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 supported by JDRF (nPOD: 5-SRA-2018-557-Q-R) and The Leona M. &#38; Harry B. Helmsley Charitable Trust (Grant#2018PG-T1D053, G-2108-04793). 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&#160;http://www.jdrfnpod.org/for-partners/npod-partners/. The authors are grateful to the donors and their families for their invaluable contribution. This work was supported by the American Diabetes Association 4-22-PDFPM (J.K.P.) and the Leona M. and Harry B. Helmsley Charitable Trust (grant 2015-PG-T1D-052).

Human pancreatic sections from tissue donors of both sexes were obtained via the Network for Pancreatic Organ Donors with Diabetes (nPOD) tissue bank, University of Florida. Organ tissue from deceased individuals without identifiers have been determined to be non-human subjects research in accordance with organ donation laws and regulations and classified as Non-Human Subjects by the University of Florida Institutional Review Board (IRB; IRB no. 392-2008), waiving the need for consent. nPOD tissues specifically used for this project were approved as nonhuman by the University of Florida IRB (IRB20140093).
NOTE: For readers entirely new to the slice technique and its applications, such as perifusion and calcium imaging, it might be advisable to gain practical experience and develop basic skills using mouse or rat slices3,28 before working with human samples.
1. Preparations
NOTE: These preparations should be performed prior to tissue arrival.
<ol>
	<li>Prepare HEPES buffer by mixing 125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, 2 mM L-alanine, L-arginine, and L-glutamine. Adjust pH to 7.4 and filter sterilize.</li>
	<li>Add glucose and or other stimuli to the buffer to prepare solutions for perifusion experiment. For baseline buffer add 5.5 mM glucose. This buffer will be used for slicing procedures, slice collection, and perifusion preparations.</li>
	<li>Prepare at least two dishes for slice collection by adding aprotinin (10 &#181;g/mL) to the baseline buffer.</li>
	<li>Prepare 3.8 % low melting point agarose solution in HEPES buffer without BSA and amino acids (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES) and keep it at 37 &#176;C.</li>
	<li>Assemble the vibratome, by placing a fresh blade in the holder. If applicable set the blade angle to 15&#176; (if using Leica VT1200S). If applicable, calibrate the vibratome.</li>
	<li>Switch on the perifusion machine, select number of chambers and program protocol.</li>
	<li>Place all necessary solutions in the perifusion machine, prime the system and keep solutions heated. The machine does calculate the necessary volumes of each solution used for the protocol.</li>
</ol>
2. Tissue processing
NOTE: Slices should be generated immediately after receipt. Any delay might cause difficulties in the procedure and lead to tissue degradation.
<ol>
	<li>Place pancreas tissue in a dish with baseline buffer under a stereo microscope (Figure 1A). Gently remove connective, fibrotic, and adipose tissues using forceps and scissors.</li>
	<li>Cut tissue into multiple small pieces of approximately 0.5 cm3 (Figure 1B) using scissors or a scalpel. Blot dry pancreas pieces on tissue paper.</li>
	<li>Transfer 4 pieces into a 35 mm petri dish and fill dish with agarose solution until all pieces are fully submerged. Allow agarose to fully solidify.</li>
	<li>Carefully cut pieces out of the agarose. Run the scalpel along the edge of the dish to remove the agarose and carefully separate tissue blocks. Ensure pieces are surrounded by a thin layer of agarose.</li>
</ol>
3. Slicing
<ol>
	<li>Glue tissue pieces on the metal plate of the vibratome by placing them upside down.</li>
	<li>Mount the plate into the tray and fill the tray with baseline buffer (HEPES containing 5.5 mM baseline glucose).</li>
	<li>Set vibratome to automated slicing at 120 &#181;m and adjust start and end position.</li>
	<li>Move the blade shortly above the tissue and start slicing at slow speed (0.1 mm/s) and amplitude at 0.8 mm. Speed can be increased if tissue permits (Figure 1C).</li>
	<li>Collect slices by using curved forceps or a small brush. Accumulate slices in baseline buffer containing aprotinin (Figure 1D).</li>
	<li>Allow slices to rest for at least 1 h in baseline buffer with aprotinin. Place slices on a slow orbital shaker to allow flushing out of enzymes released by the cutting procedure.</li>
</ol>
4. Live/dead assay
NOTE: This is an optional assay that will show viability of tissue slices after procedure. However, slices cannot be reused once stained.
<ol>
	<li>Transfer a single slice in a well filled with baseline buffer.</li>
	<li>Add Fluorescein diacetate (FDA, 50 &#181;g/mL), incubate for 1 min at room temperature and protect from light.</li>
	<li>Add Propidium iodide (PI,50 &#181;g/mL), incubate for 1 min at room temperature and protect from light.</li>
	<li>Transfer slice to another well filled with PBS to wash for 1 min.</li>
	<li>Transfer slices into a Petri dish or mount on a glass slide with cover slip for imaging (Figure 2A,B).</li>
</ol>
5. Perifusion
NOTE: This protocol describes how to perform dynamic perifusion for tissue slices, however it is suitable for isolated islets as well. Islet chambers will need preparation with filter paper and bead solution before loading the islets as described in the user manual of the machine. However, islet and slice chamber can be used together in the same experiment.
<ol>
	<li>Choose 3 slices and trim the agarose down to a minimum under a stereomicroscope using brush and scalpel.</li>
	<li>Add a drop of baseline buffer (HEPES containing 5.5 mM baseline glucose) on the grid of the slice chamber and gently place one slice on the grid. Repeat for each of the 3 slices (Figure 3A). If slices are very small stack more than 3 chambers to increase the islet count. Do not place more than one slice per chamber. 
	NOTE: Number of islets per slice varies greatly between donors and depends on slice size (10-100 islets/slice). A total of 3 slices have been proven sufficient to measure both insulin and glucagon secretion.</li>
	<li>For isolated islets, use a minimum of 30 islets per column for insulin secretion. Recommended to use 100 islets for glucagon detection. Assemble individual slice chamber parts from top to bottom.</li>
	<li>Connect slice chamber to the inflow and outflow tubing&#39;s in the machine and start the protocol (Figure 3C).</li>
	<li>Use chamber heating and tray cooling pump during the protocol. Change collection plates during the protocol and store at 4 &#176;C if quantification is performed the same day or store at -80 &#176;C until then.</li>
	<li>When protocol is done, remove chambers, disassemble, and collect slices for lysis (3% HCl in absolute Ethanol) or fixation (in 4% Paraformaldehyde).</li>
	<li>Clean the machine by flushing it with water, 10% bleach, water and air.</li>
	<li>Quantify hormone secretion by using commercially available detection kits. 
	NOTE: Figure 4 shows absolute levels of insulin and glucagon secretion of 3 slices to estimate concentration ranges.</li>
</ol>
6. Calcium imaging
<ol>
	<li>Prepare Calcium dye (e.g., Fluo4-AM, Calbryte) solution according to manufacturer instruction.</li>
	<li>Dilute dye in baseline HEPES buffer solution and add aprotinin (10 &#181;g/mL).</li>
	<li>Transfer a single slice and incubate for 30-60 min on an orbital shaker at room temperature and protect from light.</li>
	<li>During incubation time, prepare slice imaging setup by filling the perifusion syringes and priming of tubings (Figure 5A,B).</li>
	<li>Connect all devices, switch on heater, and start pump flow. Recommended to use both in-flow heating and platform heater and flow rate of 0.5 mL/min.</li>
	<li>Gently place a single slice in the imaging chamber and secure using a harp.</li>
	<li>Use a low magnification objective to identify imaging area. Using reflection helps to identify islet area (Figure 5C).</li>
	<li>Switch to a higher magnification objective (e.g., 20x or 40x) and adjust position according to experimental setup. 
	NOTE: To ensure optimal cell viability and islet integrity, it is recommended that the optical sections should be 1-2 cell layers below the cutting surface.</li>
	<li>Set z-stack and or tile scan position. Set imaging interval and recording duration. Here, a resolution of at least 256 x 256 was used to allow discrimination of individual cells, an imaging interval of 5-10 s and a z-stack range (if applicable) of 30-60 &#181;m with an interval of 10 &#181;m between planes (one cell layer). See Figure 6 for detailed imaging settings. 
	NOTE: Set imaging parameter to get best resolution and maximum area but avoid bleaching the sample.</li>
	<li>Start imaging and switch solution inflow according to experiment. Once done, collect slices and fix them for immunohistochemistry.</li>
</ol>

Slicing Human Pancreas for Islet Function Research

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+network+architecture+of+adult+human+islets+promotes+distinct+cell-cell+interactions+in+situ+and+is+altered+after+transplantation.">Vessel network architecture of adult human islets promotes distinct cell-cell interactions in situ and is altered after transplantation.</a> Endocrinology. 158 (5), 1373-1385 (2017).</li><li>Marolt, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+imaging+in+intact+mouse+acinar+cells+in+acute+pancreas+tissue+slices.">Calcium imaging in intact mouse acinar cells in acute pancreas tissue slices.</a> PLoS One. 17 (6), 0268644 (2022).</li><li>Stozer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+laser+scanning+microscopy+of+calcium+dynamics+in+acute+mouse+pancreatic+tissue+slices.">Confocal laser scanning microscopy of calcium dynamics in acute mouse pancreatic tissue slices.</a> J Vis Exp. (170), e62293 (2021).</li><li>Stozer, A., Dolensek, J., Rupnik, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose-stimulated+calcium+dynamics+in+islets+of+langerhans+in+acute+mouse+pancreas+tissue+slices.">Glucose-stimulated calcium dynamics in islets of langerhans in acute mouse pancreas tissue slices.</a> PLoS One. 8 (1), e54638 (2013).</li><li>Weitz, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+pancreatic+islet+macrophages+use+locally+released+atp+to+monitor+beta+cell+activity.">Mouse pancreatic islet macrophages use locally released atp to monitor beta cell activity.</a> Diabetologia. 61 (1), 182-192 (2018).</li><li>Speier, S., Yang, S. 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The authors have nothing to disclose.

Here we present a protocol to generate viable pancreas tissue slices and their use for functional readouts like dynamic hormone secretion and functional imaging. Similar to human islet isolation, the success of the slice procedure is influenced by various factors, including donor characteristics, tissue shipment time, and tissue quality25,29. Therefore, it is crucial to carefully select tissue samples for the experiment and keep ischemia times to a minimum. In this context, other potential sources of human tissue besides cadaveric donors should be carefully considered. Including surgical donors offers the option to combine functional slice data with relevant in vivo information from the same patient, strengthening the translational relevance11. However, this tissue source brings in other factors as biopsies are usually from older patients undergoing pancreatectomy mostly due to a localized tumor. Notably, pancreas biopsies are not performed in the context of diabetes.
When performing the actual slicing procedure, timely tissue processing is critical. Slices can be stored for several hours, as outlined in this protocol, or cultured for extended periods, as described by Qadir et al.19. At present, this protocol stands as the sole method for sustaining slice viability over prolonged durations however, future efforts should assess functional changes across diverse culture times and draw comparisons with isolated islets, from the same donor.
The most critical step in the process is careful tissue preparation before embedding in agarose. Large ducts and fibrotic tissue can complicate the slicing process and potentially lead to tissue blocks breaking out of the agarose. If this occurs and the pieces remain reasonably sized, they can be reprocessed and embedded for slicing. Maintaining good tissue quality and careful processing greatly enhances the slicing procedure's efficiency and yields the maximum quantity and quality of slices. The time elapsed from tissue preparation to slice generation should not exceed 2-3 h, as longer intervals significantly impact tissue viability.
During slice perifusion, a simple yet critical step is the precise trimming of the slice to ensure a perfect fit into the chamber. This allows for the proper bathing of the tissue and uninterrupted flow. Once the protocol is initiated, it is essential to avoid further manipulation of the chambers to prevent unwanted spikes in hormone release. Sufficient buffer should be prepared, and tubing should reach the solution's bottom to prevent air suction and chambers running dry.
For calcium imaging it is important to choose the area of interest carefully, depending on experimental needs and design. It is important to minimize photobleaching by choosing imaging parameters that reduce light exposure or shorten the overall protocol time. Like dynamic hormone secretion, maintaining proper solution flow and a heated setting is crucial, as slices require near-physiological conditions for optimal function (e.g., 37 &#176;C).
Pancreatic tissue slices effectively maintain the structural integrity of the pancreas, preserving cell-cell connections between its diverse cell types. Consequently, they provide an alternative to working with isolated islets, facilitating the concurrent exploration of both endocrine and exocrine functions and their interplay. To assess the interplay of individual cell types, it is crucial to discriminate between them. Staining afterward is one option, but it has limitations in terms of available fluorescent probes and the challenge of locating exact cell layers. Therefore, it is advisable to incorporate cell-specific stimuli into the protocol to enable cell discrimination based on responses. For discriminating between cell types in mouse or human slices, effective stimuli include adrenaline for alpha cells21,30, ghrelin for delta cells31, cerulein for acinar cells5,32, bile acids for ductal cells33, and norepinephrine for vascular cells22. Similar stimuli can be employed for measuring secretory responses. While imaging studies focus on individual cells, secretion studies analyze the collective response. Hence, it is crucial to include an ample number of slices to detect the desired outcomes. The optimal quantity may vary between cell types, with three slices proving sufficient for endocrine cells; however, it is advisable to use more slices to ensure detectability rather than risk missing valuable information.
Like any other method, tissue slices have limitations that should be considered when interpreting results. Stimulus application targeting specific cell types may lead to effects on other cell types in the slice, potentially triggering feedback loops. However, those are also important to study and thus responses measured can be more representative of a physiological response. For selective cell targeting, traditional in vitro protocols may be used. Importantly acinar cells contain pancreatic enzymes that can break down proteins and digest the tissue slice, resulting in cellular degradation within a matter of hours. To maintain viability, the consistent use of trypsin inhibitors is essential when slices are in a static state, even though their application may interfere with the successful transfer of viruses employed for labeling purposes. 
Compared to islet isolation, donor variability and tissue quality can impact both the quantity and viability of the obtained slices. Insufficient viability post-slicing may result in a short lifespan and hinder the ability to culture the slices. Furthermore, islet counts can vary significantly among donors, making it challenging to estimate islet content before conducting experiments. Consequently, careful selection of donor acceptance criteria and the implementation of appropriate normalization methods, such as the percentage of hormone content for secretion or fold change to baseline, are crucial. For consistent results, it is advised to assess tissue slice viability before the experiment. Moreover, incorporating relevant control stimuli (e.g., KCl) into the experiment is recommended. In cases of individual cell analysis, such as imaging, pre-sorting cells based on their response to these control stimulations can be implemented. Despite the challenges mentioned, slices offer a valuable augmentation to current research methods. 
The protocol described can be used as starting point for several applications, and pancreas slices can be manipulated, and responses examined after a variety of stimuli. We also direct readers to numerous research studies utilizing mouse or human pancreas slices, providing valuable insights for those planning their experiments. In the future, it is possible that potential therapies may be investigated using pancreatic slices or that disease mechanisms may be modeled.

Studies on islet physiology are fundamental for understanding the pathogenesis of diabetes and developing new therapeutic approaches. So far, research has relied on isolated islets, which exposes the islets to mechanical and enzymatic stresses, likely causing changes in cell physiology; moreover, it is not possible to evaluate islet function in the context of their natural tissue environment, which is likely impacted by exocrine and vascular cells among others1. When studying pancreas from donors with T1D, there is the challenge that their islets are difficult to isolate and might become fragmented during the isolation, which might produce a selection effect on islets that may not represent the population in vivo2. Moreover, islets will be separated from their complex environment and cellular connections, especially from the infiltrating immune cells that are found in inflamed islets and are more abundant in the islet periphery. Thus, while isolated islets are a cornerstone tool in diabetes research, there are limitations. In response, we introduce a groundbreaking protocol for generating living pancreas slices, offering a solution to these challenges.
The recent development and adoption of pancreatic tissue slicing techniques is considered a breakthrough for our ability to explore the intricate biology and functions of the pancreas. This innovation has opened new avenues for dynamic studies of islet physiology and interactions among endocrine, exocrine, neural, vascular, and immune cells within their natural anatomical context. Unlike conventional approaches, this in vitro setting preserves much of the organ&#39;s cytoarchitecture, allowing for a closer approximation to its native biology. Initially developed in mice by Speier and Rupnik in 20033, this method has demonstrated its utility to assess calcium imaging, electrophysiology, and hormone secretion for both intra- and inter-cellular signaling4,5,6,7,8,9. The pancreas slice platform was then applied to the study of human pancreas tissue obtained via surgical biopsy4,10,11,12. Our group demonstrated the feasibility of obtaining and utilizing pancreas slices from cadaveric organ donors through the activity of the Network for Pancreatic Organ Donors with Diabetes (nPOD)13. nPOD provides pancreas tissues to approved investigators conducting research about human type 1 diabetes, and since adopting the pancreas slice platform nPOD routinely generates and distributes living pancreas slices14,15,16,17. Since the implementation of the pancreas slice platform in 2020, nPOD has successfully distributed tissue slices from 43 donors (including 12 donors with T1D) to numerous investigators. Using these slices, researchers performed groundbreaking research on critical aspects of islet function and explored the interplay between islets and the vasculature, nervous system, and immune cells in the context of T1D13,18,19,20,21,22,23,24. Numerous studies have highlighted the limitations of traditional approaches and underscored the significance of techniques that can capture the dynamic interplay within the pancreas25,26,27. The adaptability of slicing techniques from mouse to human pancreas, coupled with their integration into programs like the Network for Pancreatic Organ Donors with Diabetes (nPOD), exemplifies the growing acknowledgment of the method&#39;s potential to unlock valuable insights into diseases such as Type 1 diabetes.

<table><tbody><tr><td>Alanine</td><td>Sigma</td><td>A7627</td><td>amino acid</td></tr><tr><td>AlexaFluor 488 goat anti-guineapig</td><td>Invitrogen</td><td>A11073</td><td>secondary antibody</td></tr><tr><td>AlexaFluor 546 goat anti-rat</td><td>Thermo Fisher</td><td>A11077</td><td>secondary antibody</td></tr><tr><td>AlexaFluor 647 goat anti-mouse</td><td>Thermo Fisher</td><td>A21235</td><td>secondary antibody</td></tr><tr><td>Aprotinin</td><td>Sigma</td><td>A1153</td><td>inhibitor</td></tr><tr><td>Arginine</td><td>Sigma</td><td>A5006</td><td>amino acid</td></tr><tr><td>Calbryte 520 AM</td><td>AAT Bioquest</td><td>20651</td><td>Calcium dye</td></tr><tr><td>Fluo4-AM</td><td>Invitrogen</td><td>F14201</td><td>Calcium dye</td></tr><tr><td>Fluorescein diacetat</td><td>Sigma</td><td>F7378</td><td>viability marker</td></tr><tr><td>Glucagon</td><td>Sigma</td><td>G2654</td><td>primary antibody</td></tr><tr><td>Glucagon ELISA (human kit)</td><td>Mercodia</td><td>10-1271-01</td><td>ELISA for hormone detection</td></tr><tr><td>Glutamine</td><td>Sigma</td><td>G3126</td><td>amino acid</td></tr><tr><td>Insulin</td><td>Dako</td><td>A-0546</td><td>primary antibody</td></tr><tr><td>Insulin ELISA (human)</td><td>Mercodia</td><td>10-1113-01</td><td>ELISA for hormone detection</td></tr><tr><td>Low melting point agarose</td><td>Sigma</td><td>A9414</td><td></td></tr><tr><td>LSM 900</td><td>Zeiss</td><td></td><td>confocal microscope</td></tr><tr><td>Pancreas Slice Chamber</td><td>Biorep Technologies</td><td>PERI-PSC-001</td><td>slice chamber for perifucion</td></tr><tr><td>Pancreas Slice Chamber Extender Kit</td><td>Biorep Technologies</td><td>PERI-PSC-EXT</td><td>slice chamber for perifucion</td></tr><tr><td>Pancreas Slice Chamber Perforated Plate</td><td>Biorep Technologies</td><td>PERI-PSC-PP</td><td>slice chamber for perifucion</td></tr><tr><td>Perifusion system with automated tray handling</td><td>Biorep Technologies</td><td>PERI4-02-230-FA</td><td></td></tr><tr><td>Propidium iodide</td><td>Life technologies</td><td>P1304MP</td><td>dead marker</td></tr><tr><td>Semi-automatic vibratome VT1200S</td><td>Leica</td><td>14048142066</td><td></td></tr><tr><td>Somatostatin</td><td>Millipore</td><td>MAB354</td><td>primary antibody</td></tr></tbody></table>

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.

When the protocol is executed successfully, 1 g of pancreas tissue yields approximately 100-200 slices. Subsequently, these slices should undergo stereomicroscope inspection to identify those rich in islets before proceeding with functional assessments. Viability, determined by labeling with Fluorescein Diacetate (FDA) and Propidium Iodide (PI), is expected to reach 80%-90% (Figure 2A). Viability may be notably lower at the cutting surface due to cell damage during the slicing process. In viable slices, dynamic hormone secretion (Figure 2B) is observed, resulting in robust insulin release upon exposure to high glucose and membrane depolarization with Potassium chloride (KCl).
Conversely, when there are delays during the slicing procedure or the tissue quality is suboptimal, the outcomes differ significantly. The number of obtained slices may decrease, and the viability assessment may indicate a higher proportion of non-viable cells labeled with PI (Figure 2C). In these cases, the functional assessment indicates a diminished or even absent response to high glucose and membrane depolarization (Figure 2D). The data from suboptimal experiments illustrate the importance of precise execution in a timely manner and tissue quality, as they can lead to reduced tissue viability and impaired functionality. These negative outcomes serve as a valuable reminder of the critical factors to consider in the success of this method.
Common findings in calcium imaging are visualized as a heat map, as depicted in Figure 5D, or as separate traces for individual cells, as presented in Figure 5E. It is important to emphasize that the dye labels all cell types indiscriminately, hence specific stimuli are essential for distinguishing cells based on their responses. To identify viable cells, we employ KCl and sort the cells based on responses that exceed the average baseline response by more than two standard errors. Moreover, slices can be fixed and stained, enabling identification of cell types.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66468/66468fig01.jpg" /> 
Figure 1: Tissue processing and slicing. (A) 1 g of unprocessed pancreas tissue. (B) Cleaned pancreas pieces ready for agarose embedding. (C) Slicing procedure using a vibratome. (D) Freshly cut human pancreas slices. <a href="https://www.jove.com/files/ftp_upload/66468/66468fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66468/66468fig02.jpg" /> 
Figure 2: Tissue viability and insulin secretion data. (A) Representative images for viable tissue slices. Maximum intensity projection of reflected laser light (grey, top left), PI for dead cells (red, bottom left), FDA for living cells (green, bottom right), and merged images for live and dead cells (top right). Dotted line indicates an islet. Scale bar 50 &#181;m. (B) Dynamic insulin secretion of human pancreatic tissue slices from a single nondiabetic donor. The insulin kinetics show a first phase peak response after 6 min of high glucose stimulation, followed by a plateau second phase. Data is normalized to the average baseline secretion at 5.5 mM glucose (stimulation index, fold change). Secretion has been performed on slices from the same donor as shown in (A). (C) Representative images for non-viable tissue slices. Maximum intensity projection of reflected laser light (grey, top left), PI for dead cells (red, bottom left), FDA for living cells (green, bottom right), and merged images for live and dead cells (top right). Dotted line indicates an islet. Scale bar 50 &#181;m. (D) Dynamic insulin secretion of human pancreatic tissue slices from a single nondiabetic donor. The insulin kinetics demonstrate a clear loss of both glucose stimulated insulin secretion and membrane depolarization with KCl. Data is normalized to the average baseline secretion at 5.5 mM glucose (stimulation index, fold change). Secretion has been performed on slices from the same donor as shown in (C). <a href="https://www.jove.com/files/ftp_upload/66468/66468fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66468/66468fig03.jpg" /> 
Figure 3: Tissue loading for dynamic perifusion. (A) Stacked slice chambers. Individual slices are loaded on a metal grid as shown in the insert. (B) Loading of isolated islets in islet chambers. Slice and islet chambers connected to perifusion machine. <a href="https://www.jove.com/files/ftp_upload/66468/66468fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66468/66468fig04.jpg" /> 
Figure 4: Hormone secretion in slices and isolated islets. Data shown in panels (A) and (B) originate from a different donor compared to panels (C) and (D). (A) Absolute hormone secretion from 3 pancreatic tissue slices from a single nondiabetic donor. Insulin secretion is shown in green and glucagon secretion in magenta. (B) Hormone secretion shown in (A) normalized to total hormone content (% of content). Content is measured from all 3 slices used in the experiment. (C) Dynamic insulin secretion of isolated islets (100 islets) and pancreatic tissue slices (3 slices) from the same nondiabetic donor. Data is shown in absolute numbers (mU/L). (D) Insulin secretion shown in (C) normalized to total insulin content (% of content). <a href="https://www.jove.com/files/ftp_upload/66468/66468fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66468/66468fig05.jpg" /> 
Figure 5: Imaging setup and expected results. (A) Setup for functional imaging with an upright confocal microscope and a perifusion setup. (B) Imaging chamber connected to inflow and outflow. (C) Z stack of confocal images of an islet within a tissue slice from a healthy human donor showing reflected light (top) and Fluo4 signal (bottom). Reflection is used to identify islets within the slice (dotted line). (D) Heatmap showing in vitro Ca2+ dynamics of islet cells expressed as the fluorescent intensity of Fluo4 normalized to the basal signal intensity at 5.5 mM glucose and stimulation with high glucose (16.7 mM). Each row represents a single cell followed over time in the x-axis and their response in magnitude change (%) of the fluorescent intensity over baseline (dF/F) shown in the color scale from blue (low intensity) to red (high intensity). (E) Representative traces of 4 individual cells showing a response to high glucose. <a href="https://www.jove.com/files/ftp_upload/66468/66468fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66468/66468fig06.jpg" /> 
Figure 6: Calcium imaging settings. Screenshot of the software of representative settings chosen to perform time-lapse recordings of 40 &#181;m (XYZT imaging). <a href="https://www.jove.com/files/ftp_upload/66468/66468fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Advancing Type 1 Diabetes Research Using Innovative Pancreatic Slice Platforms at JoVE.com

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.

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 ...

generating-human-pancreatic-tissue-slices-to-study-endocrine-exocrine

Research

JoVE Journal

Biology

1.4K Views.  Arthur Riggs Diabetes and Metabolism Research Institute. Onze focus ligt op onderzoek naar diabetes type één, met de nadruk op therapieën die het immuunsysteem reguleren en bèta-/alfacelfuncties herstellen. We hebben een uniek studieplatform ontwikkeld met behulp van levende menselijke alvleesklierplakken van orgaandonaties, als aanvulling op onderzoek naar geïsoleerde eilandjes. Deze aanpak maakt een gedetailleerde analyse van vitale cellulaire interacties en functies mogelijk.Onze samenwerking met een netwerk voor pancreasorgaandonore...

Video: Auteur in de schijnwerpers: onderzoek naar diabetes type 1 bevorderen met behulp van innovatieve platforms voor pancreassegmenten

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Developmental Biology

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Medicine

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell types include enzyme-secreting acinar cells, an arborized ductal system responsible for the transportation of enzymes to the gut, and hormone-producing endocrine cells. 
Endocrine beta-cells are the sole cell type in the body that produce insulin to lower blood glucose levels. Diabetes, a disease characterized by a loss or the dysfunction of beta-cells, is reaching epidemic proportions. Thus, it is essential to establish protocols to investigate beta-cell development that can be used for screening purposes to derive the drug and cell-based therapeutics. While the experimental investigation of mouse development is essential, in vivo studies are laborious and time-consuming. Cultured cells provide a more convenient platform for screening; however, they are unable to maintain the cellular diversity, architectural organization, and cellular interactions found in vivo. Thus, it is essential to develop new tools to investigate pancreatic organogenesis and physiology.
Pancreatic epithelial cells develop in the close association with mesenchyme from the onset of organogenesis as cells organize and differentiate into the complex, physiologically competent adult organ. The pancreatic mesenchyme provides important signals for the endocrine development, many of which are not well understood yet, thus difficult to recapitulate during the in vitro culture. Here, we describe a protocol to culture three-dimensional, cellular complex mouse organoids that retain mesenchyme, termed pancreatoids. The e10.5 murine pancreatic bud is dissected, dissociated, and cultured in a scaffold-free environment. These floating cells self-assemble with mesenchyme enveloping the developing pancreatoid and a robust number of endocrine beta-cells developing along with the acinar and the duct cells. This system can be used to study the cell fate determination, structural organization, and morphogenesis, cell-cell interactions during organogenesis, or for the drug, small molecule, or genetic screening.

Generation of Scaffold-free, Three-dimensional Insulin Expressing Pancreatoids from Mouse Pancreatic Progenitors In Vitro

Here, we present a protocol to generate insulin expressing 3D murine pancreatoids from free-floating e10.5 dissociated pancreatic progenitors and the associated mesenchyme.

The pancreas is a complex organ composed of many different cell types that work together to regulate blood glucose homeostasis and digestion. These cell ...

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human developmental processes. Stepwise directed differentiation that recapitulates developmental processes is one of the major ways to generate pancreatic cells including pancreas/duodenum homeobox protein 1+ (PDX1+) pancreatic progenitor cells. Conventional protocols initiate the differentiation with small colonies shortly after the passage. However, in the state of colonies or aggregates, cells are prone to heterogeneities, which might hamper the differentiation to PDX1+ cells. Here, we present a detailed protocol to differentiate hPSCs into PDX1+ cells. The protocol consists of four steps and initiates the differentiation by seeding dissociated single cells. The induction of SOX17+ definitive endoderm cells was followed by the expression of two primitive gut tube markers, HNF1&#946; and HNF4&#945;, and eventual differentiation into PDX1+ cells. The present protocol provides easy handling and may improve and stabilize the differentiation efficiency of some hPSC lines that were previously found to differentiate inefficiently into endodermal lineages or PDX1+ cells.

Efficient Generation of Pancreas/Duodenum Homeobox Protein 1+ Posterior Foregut/Pancreatic Progenitors from hPSCs in Adhesion Cultures

Here, we present a detailed protocol to differentiate human pluripotent stem cells (hPSCs) into pancreas/duodenum homeobox protein 1+ (PDX1+) cells for the generation of pancreatic lineages based on the non-colony type monolayer growth of dissociated single cells. This method is suitable for producing homogenous hPSC-derived cells, genetic manipulation and screening.

Human pluripotent stem cell (hPSC)-derived pancreatic cells are a promising cell source for regenerative medicine and a platform to study human ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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 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 ...

Immunology and Infection

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

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 ...

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 ...

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

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