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

Summary

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

Abstract

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.

Introduction

Involvement of the pancreas is pathognomonic to diseases such as pancreatitis, T1D, and T2D^1,2,3. 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 procedures^5,6,7. Pancreatic tissue slices from human....

Protocol

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.......

Discussion

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.......

Materials

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