><td>H201</td><td></td></tr><tr><td>Stereoscope</td><td>Leica</td><td>IC90 E MSV266</td><td></td></tr><tr><td>SYTOX Blue Dead Cell Stain</td><td>Invitrogen</td><td>S34857</td><td>blue-fluorescent nucleic acid stain</td></tr><tr><td>Transfer Pipet</td><td>Falcon</td><td>357575</td><td>Falcon&amp;trade; Plastic Disposable Transfer Pipets</td></tr><tr><td>Valve Control System</td><td>Warner Instruments</td><td>VCS-8</td><td>System for dynamic stimulation recordings</td></tr><tr><td>Vibratome VT1000 S</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>FSGPD02</td><td>Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02</td></tr></tbody></table>

observing islet function and islet-immune cell interactions in live pancreatic tissue slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This protocol will yield live pancreatic tissue slices suitable for both functionality studies and immune cell recordings. Slice appearance in both brightfield and under reflected light are shown in Figure 1A,B. As discussed, islets can be found in slices using reflected light due to their increased granularity that occurs because of their insulin content (Figure 1C) and are clearly observed compared to the background tissue when reflected light...

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Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

observing-islet-function-islet-immune-cell-interactions-live

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JoVE Journal

Immunology and Infection

3.9K Views.  University of Florida. Ons protocol zal helpen bij het beantwoorden van de impact die immuuncelinteracties hebben op de eilandjesfunctionaliteit. De methode voor het snijden van levend pancreasweefsel maakt het mogelijk om de fysiologie en functie van eilandjes te bestuderen met behoud van de weefsels die ten grondslag liggen aan pathologieën en de inheemse micro-omgeving. Deze methode zou inzicht kunnen geven in de ziekteprocessen van pancreasziekten, zoals pancreatitis, diabetes type I en diabetes type II.

Video: Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

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, ...

Medicine

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Biology

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

Slicing Human Pancreas for Islet Function Research

Generating Human Pancreatic Tissue Slices to Study Endocrine and Exocrine Pancreas Physiology

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the pancreas endocrine and exocrine physiology and interplay. Much has been learned from the study of isolated pancreatic islets, but this prevents examining their function and interactions in the context of the whole tissue. Pancreas slices provide a unique opportunity to explore the physiology of normal, inflamed, and structurally damaged islets within their native environment, in turn allowing the study of interactions between endocrine and exocrine compartments to better investigate the complex dynamics of pancreatic tissue. Thus, the adoption of the living pancreas slice platform represents a significant advancement in the field. This protocol describes how to generate living tissue slices from deceased organ donors by tissue embedding in agarose and vibratome slicing as well as their utilization to assess functional readouts such as dynamic secretion and live cell imaging.

Author Spotlight: Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms

This protocol describes how to generate human pancreas slices from deceased organ donors to study cell function under near-physiological conditions. This innovative approach enables the investigation of normal and structurally damaged islets and the intricate interplay between endocrine and exocrine compartments.

It is crucial to study the human pancreas to understand the pathophysiological mechanisms associated with type 1 (T1D) and 2 diabetes (T2D) as well as the ...

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

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