观察活胰腺组织切片中的胰岛功能和胰岛-免疫细胞相互作用
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
Summary
本研究介绍了活胰腺组织切片在胰岛生理学和胰岛免疫细胞相互作用研究中的应用。
Abstract
活的胰腺组织切片允许在完整的胰岛微环境中研究胰岛的生理学和功能。切片由嵌入琼脂糖中的活人和小鼠胰腺组织制备，并使用振动切片机切割。这种方法允许组织保持活力和功能，除了保留潜在的病理，如1型（T1D）和2型糖尿病（T2D）。切片方法通过维持构成胰腺内分泌和外分泌组织的复杂结构和各种细胞间相互作用，为胰腺的研究提供了新的方向。该协议展示了如何对胰腺切片内的活内源性免疫细胞进行染色和延时显微镜检查，以及胰岛生理学评估。此外，可以使用主要的组织相容性复合物多聚体试剂来改进这种方法，以识别胰岛细胞抗原特异性的免疫细胞群。
Introduction
胰腺受累是胰腺炎、T1D 和 T2D1，2，3 等疾病的特征性特征。孤立胰岛功能的研究通常涉及将胰岛从周围环境中移除4。开发了活胰腺组织切片方法，以允许研究胰腺组织，同时保持完整的胰岛微环境并避免使用应激的胰岛分离程序5，6，7。来自人类供体组织的胰腺组织切片已被成功用于研究T1D，并且除了免疫细胞浸润之外，还显示出β细胞丢失和功能障碍的过程8，9，10，11，12，13。活胰腺组织切片法可应用于小鼠和人胰腺组织5，6，8。来自器官捐献者组织的人类胰腺组织切片是通过与糖尿病胰腺器官捐献者网络（nPOD）合作获得的。小鼠切片可以从各种不同的小鼠品系中生成。
该协议将侧重于非肥胖糖尿病重组激活基因-1-null（NOD.Rag1-/-） 和 T 细胞受体转基因 （AI4） （NOD.Rag1-/-.AI4 α/β）小鼠品系。点头。Rag1-/- 小鼠由于重组激活基因1（Rag1）14的破坏而无法发育T细胞和B细胞。点头。Rag1-/-.AI4 α/β 小鼠被用作加速型1型糖尿病的模型，因为它们产生靶向胰岛素表位的单个T细胞克隆，导致胰岛持续浸润和快速疾病发展15。此处的方案描述了通过应用共聚焦显微镜方法使用活人胰腺切片进行功能和免疫学研究的程序。本文描述的技术包括活力评估，胰岛鉴定和定位，胞质Ca2 + 记录，以及免疫细胞群的染色和鉴定。
Protocol
注意：所有使用小鼠的实验方案均已获得佛罗里达大学动物护理和使用委员会（201808642）的批准。来自两性组织供体的人类胰腺切片是通过佛罗里达大学糖尿病胰腺器官供体网络（nPOD）组织库获得的。根据器官捐赠法律法规，与nPOD合作的认证器官采购组织从尸体器官捐献者身上摘取了人类胰腺，并被佛罗里达大学机构审查委员会（IRB No. 392-2008）归类为”非人类受试者”，免除了同意的需要。专门?…
Representative Results
该协议将产生适合功能研究和免疫细胞记录的活胰腺组织切片。明场和反射光下的切片外观如图 1A，B所示。如前所述，胰岛可以使用反射光在切片中找到，因为它们的颗粒度增加，由于其胰岛素含量而发生（图1C），并且在使用反射光时与背景组织相比被清楚地观察到。应在切片生成后评估生存能力，如果超过20%的胰岛不可行，则不应记?…
Discussion
该协议的目的是解释胰腺切片的产生以及在功能和免疫学研究中使用切片所需的程序。使用活胰腺切片有很多好处。然而，有几个关键步骤对于组织在所描述的实验方案期间保持活力和有用至关重要。必须快速工作。应尽量减少注射胰腺和在振动切片机上产生切片之间的时间长度，以保持组织的活力。通过在切片之前将胰腺保持在冷ECS中，而不是室温ECS，还可以提高生存能力。重要的是，切片不?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作由NIH拨款R01 DK123292，T32 DK108736，UC4 DK104194，UG3 DK122638和P01 AI042288资助。这项研究是在糖尿病胰腺器官捐献者网络（nPOD;RRID：SCR_014641），由JDRF（nPOD：5-SRA-2018-557-Q-R）和Leona M. & Harry B. Helmsley慈善信托基金（Grant #2018PG-T1D053）赞助的1型糖尿病合作研究项目。所表达的内容和观点是作者的责任，并不一定反映nPOD的官方观点。与nPOD合作提供研究资源的器官采购组织（OPO）列在 http://www.jdrfnpod.org/for-partners/npod-partners/。感谢佛罗里达大学的Kevin Otto博士提供用于生成小鼠切片的振动切片机。
Materials
| #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 |
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Cite This Article
Huber, M. K., Drotar, D. M., Hiller, H., Beery, M. L., Joseph, P., Kusmartseva, I., Speier, S., Atkinson, M. A., Mathews, C. E., Phelps, E. A. Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices. J. Vis. Exp. (170), e62207, doi:10.3791/62207 (2021).