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Pancreatic Acinar Cell 3-Dimensional Culture
胰腺腺泡细胞的三维培养   

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参见作者原研究论文

本实验方案简略版
Oncogene
Apr 2013

Abstract

Normal pancreatic acinar cells are difficult to maintain on traditional plastic culture surfaces due to their physical properties of housing large quantities of digestive enzymes and the formation of intercellular tight junctions and gap junctions (Apte and Wilson 2005; Rukstalis et al., 2003). However, placing primary acinar cells within a 3-dimensional matrix (3D-culture) maintains the cells for sufficient time so that they can be monitored for physiological changes to different stimuli. We have used a modified collagen 3D-culture system that has been adapted from Means et al. (2005) to model the very early events associated with pancreatic cancer development. In this model, KrasG12D-expressing pancreatic acinar cells, or wildtype acinar cells treated with EGFR-dependent growth factors (i.e., TGFα), convert to ductal cysts that mimic the acinar-to-ductal metaplasia (ADM) stage that precedes formation of Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Ductal Adenocarcinoma (PDAC) (Means et al., 2005; Shi et al., 2013).

Keywords: Tissue culture (组织文化), Differentiation (微分), Pancreas (胰腺)

Materials and Reagents

  1. Rat Tail Collagen I (Life Technologies, Invitrogen™, catalog number: A10483 )
  2. Fetal Bovine Serum (FBS)
  3. Pen-Strep
  4. Hanks Balanced Salt Solution (HBSS) (Life Technologies, Gibco®, catalog number: 14065 )
  5. E.Z.N.Z Total RNA Kit I (Omega Bio-Tek, catalog number: R6834-01 )
  6. BME-phenol blue
  7. Xylene
  8. 10x RPMI 1640 medium (Life Technologies, Gibco®, catalog number: 31800-022 ) (see Recipes)
  9. 4.2% NaHCO3 (see Recipes)
  10. Collagenase P (Roche, catalog number: 11213857001 ) (see Recipes)
  11. 100x Soybean Trypsin Inhibitor (STI) (Sigma-Aldrich, catalog number: T6522 ) (see Recipes)
  12. 2,000x Dexamethasone (Dex) (Sigma-Aldrich, catalog number: D4902 ) (see Recipes)
  13. 3D-culture medium (see Recipes)

Equipment

  1. Centrifuge (Beckman Coulter, model: GS-15R or equivalent, rotor S4180)
  2. Standard 5% CO2 tissue culture incubator
  3. 24-well tissue culture plates
  4. 37 °C shaker
  5. 6 cm culture dish
  6. Dissection scissors
  7. 15 ml plastic screw cap tube
  8. Laminar flow hood
  9. Rocking platform
  10. Tissue processor
  11. Polypropylene Mesh (105 μm & 500 μm sizes) (Spectra/Mesh) (Fisher Scientific, catalog number: 146436 and 146418 )
    Note: Cut meshes into 10 x 10 cm squares and sterilize by autoclaving.
  12. Homogenizer spin column (Omega Bio-Tek, catalog number: HCR003 )
  13. Tissue-Teck Biopsy Uni-cassette (Sakura, catalog number: 4087)
  14. Falcon tube (15 ml and 50 ml)
  15. Nanodrop spectrophotometer

Procedure

The following procedures for one mouse pancreas

Notes:

  1. All procedures are performed in a laminar flow hood under sterile conditions.
  2. For each pancreas prepare 30 ml Cold HBSS + 5% FBS and 20 ml HBSS + 30% FBS.

  1. Prepare 24-well tissue culture plates (you will need 250 μl collagen mix per well)
    1. On ice, mix rat tail collagen in a ratio of 0.9 ml collagen: 0.1 ml 10x RPMI 1640 medium. Add 80-100 μl 4.2% NaHCO3 per 1 ml mixture (usually a light pink/yellowish color will be achieved).
      Note: The volume of NaHCO3 should be tested and adjusted for each batch of collagen.
    2. Place each 24-well plate on ice and pipette 250 μl of collagen gel mixture into each well, assuring that the gel covers the bottom of each well. Repeat so as to have a separate plate for each day of harvest. Cover and place in a 37 °C, 5% CO2 incubator for at least 1 h to solidify.

  2. Isolate cells
    1. Sacrifice each mouse following institutional IACUC protocols.
    2. Put the mouse on a dissection plate in a supine position, spray with 70% EtOH and open the abdominal cavity to expose the pancreas.
    3. Resect the pancreas and place into ~20 ml of HBSS in a 6 cm culture dish on ice. Swirl to wash, and decant liquid.
    4. Add 5 ml cold HBSS and mince the pancreas quickly with dissection scissors.
    5. Transfer the pancreas material into a 15 ml plastic screw cap tube. Add 100 μl 10 mg/ml collagenase P to the tissue suspension and mix gently.
    6. Wrap Parafilm around the cap and shake at 225 rpm in a 37 °C shaker for 15-20 min, until most of the tissue clumps are gone and the suspension look cloudy (examine by eye every 5 min to prevent over- or under-digestion).
    7. Add 5 ml cold HBSS + 5% FBS and centrifuge 2,000 rpm, 2 min at 4 °C. Aspirate supernatant.
    8. Resupsend the pellet 3 times in 5 ml cold HBSS + 5% FBS, spinning at 1,500 rpm, 2 min between rinses.
    9. After the final wash, resuspend the pellet in 5 ml HBSS + 5% FBS.
    10. With sterilized scissors, cut the tip of the1,000 μl pipette tip to make the opening wider and then gently pipet the cell suspension through a sterile 500 μm mesh.
    11. Pipet an additional 5 ml HBSS + 5% FBS to wash all remaining cells through the mesh.
    12. Repeat by pipetting the cell suspension through a 105 μm mesh.
    13. Slowly pipet the cell suspension on top of a 50 ml tube containing 20 ml HBSS + 30% FBS.
    14. Centrifuge 1,000 rpm, 2 min, 4 °C to pellet individual acini clusters.
    15. Aspirate supernatant.

  3. Plating cells
    1. Resuspend the cell pellet in 8-10 ml (volume adjusted according to the size of pancreas) in 3D-culture medium.
    2. Mix the collagen as above step A1.
    3. Mix equal parts of the cell suspension and collagen mix.
    4. Immediately plate the cell suspension in the collagen-coated wells, 0.5 ml/well (see Figure 1).


      Figure 1. 3D-culture setup

    5. Allow the collagen-cell mixture to solidify ~1 h at 37 °C, 5% CO2, and then add 1 ml warm 3D-culture medium (with or without growth factors inhibitors).
    6. Change the medium on days 1 and 3. For KrasG12D-expressing acinar cells, over 90% of ductal cysts should form by day 5 (see Figure 2). Similarly, wild-type acinar cells provided 50 ng/ml TGFα following plating will form ductal cysts by day 5, with a conversion rate of about 70%-80% (see Figure 2) (Shi et al., 2013).


      Figure 2. Pancreatic acinar cells in 3D-culture

  4. RNA/protein prep from acinar cell 3D-culture
    1. Dilute collagenase P (10 mg/ml) 1:50 in 1x HBSS (room temperature). Prepare 4 ml solution in a 15 ml falcon tube for each sample.
    2. Wash cells/collagen disc in culture well once with 1x HBSS. Transfer collagen disc to a 15 ml falcon tube with a spatula. Pool 3 or 4 wells together to obtain enough cells.
    3. Digest collagen for ~ 30 min in a 37 °C shaker, 250 rpm. Check every 10 or 15 min.
    4. After all of the collagen is digested, pellet the cells at 2,000 rpm for 2 min at 4 °C.
    5. Remove the supernatant, resuspend cells with 1 ml cold HBSS and transfer to a 1.5 ml centrifuge tube.
    6. Centrifuge at 5,000 rpm for 2 min at 4 °C. Carefully remove the supernatant. Loosen the pellet by flicking the bottom of the tube with your finger tips.
    7. OPTION 1: RNA Prep. Follow the E.Z.N.A. Total RNA Mini Kit protocol. Homogenize using a homogenizer column. Resuspend the final RNA in 40 μl ddH2O. Measure the RNA concentration using a nanodrop spectrophotometer. Store at -20 °C.
    8. OPTION 2: Protein Prep. Prepare 100 μl 4x Sample Buffer (BME-phenol blue) + 1:50 protease inhibitor cocktail + 1:100 phosphatase inhibitor cocktail 1 + 1:100 phosphatase inhibitor cocktail 2 + 1:200 Na3VO4 for each sample. Lyse the cells by pipetting and sonicate 15 seconds. Store at -20 °C.

  5. Collagen disc fixation
    1. Fix the collagen discs in the wells with 10% neutral buffered formalin for 30 min at room temperature, and then separate the discs from the plate wall using a yellow 200 μl pipette tip and transfer the discs into scintillation vials containing 10% neutral buffered formalin. Fix on rocker at RT overnight.
    2. Transfer the fixed collagen discs from the scintillation vials into standard biopsy cassettes.
    3. Gradually dehydrate the collagen discs by incubating in 70%, 90%, and 100% EtOH and then in Xylenes in a glass container at room temperature, 2 times of 1 h incubation for each solution on a rocking platform.
    4. After Xylene clearing, transfer the cassettes into the paraffin tank of the tissue processor. Let the collagen discs go through the last two steps of paraffin incubation (1 h each at 65 °C) and then keep them in paraffin tank until it is time for making paraffin blocks.
      1. IMPORTANT: Keep cassettes horizontal or the gels will "flow" into the corners and will be difficult to embed.
      2. Collagen discs can be processed through an automatic tissue processor. However, the freshness (cleanliness) of the solutions inside the processor greatly affects the outcome (the collagen discs will be hard and shrunken when solutions in the processor become old). Manual processing with small volumes of fresh solutions will keep the collagen discs in their original shape after processing.

Recipes

  1. 10x RPMI 1640 medium
    Dissolve 1 package of RPMI 1640 powder in 100 ml ddH2O
    Add 2 g NaHCO3, pH to 7.2
    Filter sterilize
    Store at 4 °C
    Make fresh solution every 2 months
  2. 4.2% NaHCO3
    Dissolve 4.2 g NaHCO3 in 100 ml ddH2O
    Filter sterilize
    Store at 4 °C
  3. Collagenase P
    Dissolve in 10 ml ddH2O to make a 10 mg/ml stock
    Filter sterilize, aliquot and store at -80 °C
    Thaw a new vial for each acinar prep
  4. 100x Soybean Trypsin Inhibitor (STI)
    Dissolve in ddH2O to make a 10 mg/ml stock
    Filter sterilize, aliquot and store at -20 °C
  5. 2,000x Dexamethasone (Dex)
    Dissolve 25 mg in 12.5 ml of 100% EtOH to generate a 2 mg/ml stock
    Aliquot and store at -20 °C
  6. 3D-culture medium
    RPMI 1640
    1% FBS
    1% Pen-Strep
    0.1 mg/ml STI
    1 μg/ml Dex

Acknowledgments

This protocol was adapted from Shi et al. (2013).

References

  1. Apte, M. and Wilson, J. (2005). The importance of keeping in touch: regulation of cell-cell contact in the exocrine pancreas. Gut 54(10): 1358-1359.
  2. 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.
  3. Rukstalis, J. M., Kowalik, A., Zhu, L., Lidington, D., Pin, C. L. and Konieczny, S. F. (2003). Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1. J Cell Sci 116(16): 3315-3325.
  4. Shi, G., DiRenzo, D., Qu, C., Barney, D., Miley, D. and Konieczny, S. (2013). Maintenance of acinar cell organization is critical to preventing Kras-induced acinar-ductal metaplasia. Oncogene 32(15): 1950-1958.

简介

正常的胰腺腺泡细胞难以保持在传统的塑料培养表面上,因为其具有容纳大量消化酶和形成细胞间紧密连接和间隙连接的物理性质(Apte和Wilson 2005; Rukstalis等人, ,2003)。然而,将原代腺泡细胞放置在三维基质(3D培养)中保持细胞足够的时间,使得它们可以被监测对不同刺激的生理变化。我们已经使用改良的胶原三维培养系统,其已经从Means等人(2005)改编以模拟与胰腺癌发展相关的非常早期的事件。在该模型中,表达Kras G12D 的表达胰腺腺泡细胞或用EGFR依赖性生长因子(即,TGFα)处理的野生型腺泡细胞转化成导管囊肿,在胰腺上皮内瘤形成(PanIN)和胰腺导管腺癌(PDAC)形成之前的腺泡至导管化生(ADM)阶段(Means等人,2005; Shi等人,/em>,2013)。

关键字:组织文化, 微分, 胰腺

材料和试剂

  1. 大鼠尾胶原I(Life Technologies,Invitrogen TM,目录号:A10483)
  2. 胎牛血清(FBS)
  3. Pen-Strep
  4. Hanks平衡盐溶液(HBSS)(Life Technologies,Gibco ,目录号:14065)
  5. E.Z.N.Z Total RNA Kit I(Omega Bio-Tek,目录号:R6834-01)
  6. BME-酚蓝
  7. 二甲苯
  8. 10x RPMI 1640培养基(Life Technologies,Gibco ,目录号:31800-022)(参见Recipes)
  9. 4.2%NaHCO 3(参见配方)
  10. 胶原酶P(Roche,目录号:11213857001)(参见配方)
  11. 100x大豆胰蛋白酶抑制剂(STI)(Sigma-Aldrich,目录号:T6522)(参见配方)
  12. 2,000x地塞米松(Dex)(Sigma-Aldrich,目录号:D4902)(参见配方)
  13. 3D培养基(见配方)

设备

  1. 离心机(Beckman Coulter,型号:GS-15R或等价物,转子S4180)
  2. 标准5%CO 2组织培养孵化器
  3. 24孔组织培养板
  4. 37℃摇床
  5. 6厘米培养皿
  6. 解剖剪刀
  7. 15毫升塑料螺旋盖管
  8. 层流罩
  9. 摇台
  10. 组织处理器
  11. 聚丙烯网(105μm&500μm尺寸)(Spectra/Mesh)(Fisher Scientific,目录号:146436和146418)
    注意:将网格切成10 x 10厘米的方块,高压灭菌。
  12. 均质器离心柱(Omega Bio-Tek,目录号:HCR003)
  13. Tissue-Teck Biopsy Uni-cassette(Sakura,目录号:4087)
  14. Falcon管(15ml和50ml)
  15. Nanodrop分光光度计

程序

以下程序为一个小鼠胰腺

注意:

  1. 所有程序在层流罩中在无菌条件下进行。
  2. 对于每个胰腺,准备30ml冷HBSS + 5%FBS和20ml HBSS + 30%FBS。

  1. 准备24孔组织培养板(你将需要250微升胶原混合每孔)
    1. 在冰上,以0.9ml胶原:0.1ml 10x RPMI 1640培养基的比例混合大鼠尾胶原。 每1ml混合物中加入80-100μl4.2%NaHCO 3(通常为浅粉色/淡黄色)。
      注意:应对每批胶原测试并调整NaHCO的量
    2. 将每个24孔板在冰上,吸取250微升胶原凝胶混合物到每个孔,确保凝胶覆盖每个孔的底部。 重复,以便有一个单独的板的每一天的收获。 盖上并置于37℃,5%CO 2培养箱中至少1小时以固化。

  2. 分离细胞
    1. 根据制度IACUC协议牺牲每只小鼠。
    2. 将小鼠放在仰卧位的解剖板上,用70%乙醇喷雾,打开腹腔暴露胰腺。
    3. 切除胰腺,并置于〜20毫升的HBSS在6厘米的培养皿在冰上。 旋转洗涤,倾析液体。
    4. 加入5毫升冷HBSS,并用解剖剪刀迅速切碎胰腺
    5. 将胰腺材料转移到15毫升塑料螺旋盖管。 添加100微升10毫克/毫升胶原酶P的组织悬浮液,轻轻混合
    6. 在帽子周围包裹石蜡膜并在37℃振荡器中以225rpm振摇15-20分钟,直到大多数组织块消失并且悬浮液看起来混浊(每5分钟通过眼睛检查以防止过度消化或消化不足 )。
    7. 加入5ml冷HBSS + 5%FBS并在4℃下离心2,000rpm,2分钟。 吸出上清液。
    8. 在5ml冷HBSS + 5%FBS中重新研磨沉淀3次,以1500rpm旋转,漂洗之间2分钟。
    9. 最后一次洗涤后,将沉淀重悬在5ml HBSS + 5%FBS中
    10. 用无菌剪刀,剪切1000微升移液器吸头的尖端,使开口更宽,然后轻轻地吸取细胞悬液通过无菌的500微米网格。
    11. 吸取额外的5毫升HBSS + 5%FBS,以洗涤所有剩余的细胞通过网
    12. 重复步骤:将细胞悬浮液通过105μm网孔
    13. 缓慢吸取细胞悬浮液在含有20ml HBSS + 30%FBS的50ml管的顶部
    14. 离心1,000 rpm,2分钟,4°C颗粒单个腺泡簇。
    15. 吸出上清液。

  3. 电镀细胞
    1. 在3D培养基中以8-10ml(根据胰腺的大小调整体积)重悬细胞沉淀。
    2. 如上步骤A1混合胶原。
    3. 混合等份的细胞悬浮液和胶原蛋白混合物。
    4. 立即将细胞悬液置于胶原包被的孔中,0.5ml /孔(参见图1)

      图1. 3D文化设置

    5. 允许胶原细胞混合物在37℃,5%CO 2下固化约1小时,然后加入1ml温热的3D-培养基(含或不含生长因子抑制剂)。
    6. 在第1天和第3天更换培养基。对于Kras 表达腺泡细胞,在第5天应形成超过90%的导管囊肿(参见图2)。类似地,在铺板后提供50ng/mlTGFα的野生型腺泡细胞在第5天将形成导管囊肿,转化率为约70%-80%(参见图2)(Shi等人。 >,2013)。


      图2. 3D文化中的胰腺腺泡细胞

  4. RNA /蛋白制备从腺泡细胞3D文化
    1. 在1x HBSS(室温)中稀释胶原酶P(10mg/ml)1:50。 在每个样品的15 ml falcon管中制备4 ml溶液
    2. 用1x HBSS洗涤培养基中的细胞/胶原盘一次。 用刮刀将胶原盘转移到15 ml falcon管中。 池3或4井一起获得足够的细胞
    3. 在37℃振荡器中,以250rpm,将胶原蛋白消化约30分钟。 每10或15分钟检查一次。
    4. 在所有胶原消化后,在4℃以2,000rpm离心细胞2分钟
    5. 取出上清液,用1ml冷HBSS重悬细胞,并转移到1.5ml离心管中
    6. 在4℃下以5,000rpm离心2分钟。 小心取出上清液。 通过用指尖轻轻摇动管的底部,松开颗粒。
    7. 选项1:RNA制备。 按照E.Z.N.A. 总RNA迷你Kit协议。 使用均化器柱匀化。 将最终的RNA重悬在40μlddH 2 O中。 使用nanodrop分光光度计测量RNA浓度。 储存于-20°C。
    8. 选项2:蛋白制备。 制备100μl4x样品缓冲液(BME-苯酚蓝)+ 1:50蛋白酶抑制剂混合物+ 1:100磷酸酶抑制剂混合物1 + 1:100磷酸酶抑制剂混合物2 + 1:200 Na 3 VO + sub> 4 。 通过吸取和声波处理细胞15秒。 储存于-20°C。

  5. 胶原盘固定
    1. 在室温下用10%中性缓冲福尔马林在孔中固定胶原盘30分钟,然后使用黄色200μl移液管吸头将盘与板壁分离,并将盘转移到含有10%中性缓冲福尔马林的闪烁瓶中。在室温下在摇床上固定过夜。
    2. 将固定的胶原盘从闪烁瓶转移到标准活检盒中。
    3. 通过在70%,90%和100%EtOH中然后在室温下在玻璃容器中的二甲苯中孵育来逐渐脱水胶原盘,在摇摆平台上对于每种溶液孵育2小时1次。
    4. 在二甲苯清除后,将盒转移到组织处理器的石蜡罐中。让胶原盘经历石蜡培养的最后两个步骤(每个在65℃下1小时),然后将它们保持在石蜡槽中,直到制备石蜡块为止。
      1. 重要事项:保持纸盒水平或者凝胶会"流入"角落,很难嵌入
      2. 胶原盘可以通过自动组织处理器处理。 然而,处理器内部的溶液的新鲜度(清洁度)极大地影响结果(当处理器中的溶液变老时,胶原盘将是硬的并且收缩)。 用小体积的新鲜溶液手动处理将使胶原盘在加工后保持其原始形状

食谱

  1. 10x RPMI 1640培养基
    将1包RPMI 1640粉末溶解在100ml ddH 2 O中 加入2g NaHCO 3,pH为7.2 过滤灭菌
    存储在4°C
    每2个月清新溶液
  2. 4.2%NaHCO 3/v/v 将4.2g NaHCO 3溶解在100ml ddH 2 O中。
    过滤灭菌
    存储在4°C
  3. 胶原酶P
    溶解在10ml ddH 2 O中以制备10mg/ml原液
    过滤灭菌,分装并储存于-80℃ 解冻每个腺泡制备新的小瓶
  4. 100x大豆胰蛋白酶抑制剂(STI)
    溶解在ddH 2 O中以制备10mg/ml原液
    过滤灭菌,分装并储存在-20°C
  5. 2,000x地塞米松(Dex)
    将25mg溶于12.5ml的100%EtOH中以产生2mg/ml原液
    等分并存储在-20°C
  6. 3D培养基
    RPMI 1640
    1%FBS
    1%Pen-Strep
    0.1 mg/ml STI
    1μg/ml Dex

致谢

该协议改编自Shi等人。(2013)。

参考文献

  1. Apte,M。和Wilson,J。(2005)。 保持联系的重要性:调节外分泌胰腺中的细胞 - 细胞接触。 Gut 54(10):1358-1359。
  2. 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。
  3. Rukstalis,J.M.,Kowalik,A.,Zhu,L.,Lidington,D.,Pin,C.L。和Konieczny,S.F。(2003)。 Connexin32的外分泌特异性表达依赖于碱性螺旋 - 环 - 螺旋转录因子Mist1。 a> J Cell Sci   116(16):3315-3325。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Qu, C. and Konieczny, S. F. (2013). Pancreatic Acinar Cell 3-Dimensional Culture. Bio-protocol 3(19): e930. DOI: 10.21769/BioProtoc.930.
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