前眼室人类胰岛移植和贡献宿主细胞的纵向维沃成像和定量
Summary
该协议的目标是持续监测人类胰岛移植过程和贡献宿主与供体细胞的动态。这是通过移植人类小岛到NOD的眼部（ACE）前室来完成的。（Cg）-Gt（ROSA）26Sortm4–Rag2-/-鼠标接收者，然后是重复的 2 光子成像。
Abstract
成像β细胞是了解胰岛移植的关键一步。虽然在体内已经开发和利用了用于β细胞生物学记录的不同成像 平台，但它们在允许单细胞分辨率和连续纵向记录方面是有限的。由于角膜的透明度，小鼠眼前室（ACE）非常适合研究人类和小鼠胰岛细胞生物学。以下是如何使用这种方法对单个人类小岛移植进行连续纵向记录和再血管化的描述。使用NO NOD将人类小岛嫁接插入ACE。（Cg）-Gt（ROSA）26Sortm4–Rag2-/-小鼠作为接受者。这允许调查接受者与供体细胞的扩大和接受者细胞在促进移植物的封装和血管化方面的贡献。此外，还概述了一种分步方法，用于图像分析和定量的小岛体积或分段血管和小岛胶囊形成接受细胞。
Introduction
糖尿病描述一组代谢性疾病，其特征是血糖水平升高，其特征是胰腺胰岛β细胞损失或功能障碍导致胰岛素生产不足，通常伴有胰岛素抵抗。1型（T1D）和2型糖尿病（T2D）是复杂的疾病，其中β细胞的渐进功能障碍导致疾病发展。T1D是由对β细胞的自身免疫攻击引起的，而T2D被认为是由代谢因素驱动的，尽管有越来越多的证据表明低级全身炎症1。移植供体人类小岛，特别是T1D患者，提供生理血糖控制的潜力。然而，组织捐献者短缺和小岛移植不良使得小岛移植成为主流治疗选择。由于缺氧、炎症、免疫原源环境2，3，功能小岛移植在移植后（24~48小时）的直接转移期丢失了很大一部分功能性小岛移植物。为了评估干预方法的效率，以提高小岛的生存，持续监测这种移植是必要的。
在体内技术，以图像和跟踪移植后人类胰腺胰岛的命运仍然是一个挑战，糖尿病研究,4，5。迄今为止，非侵入性成像技术，包括正电子发射断层扫描（PET）、磁共振成像（MRI）或超声波（美国）等，在实验条件下对移植小岛进行定量和功能评估的潜力。然而，鉴于小小小的小小小小，这些模式的定量测量由于分辨率不足而受到影响。眼前腔室（ACE）作为观察的移植点，是一种很有前途的无创成像解决方案，提供有效的更高的空间分辨率和长期长时间的频繁监测。这种方法已被成功地用于研究小鼠小岛生物学（见杨等人7），自身免疫免疫反应8，以及人类小岛移植9，10。109
在这里，ACE移植方法与2光子成像方法相结合，通过连续和重复记录单个胰岛移植，在移植后长达10个月，研究人类胰岛移植过程的动态。成像深度更大、整体光漂白和照片损伤减少的多光子成像特性克服了共声显微镜11的成像局限性。荧光成像的定量涉及几个阶段，包括小岛样品制备、小岛移植、图像采集、图像滤波以去除小岛噪声或背景、分割、定量和数据分析。最具挑战性的步骤通常是将图像分割或分割为多个部分或区域。这可能涉及将信号与背景噪声分离，或根据颜色或形状的相似性聚类体素区域，以检测和标记表示小岛血管的 3D 体积的体素。一旦分段，数据对象卷大小等统计信息通常很容易提取。提供一种用于对成像数据进行量化和提取的方法，如分段和数据可视化。特别注意去除人类小岛中的自荧光，区分小岛血管和小岛胶囊形成接受细胞。
Protocol
瑞典隆德地区伦理委员会根据《关于人类研究伦理审查法》批准了这项研究。动物实验严格按照瑞典动物实验伦理进行，并经马尔默和隆德伦理委员会批准。6 至 8 周大免疫缺陷 NOD。（Cg）-gt（ROSA）26Sortm4-Rag2-/- （NOD.罗莎 -番茄Rag2-/-）接受小鼠被用作人类小岛10的移植接受者。 1. 岛移植准备 C…
Representative Results
非标记的人类小岛被移植到8周大的女性NOD的ACE中。（Cg）-gt（ROSA）26Sortm4-Rag2-/-（NOD.罗莎- 番茄Rag2=/=）接受小鼠。为了防止人体组织排斥，免疫缺陷的Rag2敲除小鼠被选为接受者。在这些转基因小鼠中，所有细胞和组织都表示一种以膜为目标的番茄荧光蛋白（mT），可以清楚地识别接受者和供体组织。高分辨率 2 光子显微镜对小岛移植物的?…
Discussion
提出了一种通过观察接受者和供体组织的介入来研究人类胰岛细胞移植过程的方法。在将人类小岛植入免疫缺陷小鼠眼的前室的微创手术后，小鼠在手术后几分钟内迅速恢复。手术在一只眼睛上执行。一般来说，从植入后5~7天开始，角膜被充分治愈，可以进行病毒内成像。
在这个协议中，人类小岛嫁接的质量至关重要。人类小岛的质量以及移植结果可能因捐赠者的年龄、BMI?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究得到了瑞典研究理事会、战略研究领域 Exodiab、Dnr 2009-1039、瑞典战略研究基金会 Dnr IRC15-0067、Lund皇家物理学会、糖尿病基金会和巴恩迪亚贝特本特公司的支持。
Materials
| Anasthesia machine, e.g. Anaesthesia Unit U-400 | Agnthos | 8323001 | used for isofluran anasthesia during surgery and imaging |
| -induction chamber 1.4 L | Agnthos | 8329002 | connect via tubing to U-400 |
| -gas routing switch | Agnthos | 8433005 | connect via tubing to U-400 |
| AngioSense 680 EX | Percin Elmer | NEV10054EX | imaging agent for injection, used to image blood vessels in human islet grafts |
| Aspirator tubes assemblies | Sigma | A5177-5EA | connect with pulled capillary pipettes for manual islet picking |
| Buprenorphine (Temgesic) 0.3mg/ml | Schering-Plough Europé | 64022 | fluid, for pain relief |
| Capillary pipettes | VWR | 321242C | used together with Aspirator tubes assemblies |
| Dextran-Texas Red (TR), 70kDa | Invitrogen | D1830 | imaging agent for injection |
| Eye cannula, blunt end , 25 G | BVI Visitec/BD | BD585107 | custom made from Tapered Hydrode lineator [Blumenthal], dimensions: 0.5 x 22mm (25G x 7/8in) (45⁰), tip tapered to 30 G (0.3mm) |
| Eye gel | Novartis | Viscotears, contains Carbomer 2 mg/g | |
| Hamilton syringe 0.5 ml, Model 1750 TPLT | Hamilton | 81242 | Plunger type gas-tight syringe for islet injection |
| Head holder | |||
| -Head holding adapter | Narishige | SG-4N-S | assemled onto metal plate |
| -gas mask | Narishige | GM-4-S | |
| -UST-2 Solid Universal Joint | Narishige | UST-2 | assemled onto metal plate |
| -custom made metal plate for head-holder assembly | |||
| -Dumont #5, straight | Agnthos | 0207-5TI-PS or 0208-5-PS | attached to UST-2 (custom made) |
| Heating pad, custom made | taped to the stereotaxic platform | ||
| Human islet culture media | |||
| -CMRL 1066 | ICN Biomedicals | cell culture media for human islets | |
| -HEPES | GIBCO BRL | ||
| -L-glutamin | GIBCO BRL | ||
| -Gentamycin | GIBCO BRL | ||
| -Fungizone | GIBCO BRL | ||
| -Ciproxfloxacin | Bayer healthcare AG | ||
| -Nicotinamide | Sigma | ||
| Image analysis software | Bitplane | Imaris 9 | |
| Image Aquisition software | Zeiss | ZEN 2010 | |
| Infrared lamp | VWR | 1010364937 | used to keep animals warm in the wake-up cage |
| Isoflurane Isoflo | Abott Scandinavia/Apotek | fluid, for anesthesia | |
| Needle 25 G (0.5 x 16mm), orange | BD | 10442204 | used as scalpel |
| Petri dishes, 90mm | VWR | 391-0440 | |
| 2-Photon/confocal microscope | |||
| -LSM7 MP upright microscope | Zeiss | ||
| -Ti:Sapphire laser Tsunami | Spectra-Physics, Mai Tai | ||
| -long distance water-dipping lens 20x/NA1.0 | Zeiss | ||
| -ET710/40m (Angiosense 680) | Chroma | 288003 | |
| -ET645/65m-2p (TR) | Chroma | NC528423 | |
| -ET525/50 (GFP) | Chroma | ||
| -ET610/75 (tomato) | Chroma | ||
| -main beam splitter T680lpxxr | Chroma | T680lpxxr | Dichroic mirror to transmit 690 nm and above and reflect 440 to 650 nm size 25.5 x 36 x 1 mm |
| Polythene tubing (0.38mm ID, 1.09 mm OD) | Smiths Medical Danmark | 800/100/120 | to connect with Hamilton syringe and eye canula |
| Stereomicroscope | Nikon | Model SMZ645, for islet picking | |
| Stereomicroscope (Flourescence) | for islet graft imaging | ||
| -AZ100 Multizoom | Nikon | wide field and long distance | |
| -AZ Plan Apo 1x | Nikon | ||
| -AZ Plan Apo 4x | Nikon | ||
| -AZ-FL Epiflourescence with C-LHGFI HG lamp | Nikon | ||
| -HG Manual New Intensilight | Nikon | ||
| -Epi-FL Filter Block TEXAS RED | Nikon | contains EX540-580, DM595 and BA600-660 | |
| -Epi-FL Filter Block G-2A | Nikon | (EX510-560, DM575 and BA590) | |
| -Epi-FL Filter Block B-2A | Nikon | (EX450-490, DM505 and BA520) | |
| -DS-Fi1 Colour Digital Camera (5MP) | Nikon | ||
| Syringe 1-ml, Omnitix | Braun | 9161406V | for Buprenorphine injection, used with 27 G needle |
| Surgical tape | 3M |
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Citar este artigo
Nilsson, J., Holmberg, D., Schmidt-Christensen, A. Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber. J. Vis. Exp. (160), e61234, doi:10.3791/61234 (2020).