JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Immunology and Infection

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

Published: April 12th, 2021

DOI:

10.3791/62207

1Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, 2Paul Langerhans Institute Dresden (PLID) of the Helmholtz Zentrum München at the University Clinic Carl Gustav Carus of Technische Universität Dresden, 3Institute of Physiology, Faculty of Medicine, Technische Universität Dresden, 4German Center for Diabetes Research (DZD), 5J. Crayton Pruitt Family Department of Biomedical Engineering, University of Florida

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 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.

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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NOTES: All experimental protocols using mice were approved by the University of Florida Animal Care and Use Committee (201808642). 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. Human pancreata were harvested from cadaveric organ donors by certified organ procurement organizations partnering with nPOD in accordance with organ donation laws and regulations and classified as "Non-Human Subjects.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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. & 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-part....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
#3 Style Scalpel Handle Fisherbrand 12-000-163
1 M HEPES Fisher Scientific BP299-100 HEPES Buffer, 1M Solution
10 cm Untreated Culture Dish Corning 430591
10 mL Luer-Lok Syringe BD 301029 BD Syringe with Luer-Lok Tips
27 G Needle BD BD 305109 BD General Use and PrecisionGlide Hypodermic Needles
35 mm coverglass-bottom Petri dish Ibidi 81156 µ-Dish 35 mm, high
50 mL syringe BD 309653
8-well chambered coverglass Ibidi 80826 µ-Slide 8 Well
APC anti-mouse CD8a antibody Biolegend 100712
BSA Fisher Scientific 199898
Calcium chloride Sigma C5670 CaCl2
Calcium chloride dihydrate Sigma C7902 CaCl2 (dihydrate)
Compact Digital Rocker Thermo Fisher Scientific 88880020
Confocal laser-scanning microscope Leica SP8 Pinhole = 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 × 512 pixel resolution
D-(+)-Glucose Sigma G7021 C6H12O6
ddiH2O
Dithizone Sigma-Aldrich D5130-10G
DMSO Invitrogen D12345 Dimethyl sulfoxide
Ethanol Decon Laboratories 2805
Falcon 35 mm tissue culture dish Corning 353001 Falcon Easy-Grip Tissue Culture Dishes
FBS Gibco 10082147
Feather No. 10 Surgical Blade Electron Microscopy Sciences 7204410
fluo-4-AM Invitrogen F14201 cell-permeable Ca2+ indicator
Gel Control Super Glue Loctite 45198
Graefe Forceps Fine Science Tools 11049-10
Hardened Fine Scissors Fine Science Tools 14090-09
HBSS Gibco 14025092 Hanks Balanced Salt Solution
HEPES Sigma H4034 C8H18N2O4S
Ice bucket Fisherbrand 03-395-150
Isoflurane Patterson Veterinary NDC 14043-704-05
Johns Hopkins Bulldog Clamp Roboz Surgical Store RS-7440  Straight; 500-900 Grams Pressure; 1.5" Length
Kimwipes Kimberly-Clark Professional 34705 Kimtech Science™ Kimwipes™ Delicate Task Wipers, 2-Ply
LIVE/DEAD Viability/Cytotoxicity Kit Invitrogen L3224 This kit contains the calcein-AM live cell dye.
Low glucose DMEM Corning 10-014-CV
Magnesium chloride hexahydrate Sigma M9272 MgCl2 (hexahydrate)
Magnesium sulfate heptahydrate Sigma M2773 MgSO4 (heptahydrate)
Magnetic Heated Platform Warner Instruments PM-1 Platform for imaging chamber for dynamic stimulation recordings
Microwave GE JES1460DSWW
Nalgene Syringe Filter Thermo Fisher Scientific 726-2520
No.4 Paintbrush Michaels 10269140
Open Diamond Bath Imaging Chamber Warner Instruments RC-26 Imaging chamber for dynamic stimulation recordings
Oregon Green 488 BAPTA-1-AM Invitrogen O6807 cell-permeable Ca2+ indicator
Overnight imaging chamber Okolab H201-LG
PBS Thermo Fisher Scientific 20012050 To make agarose for slice generation
PE-labeled insulin tetramer Emory Tetramer Research Core sequence YAIENYLEL
Penicillin Streptomycin Gibco 15140122
Potassium chloride Sigma P5405 KCl
Potassium phosphate monobasic Sigma P5655 KH2PO4
Razor Blades Electron Microscopy Sciences 71998 For Vibratome; Double Edge Stainless Steel, uncoated
RPMI 1640 Gibco 11875093
SeaPlaque low melting-point agarose Lonza 50101 To make agarose for slice generation
Slice anchor Warner Instruments 64-1421
Slice anchor (dynamic imaging) Warner Instruments 640253 Slice anchor for dynamic imaging chamber
Sodium bicarbonate Sigma S5761 NaHCO3
Sodium chloride Sigma S5886 NaCl
Sodium phosphate monohydrate Sigma S9638 NaH2PO4 (monohydrate)
Soybean Trypsin Inhibitor Sigma T6522-1G Trypsin inhibitor from Glycine max (soybean)
Stage Adapter Warner Instruments SA-20MW-AL To fit imaging chamber for dynamic stimulation recordings on the microscope stage
Stage-top incubator Okolab H201
Stereoscope Leica IC90 E MSV266
SYTOX Blue Dead Cell Stain Invitrogen S34857 blue-fluorescent nucleic acid stain
Transfer Pipet Falcon 357575 Falcon™ Plastic Disposable Transfer Pipets
Valve Control System Warner Instruments VCS-8 System for dynamic stimulation recordings
Vibratome VT1000 S Leica VT1000 S
Water bath Fisher Scientific FSGPD02 Fisherbrand Isotemp General Purpose Deluxe Water Bath GPD 02

  1. Uc, A., Fishman, D. S. Pancreatic disorders. Pediatric Clinics of North America. 64 (3), 685-706 (2017).
  2. Bluestone, J. A., Herold, K., Eisenbarth, G. Genetics, pathogenesis and clinical interventions in type 1 diabetes. Nature. 464 (7293), 1293-1300 (2010).
  3. Taylor, R. Type 2 diabetes: etiology and reversibility. Diabetes Care. 36 (4), 1047-1055 (2013).
  4. Meier, R. P., et al. Islet of Langerhans isolation from pediatric and juvenile donor pancreases. Transplant International. 27 (9), 949-955 (2014).
  5. Marciniak, A., et al. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology. Nature Protocols. 9 (12), 2809-2822 (2014).
  6. Panzer, J. K., Cohrs, C. M., Speier, S. Using pancreas tissue slices for the study of islet physiology. Methods in Molecular Biology. 2128, 301-312 (2020).
  7. Speier, S., Rupnik, M. A novel approach to in situ characterization of pancreatic beta-cells. Pflugers Archive. 446 (5), 553-558 (2003).
  8. Panzer, J. K., et al. Pancreas tissue slices from organ donors enable in situ analysis of type 1 diabetes pathogenesis. JCI Insight. 5 (8), 134525 (2020).
  9. Dolai, S., et al. Pancreatitis-induced depletion of syntaxin 2 promotes autophagy and increases basolateral exocytosis. Gastroenterology. 154 (6), 1805-1821 (2018).
  10. Dolai, S., et al. Pancreas-specific SNAP23 depletion prevents pancreatitis by attenuating pathological basolateral exocytosis and formation of trypsin-activating autolysosomes. Autophagy. , 1-14 (2020).
  11. Qadir, M. M. F., et al. Long-term culture of human pancreatic slices as a model to study real-time islet regeneration. Nature Communications. 11 (1), 3265 (2020).
  12. Cohrs, C. M., et al. Dysfunction of persisting β cells is a key feature of early type 2 diabetes pathogenesis. Cell Reports. 31 (1), 107469 (2020).
  13. Liang, T., et al. Ex vivo human pancreatic slice preparations offer a valuable model for studying pancreatic exocrine biology. Journal of Biological Chemistry. 292 (14), 5957-5969 (2017).
  14. Shultz, L. D., Ishikawa, F., Greiner, D. L. Humanized mice in translational biomedical research. Nat Reviews. Immunology. 7 (2), 118-130 (2007).
  15. Lamont, D., et al. Compensatory mechanisms allow undersized anchor-deficient class I MHC ligands to mediate pathogenic autoreactive T cell responses. Journal of Immunology. 193 (5), 2135-2146 (2014).
  16. Fish, R., Danneman, P. J., Brown, M., Karas, A. . Anesthesia and analgesia in laboratory animals. , (2011).
  17. Clark, S. A., Borland, K. M., Sherman, S. D., Rusack, T. C., Chick, W. L. Staining and in vitro toxicity of dithizone with canine, porcine, and bovine islets. Cell Transplantation. 3 (4), 299-306 (1994).
  18. Schindelin, J., et al. Fiji: an open-source platform for biological-image analysis. Nature Methods. 9 (7), 676-682 (2012).
  19. Monette, R., Small, D. L., Mealing, G., Morley, P. A fluorescence confocal assay to assess neuronal viability in brain slices. Brain Research Protocols. 2 (2), 99-108 (1998).
  20. Gál, E., et al. A novel in situ approach to studying pancreatic ducts in mice. Frontiers in Physiology. 10, 938 (2019).
  21. Stožer, A., Dolenšek, J., Rupnik, M. S. Glucose-stimulated calcium dynamics in islets of Langerhans in acute mouse pancreas tissue slices. PloS One. 8 (1), 54638 (2013).
  22. Stožer, A., et al. Functional connectivity in islets of Langerhans from mouse pancreas tissue slices. PLoS Computational Biology. 9 (2), 1002923 (2013).
  23. Früh, E., Elgert, C., Eggert, F., Scherneck, S., Rustenbeck, I. Glucagonotropic and glucagonostatic effects of KATP channel closure and potassium depolarization. Endocrinology. 162 (1), 136 (2021).
  24. Satin, L. S. New mechanisms for sulfonylurea control of insulin secretion. Endocrine. 4 (3), 191-198 (1996).
  25. Ren, J., et al. Slow oscillations of KATP conductance in mouse pancreatic islets provide support for electrical bursting driven by metabolic oscillations. American Journal of Physiology-Endocrinology and Metabolism. 305 (7), 805-817 (2013).
  26. Marciniak, A., Selck, C., Friedrich, B., Speier, S. Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ. PLoS One. 8 (11), 78706 (2013).
  27. Dzhagalov, I. L., Melichar, H. J., Ross, J. O., Herzmark, P., Robey, E. A. Two-photon imaging of the immune system. Current Protocols in Cytometry. , (2012).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved