Murine Pancreatic Islets Transplantation under the Kidney Capsule
肾被膜下小鼠胰岛移植
发布: 2018年03月05日第8卷第5期 DOI: 10.21769/BioProtoc.2743 浏览次数: 12070
评审: Ruth A. FranklinSylvaine YouSara Johnson
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参见作者原研究论文
The authors used this protocol in:
Feb 2006
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Abstract
Type 1 diabetes (T1D) is an autoimmune disease caused by the lack of insulin-producing pancreatic beta cells leading to systemic hyperglycemia. Pancreatic islet transplantation is a valid therapeutic approach to restore insulin loss and to promote adequate glycemic control. Pancreatic islet transplantation in mice is an optimal preclinical model to identify new therapeutic strategies aiming at preventing rejection and optimizing post-transplant immuno-suppressive/-tolerogenic therapies.
Islet transplantation in preclinical animal models can be performed in different sites such the kidney capsule, spleen, bone marrow and pancreas. This protocol describes murine islet transplantation under the kidney capsule. This is a widely accepted procedure for research purposes. Stress caused in the animals is minimal and it leads to reliable and reproducible results.
Pancreatic islets (胰岛)
Islet transplantation (胰岛移植)
Murine model (小鼠模型)
Background
Many alternative sites for islet implantation have been reported so far in small animal models and the ideal site must be selected according to the technical advantages of the procedure to be used and for the purpose of the experiments. Bearing in mind that the kidney capsule is an extravascular site and it is not immunoprotected, pancreatic islet transplantation under the kidney capsule remains a surgical procedure with low mortality rates leading to hyperglycemia reversion within a few days. In addition, transplantation under the kidney capsule allows histological studies and formal demonstration of islet function (Cantarelli and Piemonti, 2011; Elisa Cantarelli et al., 2013).
Materials and Reagents
- Cotton applicators, sterile (CARLO ERBA Reagents, catalog number: 9.413 161 )
- 30 G x ½ in. needle (BD, catalog number: 305106 )
- 2 ml slip tip syringe (BD, catalog number: 302204 )
- BD INTRAMEDICTM PE 50 (BD, catalog number: B427411 )
- Petri dish, Falcon® 50 x 9 mm Sterile (Corning, Falcon®, catalog number: 351006 )
- P200 pipette tip (SARSTEDT, catalog number: 70.760.002 )
- Eppendorf tubes volume 1.5 ml (Sigma-Aldrich, catalog number: T9661-1000EA)
Manufacturer: Eppendorf, catalog number: 022363204 . - Silicone tubing adapter (2Biological Instruments, catalog number: SFM3-1550 )
- 1 ml syringe with 25 G needle (Ettore Pasquali, catalog number: 11.3500.05 )
- Suture Dermalon 5/0 19 MM (Covidien, catalog number: 1756-21 )
- Collagenase P (Roche Diagnostics, catalog number: 11213865001 )
- Betadine (MEDA PHARMA SpA, Farmacie Coli, catalog number: 023907076 )
- Avertin (Sigma-Aldrich, catalog number: T48402 )
- Sodium chloride ((NaCl) (CARLO ERBA Reagents, catalog number: FC72101100000 )
- Ketoprofen (PFIZER ITALIA Srl DIV.VET)
- RPMI 1640 medium (Lonza, catalog number: 12-167F )
- L-glutamine (Lonza, catalog number: 17-605E )
- HEPES buffer (Lonza, catalog number: 17-737E )
- Pen-Strep (Lonza, catalog number: 17-602E )
- Fetal bovine serum (Euroclone, catalog number: ECS0180L )
- Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175079 )
- Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
- Histopaque®-1077 (Sigma-Aldrich, catalog number: H8889 )
- RPMI/glutamine/HEPES/Pen-Strep/FBS (see Recipes)
- HBSS/Ca/HEPES (see Recipes)
Equipment
- Forceps (Graefe forceps, 100 mm, curved (ProSciTech, catalog number: T131C ), Tweezers, style 3 (ProSciTech, catalog number: T03-212 )
- Scissor (ProSciTech, catalog number: TS103-200SB )
- Surgical Shaver (2Biological Instruments, catalog number: 2BTOSRC )
- Cautery (Global medical solutions, catalog number: BAA00 )
- Thermostatic bath (Thermo Fisher Scientific, Thermo ScientificTM, model: TSGP02 )
- Inverted microscope (Compact, Modular Stereo, Leica, model: Leica M60 )
- Incubator at 37 °C with 5% CO2, relative humidity ambient to 80% [e.g., Series II 3110 Water-Jacketed CO2 incubators (Thermo Fisher Scientific, Thermo ScientificTM, model: FormaTM II 3110 Series )]
- Pipetman P20/P200/P1000 (Gilson)
- Microsyringe 25 μl Hamilton syringe (Hamilton, catalog number: 80401 )
- Heating pad 25 x 40 cm, Two Temperature Range (2Biological Instruments, LCPH)
- Herasafe KS, Class II biological safety cabinet with UV surface disinfection irradiator (Thermo Fisher Scientific, Thermo ScientificTM, model: HerasafeTM KS II , catalog number: 51022481)
Software
- Prism software (GraphPad, USA)
Procedure
文章信息
版权信息
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Jofra, T., Galvani, G., Georgia, F., Silvia, G., Gagliani, N. and Battaglia, M. (2018). Murine Pancreatic Islets Transplantation under the Kidney Capsule. Bio-protocol 8(5): e2743. DOI: 10.21769/BioProtoc.2743.
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分类
免疫学 > 动物模型 > 小鼠
细胞生物学 > 细胞移植 > 同种异体移植
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