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Isolation, Culture and Differentiation of Primary Acinar Epithelial Explants from Adult Murine Pancreas
从成年鼠胰腺中分离、培养和分化原代腺泡上皮外植体   

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Abstract

The adult pancreas possesses an intrinsic developmental plasticity whereby acinar cells can convert into ductal structures under some pathological conditions. Acinar tissue can be isolated from murine pancreas and kept in three-dimensional collagen culture. Acinar to ductal metaplasia can be induced in primary acinar epithelial explants by treatment with growth factors. This method can be utilized in ex vivo studies involving pancreatic epithelial differentiation.

Keywords: Pancreatic acini(胰腺腺泡细胞), Acinar-ductal-metaplasia(腺泡导管化生), 3-dimensional culture(三维培养)

Materials and Reagents

  1. Mice (4-7 weeks old). As a positive control you can always use wild type mice and induce differentiation of acinar explants by treatment with EGF as described. Depending on your application you may use transgenic mice.
  2. McCoy′s 5A medium (Sigma-Aldrich, catalog number: M8403 )
  3. Waymouth′medium MB 752/1 medium (Genaxxon, catalog number: C4119 )
  4. BSA (Sigma-Aldrich, catalog number: A4503 )
  5. Soybean trypsin inhibitor (SBTI) (Sigma-Aldrich, catalog number: T6522 )
  6. Collagenase Type VIII (Sigma-Aldrich, catalog number: C2139 )
  7. 80% EtOH
  8. Penicillin-streptomycin (P/S) (Life Technologies, Gibco®, catalog number: 15140-122 )
  9. Amphotericin B (not critical, for example from Biochrom, catalog number: A2610 )
  10. FCS (Life Technologies, Gibco®, catalog number: 10270 )
  11. Bovine pituary extract (BPE) (Life Technologies, Gibco®, catalog number: 13028-014 )
  12. Insulin, Transferrin, Selenium, Ethanolamine solution (ITS-X) (Life Technologies, Gibco®, catalog number: 51500 )
  13. Rat Tail Collagen Type I (BD Biosciences, catalog number: 354236 )
  14. Sterile 10x PBS (BIOCHROM, catalog number: 182-50 )
  15. Sterile dH2O
  16. Sterile 1 N NaOH
  17. rhTGFa (R&D systems, catalog number: 239-A )
  18. mEGF (BD Biosciences, catalog number: 354010 )
  19. SBTI stock solution (see Recipes)
  20. Collagenase VIII stock solution (see Recipes)
  21. Wash solution (see Recipes)
  22. 0.1% Culture medium (Waymouth′s medium) (Genaxxon, catalog number: C4119) (see Recipes)
  23. 30% Culture medium (Waymouth′s medium) (Genaxxon, catalog number: C4119) (see Recipes)

Equipment

  1. Stericups filter units (EMD Millipore)
  2. 50 ml Falcon tubes
  3. Tissue culture dishes (10 mm)
  4. 6-well plates
  5. 24- or 48-well plates
  6. Dissecting forceps and scissors
  7. Disposable scalpels
  8. 100 μM cell strainer (BD Biosciences, Falcon®, catalog number: 352360 )
  9. Syringe plungers

Procedure

I.   On the bench:

Mice should be between 4 and 7 weeks of age. Isolation of acinar issue does not work with older mice. Because time is a critical step for the viability of acinar cells, we usually do not process more than 2 mice at the same time.
From one mouse it is possible to obtain about 1,000 acinar-explants. Acinar tissue can be seeded for example in 15 wells of a 48-well plate, thus the obtimal denisty for differetiation of acinar tissue is obtained when you see about 30 acinar explants in a well through a 10x objetive. If density is too high differentiation will take longer, if too low acinar cells will die.

  1. Put forceps and scissors into 80% EtOH.
  2. Anesthetize the mouse with isofluorane, sacrifice the mouse by cervical dislocation and resect the pancreas as fast and possible (time is a critical step).
  3. Place the resected pancreas into a tissue culture dish containing ice cold sterile PBS.


II.  Up to now all protocol steps should be performed under sterile conditions.

Centrifugation steps at 720 x g (18 °C) are a critical step!

  1. In the hood, transfer the pancreas into a culture dish containing 10 ml of PBS.
  2. Immediately put the pancreas in culture dish with 5 ml collagenase VIII solution (see Recipe 4) and cut the organ in very small pieces (less then 1 mm of diameter) with a scalpel within 2 min. Pipette the tissue up and down.
  3. Incubate the dish at 37 °C for 10 min. Shake from time to time.
  4. Transfer the sample into a 50 ml falcon. Pipette the solution 3x up and down.
  5. Wash the dish with 10 ml wash solution and transfer to the 50 ml falcon.
  6. Centrifuge, 720 x g, 5 min, 18 °C.
  7. Carefully remove the supernatant.
  8. Resuspend the pellet in 5 ml collagenase VIII solution. Put in a culture dish. Pipette the tissue up and down.
  9. Incubate at 37 °C for 10 min. Shake from time to time.
  10. Aspirate the sample and filter through a 100 μm nylon cell strainer positioned on a 50 ml falcon.
  11. Macerate the tissue pieces through the cell strainer with a syringe plunger.
    Note: This step is difficult to describe, try to press the tissue through the strainer with the syringe plunger without applying a shear stress. It is possible you have to optimize this step in your hands.
  12. Wash the mesh with 10 ml wash solution to carefully collect any remaining cell.
  13. Centrifuge, 720 x g, 5 min, 18 °C.
  14. Carefully remove the supernatant.
  15. Aspirate the pellet in 20 ml wash solution, and transfer the solution in a fresh tube. Do not aspirate up and down!
  16. Centrifuge, 720 x g, 5 min, 18 °C.
  17. Resuspend the pellet in 2 ml culture medium 30% and transfer it on a well of a 6-well culture dish.
  18. Check the quality of isolated acinar tissue under the microscope. Isolation is good if you see clusters of acinar cells (epithelial explants, the morphology is the same as in the Ctr. of the figure below) and only rare isolated acinar cells swimming around. Sometimes damaged acinar cells agglomerate together, remove such sticky agglomerates with a forceps.
  19. Let the isolated acini recover for 60 min in the incubator (37 °C with 5% CO2).
  20. Meanwhile precoat a cell culture plate of the desired size with collagen (see below). Calculate the required volume of collagen - medium mixture required for step 25. The volume depends from your application. Calculated 100 μl volume/well for a 48-well plate or 80 μl volume/well for an 8 well chamber slide. Typically you will seed the acinar explants obtained from one wild type mice in 15 wells of a 48 well plate. In this case you need 100 μl x 15/2 = 750 μl collagen and 100 μl x 15/2 = 750 μl medium 0.1%.
  21. Collect the acinar suspension from the plate and transfer it to a fresh 15 ml tube.
  22. Centrifuge acinar suspension, 720 x g, 5 min, 18 °C.
  23. Carefully aspirate the supernatant.
  24. Resuspend the pellet in a mixture of collagen and culture medium 0.1% (1:1 vol. /vol.)
  25. Pipet the acini/collagen-medium suspension mixture into each coated well in the plate (at least in triplicate). For the typical 48-well example you pipet 100 μl of the mixture in 15 wells.
  26. Wait until solidification of the collagen (about 30 min)
  27. Add some more collagen (100 μl/well for the 48 well plate). You may skip this step when you will perform immunofluorescence as a downstream application. Wait until solidification (about 30 min)
  28. Add culture medium 0.1% with/without the desired supplements to each well. Typically 400 μl/well for a 48 well plate.
  29. To induce transdifferentiation of acinar explants, add TGFa (10 ng ml-1) or EGF (25 ng ml-1) into the culture medium 0.1% at day 1-3-5 where day 1 is the day of isolation.
    Note:
    1) We usually quantify transdifferentiation of wild type acinar explants after EGF treatment at day 5 (Figure 1).

    2) Culture can be kept until day 8. For longer periods of culture you have to reseed the acinar explants into fresh collagen because cultures become acidic and collagen breaks down.


III. Gelation Procedure for Rat Tail Collagen I, 2.5 mg ml-1

  1. Place on ice: Collagen, sterile 10x PBS, sterile water, sterile 1 N NaOH, falcon tubes. Keep all reagents on ice.
  2. Calculate the total amount of collagen (final concentration of 2.5 mg ml-1) needed with the formula: (final Vol. x 2.5)/ (collagen concentration in the bottle). You need a 100/120 μl collagen layer/well for a 48-well plate or 80 μl collagen layer/well for a 8 well chamber slide.
  3. Prepare a tube on ice with the following volume of 10x PBS: (final Vol.)/10.
  4. Add the following volume of 1 N NaOH to the tube containing 10x PBS: (Vol. collagen to be added) x 0.023 ml.
  5. Add to the 10x PBS/1 N NaOH the following volume of sterile ice-cold water: (final Vol.) - (Vol. collagen) - (Vol. 10x PBS) - (Vol. 1 N NaOH).
  6. Mix the contents of tube and hold in ice.
  7. Add the calculated volume of collagen and mix. Leave on ice (stable for 2-3 h) until ready for use.
  8. Coat tissue culture dishes with collagen (use 24/48 wells for quantification of transdifferentiation, protein or RNA extraction, or use 8 well chamber slides for later staining applications).
  9. Allow collagen to solidify at 37 °C (about 30 min)
    Note: Downstream applications include immunofluorescence, protein extraction, RNA extraction and cytotoxicity assay.


    Figure 1. Example of acinar explants at day 5. Acinar explants from a wild type mouse were isolated and cultivated for 5 days in collagen as described. Ctr (medium only): a typical acinar explant is composed by a sphere of several acinar cells. EGF: in presence of 25 ng/ml of EGF cell clusters get a more flattened morphology and differentiate to a duct-like structure characterized by a lumen (arrow) lined by several cells.

Recipes

  1. SBTI stock solution
    10 mg/ml in McCoy medium, store at -20 °C.
  2. (optional) Collagenase VIII stock solution
    9.6 mg/ml in McCoy medium (not fully dissolved), store at -20 °C.
  3. Wash solution (40 ml/mouse)
    McCoy′s medium
    0.1% BSA (sterile filtered)
    0.2 mg/ml SBTI (Stock 1:50)
  4. Collagenase VIII solution (10 ml/mouse)
    McCoy′s medium
    0.1% BSA (sterile filtered)
    0.2 mg/ml SBTI (Stock 1:50)
    1.2 mg/ml Collagenase VIII (critical step, add collagenase just before use, dilute the collagenase VIII stock solution 1:8)
  5. 0.1% culture medium
    Waymouth′s medium
    0.1% BSA (sterile filtered)
    0.2 mg/ml SBTI (Stock 1:50)
    P/S 1:200 (optional, not necessary for short term culture)
    0.25 μg/ml Amphotericin B (antifungal, optional)
    ITS-X 1:100
    BPE: 50 μg/ml (dilute according the batch concentration) (Aliquots, -20 °C)
    0.1% FCS
  6. 30% culture medium
    0.1% culture medium
    30% FCS

Acknowledgments

This protocol for acinar epithelial explants from adult murine pancreas was established by modification previously published protocols ((Lisle and Logsdon, 1990; Githens et al., 1994; Means et al., 2005). The work was supported by grants from the Wilhelm Sander Stiftung (2010.021.1).

References

  1. Ardito, C. M., Grüner, B. M., Takeuchi, K. K., Lubeseder-Martellato, C., Teichmann, N., Mazur, P. K., DelGiorno, K. E., Carpenter, E. S., Halbrook, C. J. and Hall, J. C. (2012). EGF receptor is required for KRAS-induced pancreatic tumorigenesis. Cancer Cell 22(3): 304-317.
  2. Githens, S., Schexnayder, J. A., Moses, R. L., Denning, G. M., Smith, J. J. and Frazier, M. L. (1994). Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. In Vitro Cell Dev Biol Anim 30A(9): 622-635.
  3. Heid, I., Lubeseder–Martellato, C., Sipos, B., Mazur, P. K., Lesina, M., Schmid, R. M. and Siveke, J. T. (2011). Early requirement of Rac1 in a mouse model of pancreatic cancer. Gastroenterology 141(2): 719-730. e717.
  4. Lisle, D., Ratcliffe, J. F., Faoagali, J. and Cherian, S. (1990). Bacterial contamination of contrast media stored after opening. Br J Radiol 63(751): 532-534.
  5. Means, A. L., Meszoely, I. M., Suzuki, K., Miyamoto, Y., Rustgi, A. K., Coffey, R. J., Wright, C. V., Stoffers, D. A. and Leach, S. D. (2005). Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 132(16): 3767-3776.

简介

成人胰腺具有固有的发育可塑性,其中腺泡细胞可以在一些病理条件下转化为导管结构。 腺泡组织可以从鼠胰腺中分离并保存在三维胶原培养物中。 腺泡到导管化生可以通过用生长因子治疗在原发性腺泡上皮外植体中诱导。 该方法可以用于涉及胰腺上皮分化的离体研究。

关键字:胰腺腺泡细胞, 腺泡导管化生, 三维培养

材料和试剂

  1. 小鼠(4-7周龄)。 作为阳性对照,您可以总是使用野生型小鼠并通过如所述的EGF处理诱导腺泡外植体的分化。 根据您的应用,您可以使用转基因小鼠
  2. McCoy's 5A培养基(Sigma-Aldrich,目录号:M8403)
  3. Waymouth'medium MB 752/1培养基(Genaxxon,目录号:C4119)
  4. BSA(Sigma-Aldrich,目录号:A4503)
  5. 大豆胰蛋白酶抑制剂(SBTI)(Sigma-Aldrich,目录号:T6522)
  6. 胶原酶VIII型(Sigma-Aldrich,目录号:C2139)
  7. 80%EtOH
  8. 青霉素 - 链霉素(P/S)(Life Technologies,Gibco ,目录号:15140-122)
  9. 两性霉素B(不是关键的,例如来自Biochrom,目录号:A2610)
  10. FCS(Life Technologies,Gibco ,目录号:10270)
  11. 牛皮提取物(BPE)(Life Technologies,Gibco ,目录号:13028-014)
  12. 胰岛素,转铁蛋白,硒,乙醇胺溶液(ITS-X)(Life Technologies,Gibco ,目录号:51500)
  13. 大鼠尾胶原I型(BD Biosciences,目录号:354236)
  14. 无菌10x PBS(BIOCHROM,目录号:182-50)
  15. 无菌dH 2 O 2/b
  16. 无菌1N NaOH,
  17. rhTGFa(R& D systems,目录号:239-A)
  18. mEGF(BD Biosciences,目录号:354010)
  19. SBTI储备溶液(见配方)
  20. 胶原酶VIII储备溶液(见配方)
  21. 洗涤溶液(见配方)
  22. 0.1%培养基(Waymouth培养基)(Genaxxon,目录号:C4119)(参见配方)
  23. 30%培养基(Waymouth氏培养基)(Genaxxon,目录号:C4119)(参见配方)

设备

  1. Stericups过滤器单元(EMD Millipore)
  2. 50ml Falcon管
  3. 组织培养皿(10mm)
  4. 6孔板
  5. 24或48孔板
  6. 解剖钳和剪刀
  7. 一次性手术刀
  8. 100μM细胞过滤器(BD Biosciences,Falcon ,目录号:352360)
  9. 注射器柱塞

程序

I.   在板凳上:

小鼠应该在4和7周龄之间。腺泡问题的隔离不适用于老年小鼠。因为时间是腺泡细胞活力的关键步骤,我们通常不会同时处理超过2只小鼠。
从一只小鼠可以获得约1,000个腺泡外植体。腺泡组织可以接种在例如48孔板的15个孔中,因此当通过10×目标物在孔中看到约30个腺泡外植体时,获得腺泡组织的差异性的obtimal denisty。如果密度太高,分化将需要更长时间,如果太低的腺泡细胞死亡。

  1. 将钳子和剪刀放入80%乙醇。
  2. 用异氟烷麻醉小鼠,通过颈椎脱臼处死小鼠,并尽可能快地切除胰腺(时间是关键的一步)。
  3. 将切除的胰腺放入含有冰冷无菌PBS的组织培养皿中


II。  到目前为止,所有方案步骤应在无菌条件下进行。

在720×g(18℃)的离心步骤是关键步骤!

  1. 在罩中,将胰腺转移到含有10ml PBS的培养皿中
  2. 立即将胰腺放入具有5ml胶原酶VIII溶液的培养皿中(参见配方4),并用手术刀在2分钟内切割非常小的部分(直径小于1mm)的器官。 向上和向下吸移纸巾。
  3. 孵育皿在37℃下10分钟。 不时摇晃。
  4. 将样品转移到50毫升猎鹰。 向上和向下移液3x溶液。
  5. 用10毫升洗涤液洗涤培养皿,转移到50毫升的猎鹰
  6. 离心机,720×g,5分钟,18℃
  7. 小心地清除上清液。
  8. 将沉淀重悬在5ml胶原酶VIII溶液中。 放入培养皿。 吸取组织上下。
  9. 在37℃孵育10分钟。 不时摇晃。
  10. 吸取样品,并通过位于50ml falcon上的100μm尼龙细胞过滤器过滤
  11. 用注射器柱塞使组织片通过细胞过滤器。
    注意:这一步很难描述,尝试用注射器柱塞通过过滤器按压组织而不施加剪切应力。 您可能必须在手中优化此步骤。
  12. 用10毫升洗涤液洗涤网,仔细收集剩余的细胞
  13. 离心机,720×g,5分钟,18℃
  14. 小心地清除上清液。
  15. 在20ml洗涤溶液中吸出沉淀,并将溶液转移到新管中。 不要上下吸入!
  16. 离心机,720×g,5分钟,18℃
  17. 将沉淀重悬在2ml培养基30%中,并将其转移到6孔培养皿的孔中。
  18. 检查显微镜下孤立的腺泡组织的质量。隔离是好的,如果你看到腺泡细胞簇(上皮外植体,形态是相同的在下图中的Ctr。),只有罕见的孤立的腺泡细胞游泳。有时损伤的腺泡细胞聚集在一起,用镊子去除这种粘性团块
  19. 让孤立的腺泡在孵育器(37℃,5%CO 2)中恢复60分钟。
  20. 同时用胶原预涂所需大小的细胞培养板(见下文)。计算步骤25所需的胶原蛋白 - 培养基混合物所需的体积。体积取决于您的应用。对于48孔板计算为100μl体积/孔或对于8孔腔室载玻片计算为80μl体积/孔。通常,您将从一个野生型小鼠获得的腺泡外植体种植在48孔板的15个孔中。在这种情况下,您需要100μlx 15/2 = 750μl胶原和100μlx 15/2 = 750μl培养基0.1%。
  21. 从板中收集腺泡悬浮液,并将其转移到新鲜的15毫升管
  22. 离心acinar悬浮液,720×g/min,5分钟,18℃
  23. 小心吸出上清液。
  24. 将沉淀重悬于胶原和0.1%(1:1体积/体积)培养基的混合物中
  25. 将腺泡/胶原 - 培养基悬浮液混合物吸入板中的每个涂覆的孔中(至少一式三份)。 对于典型的48孔实例,您在15个孔中移取100μl的混合物
  26. 等待胶原凝固(约30分钟)
  27. 加入一些更多的胶原(100微升/孔的48孔板)。 当您将进行免疫荧光作为下游应用时,您可以跳过此步骤。 等待直到凝固(约30分钟)
  28. 向每个孔中加入含/不含所需补充剂的培养基0.1%。 通常对于48孔板为400μl/孔
  29. 为了诱导腺泡外植体的转分化,在第1-3天向培养基中加入TGFα(10ng ml -1 -1)或EGF(25ng ml -1 -1)至培养基0.1% -5,其中第1天是隔离日。
    注意:
    1)我们通常量化野生型腺泡外植体在EGF处理后第5天的转分化(图1)。
    2)培养可以保持到第8天。对于更长的培养时间,你必须将腺泡外植体重新投入到新鲜胶原蛋白中,因为培养物变成酸性,胶原蛋白分解。


III。 大鼠尾胶原I的胶凝方法,2.5mg ml -1 sup

  1. 置于冰上:胶原,无菌10x PBS,无菌水,无菌1N NaOH,falcon管。 将所有试剂置于冰上。
  2. 用下式计算所需的胶原蛋白的总量(最终浓度为2.5mg ml -1 -1):(最终体积×2.5)/(瓶中的胶原浓度)。 对于48孔板,您需要100/120μl胶原层/孔,对于8孔室玻片,需要80μl胶原层/孔。
  3. 在冰上用以下体积的10×PBS:(最终体积)/10制备管
  4. 将以下体积的1N NaOH加入到含有10×PBS:(要添加的体积胶原)×0.023ml的试管中。
  5. 将10×PBS/1N NaOH加入以下体积的无菌冰冷水:(最终体积) - (体积胶原) - (体积10×PBS) - (体积1N NaOH)。
  6. 混合管中的内容物并保持在冰上
  7. 添加计算体积的胶原蛋白和混合。 放在冰上(稳定2-3小时),直到准备使用。
  8. 用胶原涂覆组织培养皿(使用24/48孔用于定量转分化,蛋白质或RNA提取,或使用8孔腔室载玻片用于随后的染色应用)。
  9. 允许胶原在37℃(约30分钟)固化
    注意:下游应用包括免疫荧光,蛋白质提取,RNA提取和细胞毒性测定。


    图1.腺泡外植体在第5天的实施例。分离来自野生型小鼠的腺泡外植体,并如所述在胶原中培养5天。 Ctr(仅培养基):典型的腺泡外植体由几个腺泡细胞的球体组成。 EGF:在25ng/ml EGF细胞簇存在下,获得更扁平的形态,并分化成以几个细胞为衬里的腔(箭头)为特征的管状结构。

食谱

  1. SBTI储液
    10 mg/ml在McCoy培养基中,储存在-20℃。
  2. (可选)胶原酶VIII储备液
    9.6mg/ml在McCoy培养基(未完全溶解)中,储存在-20℃
  3. 洗涤溶液(40ml /小鼠)
    McCoy的媒介
    0.1%BSA(无菌过滤) 0.2 mg/ml SBTI(库存1:50)
  4. 胶原酶VIII溶液(10ml /小鼠)
    McCoy的媒介
    0.1%BSA(无菌过滤) 0.2 mg/ml SBTI(库存1:50)
    1.2mg/ml胶原酶VIII(临界步骤,在使用前添加胶原酶,稀释胶原酶VIII储备液1:8)
  5. 0.1%培养基
    Waymouth的媒介
    0.1%BSA(无菌过滤) 0.2 mg/ml SBTI(库存1:50)
    P/S 1:200(可选,不需要短期文化)
    0.25μg/ml两性霉素B(抗真菌剂,可选)
    ITS-X 1:100
    BPE:50μg/ml(按批次浓度稀释)(等分试样,-20℃)
    0.1%FCS
  6. 30%培养基
    0.1%培养基
    30%FCS

致谢

用于来自成年鼠胰腺的腺泡上皮外植体的这种方案通过修改先前公开的方案来建立((Lisle和Logsdon,1990; Githens等人,1994; Means& ,2005)。这项工作得到了Wilhelm Sander Stiftung(2010.021.1)的资助。

参考文献

  1. Ardito,CM,Grüner,BM,Takeuchi,KK,Lubeseder-Martellato,C.,Teichmann,N.,Mazur,PK,DelGiorno,KE,Carpenter,ES,Halbrook,CJand Hall,JC(2012) ="_ blank"href ="http://www.ncbi.nlm.nih.gov/pubmed/22975374"> EGF受体是KRAS诱导的胰腺肿瘤发生所需的。癌细胞 22(3):304-317。
  2. Githens,S.,Schexnayder,J.A.,Moses,R.L.,Denning,G.M.,Smith,J.J.and Frazier,M.L。(1994)。 小鼠胰腺腺泡/肾小管组织产生上皮细胞培养物,其形态,生物化学和功能上无法区分小叶间导管细胞培养物。 In Vitro Cell Dev Biol Anim 30A(9):622-635。
  3. Heid,I.,Lubeseder-Martellato,C.,Sipos,B.,Mazur,PK,Lesina,M.,Schmid,RMand Siveke,JT(2011)。胰腺癌小鼠模型中Rac1的早期需求。胃肠病学 141(2):719-730。 e717。
  4. Lisle,D.,Ratcliffe,J.F.,Faoagali,J。和Cherian,S。(1990)。 开启后储存的造影剂的细菌污染。 Br J Radiol 63(751):532-534。
  5. Means,A.L.,Meszoely,I.M.,Suzuki,K.,Miyamoto,Y.,Rustgi,A.K.,Coffey,R.J.,Wright,C.V.,Stoffers,D.A。和Leach, 由腺泡细胞转分化和巢蛋白阳性中间体的产生介导的胰腺上皮可塑性。 em> Development 132(16):3767-3776。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lubeseder-Martellato, C. (2013). Isolation, Culture and Differentiation of Primary Acinar Epithelial Explants from Adult Murine Pancreas. Bio-protocol 3(13): e818. DOI: 10.21769/BioProtoc.818.
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