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Isolation and Culture of the Islets of Langerhans from Mouse Pancreas
从小鼠胰腺分离和培养胰岛的Langerhans细胞   

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Abstract

The islets of Langerhans are clusters of endocrine cells located within the pancreas. Insulin-producing beta cells are the major cell type within islets, with glucagon-producing alpha cells and somatostatin-producing delta cells the other major cell types. The beta cells are the target of immune-mediated destruction in type 1 diabetes (Graham et al., 2012). Failure of beta cell function accompanied by loss of beta cell mass is also a feature of type 2 diabetes (Wali et al., 2013). Therefore studying the biology of pancreatic islets is important to understand the pathogenesis of diabetes and to develop new therapies. Here we describe the isolation of mouse islets. This requires gentle enzymatic and mechanical digestion of the exocrine tissue and density gradient separation (Chong et al., 2004; Liu and Shapiro, 1995; Thomas et al., 1998). We then describe how islets can be cultured whole or dispersed into single cells for use in a variety of in vitro and in vivo analyses. Using this protocol reliably results in the isolation of 200-400 islets, depending on the strain of mouse.


Part I. Islet isolation


Materials and Reagents

  1. 50 ml polypropylene conical tubes (Corning, Falcon®, catalog number: 352070 )
  2. 0.2 µm syringe filter (e.g., Sartorius AG, catalog number: 16534-k )
  3. Syringe (volume dependent on volume of collagenase P prepared)
  4. Ice
  5. 2 ml slip tip syringe (will hold total volume of 3 ml) (BD, catalog number: 302204 )
  6. 30 G x ½ in. needle (BD, catalog number: 305106 )
  7. 500 micron mesh (Sefar Pty Ltd., catalog number: 06-500/38 )
    Note: cut into approximately 5 cm squares and autoclaved (Figure 1) (see Notes for alternative product)


    Figure 1. 500 µm islet mesh from Sefar Pty Ltd and 50 ml tube lid shown for size reference

  8. Collagenase P (Roche Diagnostics, catalog number: 11213865001 )
    Note: It requires testing and optimisation prior to use (see Note 1).
  9. RPMI 1640 medium (Life Technologies, Gibco, catalog number: 21870-076 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 21870-076”.
  10. Histopaque®-1077 (Sigma-Aldrich, catalog number: H8889 )
  11. Hanks balanced salt solution (HBSS) (Life Technologies, Gibco, catalog number: 14175-079 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14175-079”.
  12. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080 )
  13. HEPES (Sigma-Aldrich, catalog number: H3375-100G )
  14. HBSS/Ca/HEPES (see Recipes)

Equipment

  1. Icebox
  2. Dissecting microscope [e.g., Stereoscopic zoom microscope, (Nikon Instruments Inc., model: SMZ745 )]
  3. Fibre optic light source (e.g., AmScope Dual Goose-neck Fiber-Optic Illuminator, model: HL150-AY )
  4. Forceps [e.g., Graefe forceps, 100 mm, curved (ProSciTech Pty Ltd., catalog number: T131C-2 )]
  5. Scissors [e.g., Iris scissors, 115 mm, straight (ProSciTech Pty Ltd., catalog number: T097-2 )]
  6. Clamp [e.g., 150 mm spencer wells forceps, straight (ProSciTech Pty Ltd., catalog number: T124 )]
  7. Bench-top centrifuge at room temperature, no brake required (e.g., Thermo Fisher Scientific, HeraeusTM, model: Multifuge X1R )

Procedure

  1. Measure 5 ml per pancreas of HBSS/Ca/HEPES into a 50 ml tube.
    Measure 5 ml per pancreas of RPMI into a 50 ml tube.
    Measure 10 ml per pancreas of Histopaque into a 50 ml tube. Use multiple tubes if more than 50 ml is required but spread contents evenly across the tubes.
  2. Place all media at 37 °C in water bath.
  3. Weigh sufficient collagenase P to prepare 3 ml per mouse at the specified concentration for the batch of collagenase P (see Notes 1-3). Place weighed collagenase P into a 50 ml tube.
  4. Dissolve the collagenase P in cold (4 °C) HBSS/Ca/HEPES solution. Add the required volume of HBSS/Ca/HEPES to 50 ml tube containing weighed collagenase P and gently invert 2-3 times. Allow to stand for 1-2 min, then sterilize using a 0.2 µm filter and syringe. Keep collagenase P solution on ice from this point onwards.
  5. Place 3 ml collagenase P into a 2 ml slip tip syringe and attach a 30 G needle. Keep filled syringes on ice. Do not use a syringe with a LuerLok as the pressure when dispelling liquid is too great and the bile duct will tear.
  6. Please view Video 1 for details on how to perform the bile duct injection procedure. In brief, open the mouse abdominal cavity and cut through the peritoneal wall horizontally following the rib line and vertically using small straight scissors. Place a tissue over the upper body of the mouse with the tissue edge on the rib line. Gently press on either side of the mouse body with your fingers so the liver is exposed below the rib line. Flip the liver over onto the tissue and fold the tissue over to cover it. Wet with a small amount of ethanol and gently press down to hold everything in place.

    Video 1. Bile duct injection and pancreas dissection

  7. Place a square of autoclaved 500 µm mesh on the top of a new 50 ml tube (one per pancreas) and wet the mesh with a small amount (1-2 ml) of cold RPMI. Pour the digested pancreas through the mesh. Wash any remaining pancreas from its original tube with 30 ml cold RPMI and pour through the mesh. Cap the collection tube and return to the ice. Note that some undigested tissue may remain on top of the mesh. Repeat with other samples.
  8. Gently dissect the inflated pancreas, being careful not to cut the stomach or intestines or other abdominal organs, and place into an empty 50 ml tube. Keep on ice. Repeat steps 6-8 for other animals.
  9. When all pancreata have been resected add warm HBSS/Ca/HEPES to the tube to a total volume of 5 ml/pancreas and place at 37 °C in a water bath. The incubation time varies between batches of collagenase P and strains of mouse (see Notes 1 and 5).
  10. When the pancreata are placed in the waterbath, remove the prepared tubes of Histopaque and RPMI from the 37 °C waterbath. These should cool to room temperature during the collagenase P digestion incubation.
  11. Remove the tubes of pancreata from the waterbath and place immediately on slushy ice (ice in water). Do not swirl, shake or disturb the settled tissue.
  12. Immediately and working as quickly as possible, pour as much warm HBSS/Ca/HEPES and collagenase P mix off each pancreas as possible, without removing the pancreas from the tube. Top up each tube to 15 ml with ice-cold HBSS/Ca/HEPES. This step stops the digestion process by rapidly cooling the pancreas.
  13. Disrupt the pancreas by vigorously hand shaking the tubes for 1 min at a rate of 3 shakes per second. Hold the tubes upright and allow the pancreas to hit both the top and bottom of the tube (see Video 2). Do not invert the tubes. Place the tubes on ice.

    Video 2. Disruption of the pancreas by vigorous shaking

  14. Place a square of autoclaved 500 µm mesh on the top of a new 50 ml tube (one per pancreas) and wet the mesh with a small amount (1-2 ml) of cold RPMI. Pour the digested pancreas through the mesh. Wash any remaining pancreas from its original tube with 30 ml cold RPMI and pour through the mesh. Cap the collection tube and return to the ice. Note that some undigested tissue may remain on top of the mesh. Repeat with other samples.
  15. Allow digested pancreas to settle by gravity in the 50 ml tube on ice for 10 min.
  16. Aspirate the supernatant using a 25 ml pipette leaving 10-15 ml behind. Be careful not to disrupt the digested tissue. Top-up the tubes of digested pancreas with RPMI to a total volume of 30 ml and mix by gently inverting 2-3 times.
  17. Centrifuge tubes at 215 x g for 2 min at room temperature.
  18. Gently pour off the supernatant, being careful not to dislodge the pellet. Place tubes upside down on a paper tissue so residual media drains away. It is important to remove all media so the Histopaque is not diluted.
  19. Flick the pellets to loosen the digested pancreas. Tubes should remain at room temperature from this point. All reagents and centrifugation steps should be at room temperature (22 °C).
  20. Add 10 ml of Histopaque to each tube (must be at room temperature). Swirl gently to get an even distribution of digested pancreas. If there are clumps, gently break them up with a pipette. Avoid vigorous pipetting.
  21. Overlay each Histopaque gradient with 5 ml of RPMI (must be at room temperature). Ensure the interface is sharp and clear as this is where the islets will sit after centrifugation.
  22. Centrifuge at 850 x g for 15 min at 22 °C with the brake OFF.
  23. Gently remove the islets from the gradient using a plastic transfer pipette. The islets will be visible in a ring just below the interface of the RPMI and Histopaque (see Figure 3). Remove as much RPMI and Histopaque as required to remove the islets without disturbing the pellet. If islets that have immune cell infiltrate are being isolated from NOD mice, see Note 6 for detail on the location and removal of islets from the gradient.


    Figure 3. Location of islets at Histopaque/RPMI gradient interface. A. Schematic diagram and photograph of normal islets forming a ring just below the Histopaque/RPMI interface. B. Schematic diagram and photograph of infiltrated islets from a NOD mouse spread through the Histopaque. Schematic diagram shows transfer pipette used to harvest the islets from the gradient.

  24. Transfer the islets to a new 50 ml tube containing 30 ml RPMI (room temperature).
  25. Centrifuge at 215 x g for 2 min at room temperature.
  26. Gently pour off the supernatant and blot tubes on a paper tissue. At this point islets can be cultured or trypsinized to single cells.

Part II. Culture of intact whole islets

Materials and Reagents

  1. 94 x 15 mm PPetri dishes (Greiner Bio One International GmbH, catalog number: 633185 )
  2. FCS
  3. Glutamine
  4. Penicillin
  5. Streptomycin
  6. CMRL Medium, no glutamine (Life Technologies, Gibco, catalog number: 11530-037 ) (see Recipes)
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11530-037 ”.
  7. Complete CMRL (see Recipes)

Equipment

  1. Incubator at 37 °C with 5% CO2, relative humidity ambient to 80% [e.g., Series II 3110 Water-Jacketed CO2 incubators (Thermo Fisher Scientific, FormaTM, model: 3131 )]

Procedure

  1. Flick the islet pellet gently to resuspend and then add 5 ml of warm complete CMRL.
  2. Transfer the islets to a Petri dish by pouring. Do not use tissue culture dishes as islets will adhere.
  3. Remove residual islets by washing the tube with a further 5 ml of warm complete CMRL and pour this into the Petri dish. Do not put more than 10 ml medium into a 94 mm dish to ensure sufficient oxygenation for the islets, which are highly susceptible to hypoxia.
  4. Ensure the islets are spread within the dish and place dishes in a 5% CO2 incubator at 37 °C. Allow the islets to recover for 0.5-1 h prior to further manipulation.
  5. Under a dissecting microscope with an external light source, handpick the islets using a sterile 200 µl pipette tip into tubes or Petri dishes as required for further analysis (see Figure 4).
  6. Islets can be cultured for up to three days at 37 °C + 5% CO2.


    Figure 4. Handpicking of isolated islets for in vitro culture

  7. The islet yield varies between mouse strains. The expected yield from a C57Bl/6 mouse is 300-400 islets whereas the expected yield from a non-obese diabetic (NOD) mouse is 150-300 islets depending on the age of the mouse (see Note 6).

Part III. Trypsinization of islets to single cells

Materials and Reagents

  1. 1 ml Gilson pipette
  2. 0.2 µm syringe filter (e.g., Sartorius AG, catalog number: 16534-k )
  3. FCS
  4. Glutamine
  5. Penicillin
  6. Streptomycin
  7. Dulbecco’s phosphate buffered saline
  8. Bovine trypsin
  9. EDTA
  10. CMRL Medium, no glutamine (Life Technologies, Gibco, catalog number: 11530-037 ) (see Recipes)
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11530-037 ”.
  11. Complete CMRL 1066 (see Recipes)
  12. Trypsin for islets (see Recipes)

Equipment

  1. 37 °C waterbath (e.g., Ratek Instruments, model: WB14 )
  2. Incubator at 37 °C with 5% CO2, relative humidity ambient to 80% [e.g., Series II 3110 Water-Jacketed CO2 incubators (Thermo Fisher Scientific, FormaTM, model: 3131 )]

Procedure

  1. Please view Video 3 for trypsinization of islets.
  2. Add 300 µl trypsin to the islet pellet.
  3. Incubate for 5 min at 37 °C in a waterbath.
  4. Pipette the islets up and down 10 times using a 1 ml Gilson pipette. The islets should disperse and this can be visualized. See Video 3.
  5. Add 3-5 ml complete CMRL to stop the trypsin reaction.
  6. Centrifuge at 305 x g for 5 min to pellet the single cells.
  7. Pour off the media and replace with 1 ml of complete CMRL.
  8. Incubate the tube containing trypsinized islets at 37 °C in a 5% CO2 incubator for 1-2 h to allow recovery of the cells before further experiment/analysis.

    Video 3. Trypsinization of islets to single cells

Notes

  1. There is significant variation in collagenase P activity of each enzyme lot number. The variation alters the concentration used and the time of the digestion. An ideal batch of enzyme should have a collagenase P activity of 1.6-2.2 U/mg to be used at a concentration range of 0.3 mg/ml-0.55 mg/ml and incubation time of 15-17 min. The balance between collagenase activity and trypsin activity also varies widely and for efficient islet isolation, it is important that the trypsin activity is not too high. Trypsin activities within the range of 2.63-4.3 U/mg provide optimal results. The trypsin activity must be < 5 U/mg.
    When purchasing a new lot number of collagenase P conduct thorough testing, including titration of concentration and incubation time.
  2. Collagenase P should be brought to room temperature prior to weighing to prevent condensation and damage to the product. If a large quantity is purchased, place a small amount in a new bottle as a ‘working batch’ so as not to lose activity of the whole stock.
  3. Wear a mask and gloves when weighing Collagenase P, as it is both a respiratory and skin irritant.
  4. The length and fragility of the bile duct varies with different strains of mice. C57Bl/6 and genetic derivatives have shorter and more fragile bile ducts than other strains. Be very careful when clamping and inserting the needle into the duct and inject Collagenase P solution slowly. Bile ducts in the obese diabetic mouse model Leprdb/db are very fragile and require extra care.
  5. The above protocol can be used to isolate islets from any mouse strain, however, there are slight variations in digestion incubation time depending on which strain is used. C57Bl/6 islets are more prone to over digestion than Balb/C or NOD mouse islets and incubation times for C57Bl/6 mice and genetic variations of this strain should always be reduced by approximately 1 min.
  6. The NOD mouse is animal model of type 1 diabetes that develops progressive infiltration of islets by immune cells beginning at approximately 4 weeks of age (Graham et al., 2012). The presence of immune cells alters the position of the islets within the density gradient. Islets containing immune cells will spread out and appear well below the Histopaque/RPMI interface (see Figure 3). The immune-mediated destruction of the islets in NOD mice also decreases the islet yield in older mice.
  7. Cell strainers, for example PluriStrainer 500 µm from pluriSelect (https://www.pluriselect.com/product-details/product/pluristrainer-500-um-cell-strainer.html) could be used as an alternative to the 500 µm mesh used in Part I. step 14 of the islet isolation procedure. However these have not been tested in our laboratory.

Recipes

  1. HBSS/Ca/HEPES
    1 L of Hanks balanced salt solution (HBSS)
    2 mM CaCl2
    20 mM HEPES
    Combine all reagents in a sterile biosafety cabinet
    Stored at 4 °C
  2. Complete CMRL
    500 ml CMRL 1066
    10% FCS
    2 mM glutamine
    100 U/ml penicillin
    100 µg/ml streptomycin
    Combine all reagents in a sterile biosafety cabinet
    Stored at 4 °C
  3. Trypsin for islets
    50 ml Dulbecco’s phosphate buffered saline
    5.16 mg bovine trypsin
    20 mM EDTA (0.2 ml of 0.5 M EDTA)
    Combine all ingredients
    Sterilize through a 0.2 µm syringe filter
    Aliquot and stored at -20 °C

Acknowledgments

This protocol has been developed and refined by members of our laboratory since it was first published (Chong et al., 2004; Liu and Shapiro, 1995; Thomas et al., 1998). We acknowledge all members of the group who have contributed to this process. We routinely utilize this protocol in our studies of the pathogenesis of type 1 and type 2 diabetes in mouse models (Chee et al., 2014; Graham et al., 2011; Quah et al., 2014; Wali et al., 2014; Zhao et al., 2015). This work was funded by a National Health and Medical Research Council of Australia (NHMRC) Program grant (APP1037321) and a fellowship to HET (APP1042735). The St Vincent’s Institute receives support from the Operational Infrastructure Support Scheme of the Government of Victoria.

References

  1. Chee, J., Ko, H. J., Skowera, A., Jhala, G., Catterall, T., Graham, K. L., Sutherland, R. M., Thomas, H. E., Lew, A. M., Peakman, M., Kay, T. W. and Krishnamurthy, B. (2014). Effector-memory T cells develop in islets and report islet pathology in type 1 diabetes. J Immunol 192(2): 572-580.
  2. Chong, M. M., Chen, Y., Darwiche, R., Dudek, N. L., Irawaty, W., Santamaria, P., Allison, J., Kay, T. W. and Thomas, H. E. (2004). Suppressor of cytokine signaling-1 overexpression protects pancreatic beta cells from CD8+ T cell-mediated autoimmune destruction. J Immunol 172(9): 5714-5721.
  3. Graham, K. L., Krishnamurthy, B., Fynch, S., Mollah, Z. U., Slattery, R., Santamaria, P., Kay, T. W. and Thomas, H. E. (2011). Autoreactive cytotoxic T lymphocytes acquire higher expression of cytotoxic effector markers in the islets of NOD mice after priming in pancreatic lymph nodes. Am J Pathol 178(6): 2716-2725.
  4. Graham, K. L., Sutherland, R. M., Mannering, S. I., Zhao, Y., Chee, J., Krishnamurthy, B., Thomas, H. E., Lew, A. M. and Kay, T. W. (2012). Pathogenic mechanisms in type 1 diabetes: the islet is both target and driver of disease. Rev Diabet Stud 9(4): 148-168.
  5. Liu, M. and Shapiro, M. E. (1995). A new method for isolation of murine islets with markedly improved yields. Transplant Proc 27(6): 3208-3210.
  6. Quah, H. S., Miranda-Hernandez, S., Khoo, A., Harding, A., Fynch, S., Elkerbout, L., Brodnicki, T. C., Baxter, A. G., Kay, T. W., Thomas, H. E. and Graham, K. L. (2014). Deficiency in type I interferon signaling prevents the early interferon-induced gene signature in pancreatic islets but not type 1 diabetes in NOD mice. Diabetes 63(3): 1032-1040.
  7. Thomas, H. E., Parker, J. L., Schreiber, R. D. and Kay, T. W. (1998). IFN-gamma action on pancreatic beta cells causes class I MHC upregulation but not diabetes. J Clin Invest 102(6): 1249-1257.
  8. Wali, J. A., Masters, S. L. and Thomas, H. E. (2013). Linking metabolic abnormalities to apoptotic pathways in Beta cells in type 2 diabetes. Cells 2(2): 266-283.
  9. Wali, J. A., Rondas, D., McKenzie, M. D., Zhao, Y., Elkerbout, L., Fynch, S., Gurzov, E. N., Akira, S., Mathieu, C., Kay, T. W., Overbergh, L., Strasser, A. and Thomas, H. E. (2014). The proapoptotic BH3-only proteins Bim and Puma are downstream of endoplasmic reticulum and mitochondrial oxidative stress in pancreatic islets in response to glucotoxicity. Cell Death Dis 5: e1124.
  10. Zhao, Y., Scott, N. A., Fynch, S., Elkerbout, L., Wong, W. W., Mason, K. D., Strasser, A., Huang, D. C., Kay, T. W. and Thomas, H. E. (2015). Autoreactive T cells induce necrosis and not BCL-2-regulated or death receptor-mediated apoptosis or RIPK3-dependent necroptosis of transplanted islets in a mouse model of type 1 diabetes. Diabetologia 58(1): 140-148.

简介

朗格汉斯岛是位于胰腺内的内分泌细胞簇。胰岛素生成β细胞是胰岛内的主要细胞类型,产生胰高血糖素的α细胞和生长抑素生成的δ细胞是其他主要细胞类型。 β细胞是免疫介导的1型糖尿病破坏的靶标(Graham等,2012)。 β细胞功能衰竭伴有β细胞量的丧失也是2型糖尿病的特征(Wali等,2013)。因此,研究胰岛的生物学对于了解糖尿病的发病机制和开发新的疗法是重要的。这里我们描述了小鼠胰岛的分离。这需要温和的酶促和机械消化外分泌组织和密度梯度分离(Chong et al。,2004; Liu和Shapiro,1995; Thomas et al。,1998)。然后,我们将描述胰岛如何可以整体培养或分散到单个细胞中,用于各种体外和体内分析。使用该协议可靠地导致200-400个胰岛的隔离,这取决于鼠标的应变。

第一部分。Islet隔离

材料和试剂

  1. 50ml聚丙烯锥形管(Corning,Falcon ,目录号:352070)
  2. 0.2微米注射器过滤器(例如Sartorius AG,目录号:16534-k)
  3. 注射器(体积依赖于制备的胶原酶P的体积)

  4. 2ml滑动尖端注射器(将保持总体积3ml)(BD,目录号:302204)
  5. 30G×1/2英寸针(BD,目录号:305106)
  6. 500微米筛(Sefar Pty Ltd.,目录号:06-500/38) 注意:切成大约5厘米的方块,并高压灭菌(图1)(见替代产品说明)


    图1. Sefar Pty有限公司生产的500μm胰岛网和规格参考号为
    的50ml管盖
  7. 胶原酶P(Roche Diagnostics,目录号:11213865001)
    注意:它需要在使用前进行测试和优化(见注1)。
  8. RPMI 1640培养基(Life Technologies,Gibco,目录号:21870-076) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:21870-076"
  9. Histopaque-1077(Sigma-Aldrich,目录号:H8889)
  10. Hanks平衡盐溶液(HBSS)(Life Technologies,Gibco,目录号:14175-079)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:14175-079"
  11. 氯化钙脱水物(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C5080)
  12. HEPES(Sigma-Aldrich,目录号:H3375-100G)
  13. HBSS/Ca/HEPES(参见配方)

设备

  1. 冰盒
  2. 解剖显微镜[例如,立体变焦显微镜(尼康仪器公司,型号:SMZ745)]
  3. 光纤光源(例如,AmScope Dual Goose-neck Fiber-Optic Illuminator,型号:HL150-AY)
  4. 钳子[例如,Graefe镊子,100mm,弯曲(ProSciTech Pty Ltd.,目录号:T131C-2)]
  5. 剪刀[例如,Iris scissors,115mm,straight(ProSciTech Pty Ltd.,目录号:T097-2)]
  6. 夹子[例如,直径150毫米的斯宾塞镊子(ProSciTech Pty Ltd.,目录号:T124)]
  7. 台式离心机在室温下,不需要制动(例如,Thermo Fisher Scientific,Heraeus TM ,型号:Multifuge X1R)

程序

  1. 测量每胰腺HBSS/Ca/HEPES 5ml到50ml管中。
    每管胰腺胰腺测量5ml到50ml管中 测量10ml胰腺Histopaque到50ml管中。 如果需要超过50毫升,请使用多个试管,但要将内容物均匀地涂抹在试管上
  2. 将所有培养基置于37℃水浴中
  3. 称量足够的胶原酶P,以在特定浓度下为每批胶原酶P制备3ml(参见注释1-3)。将称重的胶原酶P置于50ml管中
  4. 将胶原酶P溶解在冷(4℃)HBSS/Ca/HEPES溶液中。加入所需体积的HBSS/Ca/HEPES到含有称重的胶原酶P的50ml管中,并轻轻倒转2-3次。静置1-2分钟,然后使用0.2μm过滤器和注射器灭菌。从此时起,将胶原酶P溶液保持在冰上。
  5. 将3毫升胶原酶P放入2毫升滑头注射器,并附上一个30 G针。保持填充注射器在冰上。排出液体过多时,不要使用带有LuerLok的注射器作为压力,否则胆管会撕裂。
  6. 有关如何执行胆管注射程序的详细信息,请查看视频1。简而言之,打开鼠标腹腔,沿肋骨线水平切开腹膜壁,并用小直剪刀垂直切开。将组织放在鼠标的上半身,组织边缘在肋线上。用手指轻轻按下鼠标身体的两侧,使肝脏暴露在肋骨线下方。将肝脏翻转到组织上并将组织折叠以覆盖它。用少量乙醇润湿,轻轻按下以保持一切就位。

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    视频1.胆管注射和胰腺解剖
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  7. 将鼠标放置在解剖显微镜下,并使用弯钳镊子暴露十二指肠,并找到胆管结束十二指肠的地方(见视频1和图2)。夹住胆管进入十二指肠,并将夹子旋转到鼠标的后部,使胆管张紧。使用小的弯曲镊子在与肝脏的连接处保持胆管,并轻轻地将30G针插入导管。轻轻注入3毫升胶原酶P胆囊充气胰腺(见视频1,图2和注4)。


    图2.通过胆管注射胶原酶P. 使用30 G针头(B-C)将夹子施加到胆管,进入十二指肠(A),胶原酶P溶液通过胆管注射。的灌注的胰腺在注射胶原酶P时肿胀
  8. 轻轻地解剖膨胀的胰腺,小心不要切割胃或肠或其他腹部器官,并放入空的50毫升管。保持在冰上。对其他动物重复步骤6-8。
  9. 当所有胰腺已经切除时,将温热的HBSS/Ca/HEPES加入管中至总体积为5ml /胰腺,并置于37℃的水浴中。在胶原酶P和小鼠品系的批次之间孵育时间不同(参见注释1和5)
  10. 当胰腺置于水浴中时,从37°C水浴中取出准备好的Histopaque管和RPMI管。在胶原酶P消化培养期间,这些应该冷却至室温
  11. 从水浴中取出胰腺管,立即置于冰水(冰水)中。不要旋转,摇动或干扰沉降的组织
  12. 立即和尽快工作,尽可能多的温暖HBSS/Ca/HEPES和胶原酶P混合关闭每个胰腺,而不从管中删除胰腺。用冰冷的HBSS/Ca/HEPES将每管加热至15ml。这一步骤通过快速冷却胰腺来停止消化过程。
  13. 通过用每分钟3次振荡的速率剧烈地手动摇动管1分钟来中断胰腺。保持管直立,让胰腺击中管的顶部和底部(见视频2)。不要倒置管。将管子放在冰上。

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    视频2.通过剧烈颤抖中断胰腺
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  14. 将一个高压灭菌的500微米网格的正方形在新的50ml管(每个胰腺一个)的顶部,并用少量(1-2ml)冷RPMI润湿网。将消化的胰腺通过网格。用30 ml冷RPMI从其原来的管洗剩余的胰腺,并通过网倒入。盖上收集管,并返回到冰。请注意,一些未消化的组织可能保留在网格的顶部。与其他样品重复。
  15. 允许消化的胰腺在冰上通过重力放置在50ml管中10分钟
  16. 吸出上清液使用25毫升移液管,留下10-15毫升。小心不要破坏消化的组织。用RPMI补充消化的胰腺管至总体积为30ml,轻轻颠倒2-3次混合。
  17. 在室温下以215×g离心管离心2分钟
  18. 轻轻倒出上清液,小心不要取出沉淀。将管倒置在纸巾上,使残留的介质排出。重要的是去除所有培养基,以使Histopaque不被稀释
  19. 轻轻地球团以松开消化的胰腺。管从这一点应该保持在室温。所有试剂和离心步骤应在室温(22℃)
  20. 每管加入10ml Histopaque(必须在室温下)。轻轻旋转以获得均匀分布的消化胰腺。如果有团块,轻轻地用吸管将它们分开。避免剧烈吸液。
  21. 用5ml RPMI(必须在室温)覆盖每个Histopaque梯度。确保界面清晰,因为这是离心后胰岛所在的位置
  22. 在制动器关闭的情况下,在22℃下以850×g离心15分钟
  23. 轻轻地使用塑料移液管从梯度中删除胰岛。胰岛将在RPMI和Histopaque的界面下方的环中可见(参见图3)。根据需要去除尽可能多的RPMI和Histopaque以去除胰岛而不干扰沉淀。如果具有免疫细胞浸润的胰岛从NOD小鼠中分离,请参见注释6,了解胰岛从梯度中的位置和去除的详细信息。


    图3.胰岛在Histopaque/RPMI梯度界面处的位置。A.正常胰岛的示意图和照片,正好在Histopaque/RPMI界面下形成环。 B.来自扩散通过Histopaque的NOD小鼠的渗透胰岛的示意图和照片。示意图显示用于从梯度收获胰岛的移液管
  24. 将胰岛转移到含有30ml RPMI(室温)的新的50ml管中
  25. 在室温下以215×g离心2分钟
  26. 轻轻倒出上清液,并在纸巾上吸干管。 此时,胰岛可以培养或胰蛋白酶化为单细胞
第二部分。 完整整个胰岛的培养

材料和试剂

  1. 94×15mm培养皿(Greiner Bio One International GmbH,目录号:633185)
  2. FCS
  3. 谷氨酰胺
  4. 青霉素
  5. 链霉素
  6. CMRL Medium,无谷氨酰胺(Life Technologies,Gibco,目录号:11530-037)(参见配方)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11530-037"
  7. 完成CMRL(参见配方)

设备

  1. 在37℃,5%CO 2,相对湿度环境至80%[例如,系列II 3110水夹套CO 2培养箱 (Thermo Fisher Scientific,Forma TM,型号:3131)]

程序

  1. 轻轻冲洗胰岛沉淀轻轻重悬,然后加入5毫升温的完全CMRL
  2. 通过倾吐转移胰岛到培养皿。不要使用组织培养皿,因为胰岛会粘附。
  3. 通过用另外5ml的温的完全CMRL洗涤管来移除残余的胰岛,并将其倒入培养皿中。不要将超过10毫升的培养基放入94毫米的培养皿中,以确保对缺氧高度敏感的胰岛有足够的氧合。
  4. 确保胰岛在培养皿中扩散并将培养皿置于37℃的5%CO 2培养箱中。允许胰岛恢复0.5-1 h,然后进一步操作。
  5. 在具有外部光源的解剖显微镜下,使用无菌的200μl移液管尖端将胰岛移植到管或培养皿中以进行进一步分析(参见图4)。
  6. 胰岛可以在37℃+ 5%CO 2下培养最多三天。


    图4.孤立的胰岛在体外培养中的挑选

  7. 胰岛产量在小鼠品系之间不同。 来自C57Bl/6小鼠的预期产量为300-400个胰岛,而来自非肥胖糖尿病(NOD)小鼠的预期产量为150-300个胰岛,这取决于小鼠的年龄(参见注释6)。

第三部分。 将胰岛胰蛋白酶化为单个细胞

材料和试剂

  1. 1 ml Gilson移液器
  2. 0.2微米注射器过滤器(例如Sartorius AG,目录号:16534-k)
  3. FCS
  4. 谷氨酰胺
  5. 青霉素
  6. 链霉素
  7. Dulbecco磷酸盐缓冲盐水
  8. 牛胰蛋白酶
  9. EDTA
  10. CMRL Medium,无谷氨酰胺(Life Technologies,Gibco,目录号:11530-037)(参见配方)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:11530-037"
  11. 完成CMRL 1066(请参阅配方)
  12. 胰岛素胰蛋白酶(见配方)

设备

  1. 37℃水浴(,例如,Ratek Instruments,型号:WB14)
  2. 在37℃,5%CO 2,相对湿度环境至80%[例如,系列II 3110水夹套CO 2培养箱 (Thermo Fisher Scientific,Forma TM sup),型号:3131)]

程序

  1. 请查看视频3胰岛素胰蛋白酶消化。
  2. 向胰岛丸中加入300μl胰蛋白酶。
  3. 在37℃在水浴中孵育5分钟。
  4. 用1ml Gilson移液管吸取胰岛上下10次。 胰岛应该分散,这可以可视化。 请参阅视频3.
  5. 加入3-5ml完全CMRL以停止胰蛋白酶反应
  6. 以305×g离心5分钟以沉淀单细胞
  7. 倒出介质,更换为1ml完整的CMRL
  8. 在37℃,5%CO 2培养箱中孵育含有胰蛋白酶化胰岛的管1-2小时,以允许在进一步实验/分析之前回收细胞。

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    视频3.胰岛对单细胞的胰蛋白酶消化
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笔记

  1. 每个酶批号的胶原酶P活性存在显着变化。 该变化改变了所用的浓度和消化的时间。 理想的酶批次应该具有1.6-2.2U/mg的胶原酶P活性,以在0.3mg/ml-0.55mg/ml的浓度范围和15-17分钟的孵育时间使用。 胶原酶活性和胰蛋白酶活性之间的平衡也广泛变化并且为了有效的胰岛分离重要的是胰蛋白酶活性不是太高。在2.63-4.3U/mg范围内的胰蛋白酶活性提供最佳结果。胰蛋白酶活性必须< 5 U/mg 当购买新批号的胶原酶P进行彻底测试,包括滴定浓度和孵育时间
  2. 胶原酶P应在达到室温前称重,以防止冷凝和损坏产品。如果购买大量的产品,在新瓶子中少量放入一个"工作批次",以免丢失整个库存的活动。
  3. 在称量胶原酶P时,请戴上口罩和手套,因为它既是呼吸道刺激物又是皮肤刺激物
  4. 胆管的长度和脆性随不同的小鼠品系而变化。 C57Bl/6和遗传衍生物比其他菌株具有更短和更脆弱的胆管。在夹紧和将针插入导管时非常小心,并缓慢注射胶原酶P溶液。肥胖糖尿病小鼠模型Lepr db/db 中的胆管非常脆弱,需要特别小心。
  5. 上述方案可用于从任何小鼠品系中分离胰岛,然而,根据使用的菌株,消化温育时间有轻微的变化。 C57Bl/6胰岛比Balb/C或NOD小鼠胰岛更易于过度消化,C57Bl/6小鼠的孵育时间和该菌株的遗传变异应总是减少约1分钟。
  6. NOD小鼠是1型糖尿病的动物模型,其在大约4周龄开始由免疫细胞进行胰岛的渐进性浸润(Graham等人,2012)。免疫细胞的存在改变胰岛在密度梯度内的位置。含有免疫细胞的胰岛将扩散并出现在Histopaque/RPMI界面下方(参见图3)。 NOD小鼠中胰岛的免疫介导的破坏也降低了老龄小鼠的胰岛产量
  7. 细胞过滤器,例如pluriSelect的500μl的PluriStrainer( https://www.pluriselect.com/product-details/product/pluristrainer-500-um-cell-strainer.html )可用作500μm网格的替代品用于胰岛分离过程的第I部分。但这些都没有在我们的实验室测试。

食谱

  1. HBSS/Ca/HEPES
    1L Hanks平衡盐溶液(HBSS)
    2mM CaCl 2 2 / 20 mM HEPES
    将所有试剂混合在无菌生物安全柜中
    储存在4°C
  2. 完成CMRL
    500 ml CMRL 1066
    10%FCS
    2mM谷氨酰胺 100 U/ml青霉素
    100μg/ml链霉素 将所有试剂混合在无菌生物安全柜中
    储存在4°C
  3. 胰岛素胰蛋白酶
    50ml Dulbecco磷酸盐缓冲盐水
    5.16mg牛胰蛋白酶
    20mM EDTA(0.2ml 0.5M EDTA) 结合所有成分
    通过0.2μm注射器过滤器灭菌
    等分并保存在-20°C

致谢

该协议自我们的实验室的成员自第一次公布以来已经开发和改进(Chong等人,2004; Liu和Shapiro,1995; Thomas等人, 1998)。我们承认该小组所有为这一过程做出贡献的成员。我们常规地在我们对小鼠模型中1型和2型糖尿病的发病机理的研究中利用该方案(Chee等人,2014; Graham等人,2011; Quah等人,2014; Wali等人,2014; Zhao等人,2015)。这项工作由澳大利亚国家卫生和医学研究委员会(NHMRC)计划资助(APP1037321)和与HET的伙伴关系(APP1042735)资助。圣文森特学院获得维多利亚州政府的运营基础设施支持计划的支持。

参考文献

  1. Chees,J.,Ko,HJ,Skowera,A.,Jhala,G.,Catterall,T.,Graham,KL,Sutherland,RM,Thomas,HE,Lew,AM,Peakman,M.,Kay,TWand Krishnamurthy ,B.(2014)。  效应细胞记忆T细胞在胰岛中形成并报告1型糖尿病的胰岛病理。 J Immunol 192(2):572-580。
  2. Chong,MM,Chen,Y.,Darwiche,R.,Dudek,NL,Irawaty,W.,Santamaria,P.,Allison,J.,Kay,TWand Thomas,HE(2004) "ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15100317"target ="_ blank">细胞因子信号传导-1过表达的抑制剂保护胰腺β细胞免受CD8 +/sup> T细胞介导的自身免疫破坏。 J Immunol 172(9):5714-5721。
  3. Graham,KL,Krishnamurthy,B.,Fynch,S.,Mollah,ZU,Slattery,R.,Santamaria,P.,Kay,TWand Thomas,HE(2011)。  自身反应性细胞毒性T淋巴细胞在胰腺淋巴结中引发后在NOD小鼠的胰岛中获得更高的细胞毒效应标记物的表达。 Am J Pathol 178(6):2716-2725。
  4. Graham,KL,Sutherland,RM,Mannering,SI,Zhao,Y.,Chee,J.,Krishnamurthy,B.,Thomas,HE,Lew,AM and Kay,TW(2012)。  1型糖尿病的致病机制:胰岛既是疾病的目标也是驱动因素。 Rev Diabet Stud 9(4):148-168。
  5. Liu,M。和Shapiro,ME(1995)。  一种用于分离鼠胰岛的新方法,其产率显着提高。 Transplant Proc 27(6):3208-3210。
  6. Quah,HS,Miranda-Hernandez,S.,Khoo,A.,Harding,A.,Fynch,S.,Elkerbout,L.,Brodnicki,TC,Baxter,AG,Kay,TW,Thomas,HE和Graham,KL (2014)。  I型干扰素信号缺乏可防止早期干扰素诱导的胰岛中的基因标记,但不是NOD小鼠中的1型糖尿病。 63(3):1032-1040。
  7. Thomas,HE,Parker,JL,Schreiber,RD和Kay,TW(1998)。 
  8. Wali,JA,Masters,SL and Thomas,HE(2013)。  将代谢异常与2型糖尿病的β细胞中的凋亡途径相关联。细胞 2(2):266-283。
  9. Wali,JA,Rondas,D.,McKenzie,MD,Zhao,Y.,Elkerbout,L.,Fynch,S.,Gurzov,EN,Akira,S.,Mathieu,C.,Kay,TW,Overbergh, ,Strasser,A.和Thomas,HE(2014)。  促细胞凋亡BH3蛋白Bim和Puma是胰腺胰岛响应葡萄糖毒性的内质网和线粒体氧化应激的下游。细胞死亡5:e1124。
  10. Zhao,Y.,Scott,NA,Fynch,S.,Elkerbout,L.,Wong,WW,Mason,KD,Strasser,A.,Huang,DC,Kay,TWand Thomas,HE(2015) a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/25301392"target ="_ blank">自身反应性T细胞诱导坏死而不是BCL-2调节或死亡受体介导的细胞凋亡或RIPK3依赖性坏死细胞增生 的移植胰岛在1型糖尿病的小鼠模型中。 Diabetologia 58(1):140-148。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Graham, K. L., Fynch, S., Papas, E. G., Tan, C., Kay, T. W. and Thomas, H. E. (2016). Isolation and Culture of the Islets of Langerhans from Mouse Pancreas. Bio-protocol 6(12): e1840. DOI: 10.21769/BioProtoc.1840.
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