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MACS Isolation and Culture of Mouse Liver Mesothelial Cells
小鼠肝间皮细胞的MACS分离和培养   

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Abstract

Mesothelial cells (MCs) form a single squamous epithelial cell layer and cover the surfaces of the internal organs, as well as the walls of cavities. The isolation of MCs is of great importance to study their function and characteristics for the understanding of physiology and pathophysiology of the liver. Glycoprotein M6a (GPM6A) was originally identified as a cell surface protein expressed in neurons and recently its expression was reported in epicardium and liver MCs (Wu et al., 2001; Bochmann et al., 2010; Li et al., 2012). Here we describe a method to isolate MCs from the adult mouse liver with anti-GPM6A antibodies. Under the low glucose and serum concentration, primary MCs grow and form epithelial colonies (Figure 1).

Keywords: Antibody(抗体), Epithelial-mesenchymal transition(上皮间质转化), Fibrosis(纤维化), Gpm6a(gpm6a), Pronase(链霉蛋白酶)

Figure 1. Liver MCs 2 days in culture (20x objective)

Materials and Reagents

  1. Dulbecco’s Modified Eagle’s Medium (DMEM) Low glucose with stable L-glutamine (Thermo Fisher Scientific, catalog number: SH30021.01 )
  2. DMEM/F-12 (Thermo Fisher Scientific, catalog number: SH30023.FS )
  3. Fetal Bovine Serum (FBS) (Sigma-Aldrich, catalog number: F9665 )
  4. PBS, pH 7.4 (Sigma-Aldrich, catalog number: P3813-10PAK )
  5. Bovine Collagen Solution, Type I (Advanced BioMatrix, catalog number: 5005-B )
  6. Pronase (Roche, catalog number: 11459643001 )
  7. Antibiotic-Antimycotic (100x) (Life Technologies, catalog number: 15240-062 )
  8. BD Falcon polypropylene conical tube 50 ml (BD Biosciences, catalog number: 352070 )
  9. BD Falcon cell strainer with 70 μm nylon mesh (BD Biosciences, catalog number: 352350 )
  10. Rat anti-mouse glycoprotein M6a (GPM6A) antibodies (MBL International, catalog number: D0553 )
  11. Goat anti-rat IgG microbeads (Miltenyl Biotec, catalog number: 130-048-501 )
  12. Hydrocortisone solution (Sigma-Aldrich, catalog number: H6909-10ML )
  13. Insulin-Transferrin-Selenium-X (Life Technologies, catalog number: 41400-045 )
  14. Ketamine (Clipper Distributing Company, catalog number: NDC57319-542-02 )
  15. 5% low glucose DMEM medium (see Recipes)
  16. MC medium (see Recipes)
  17. Ketamine solution (see Recipes)

Equipment

  1. 37 °C shaker
  2. Surgery Tools: Forceps, scissors and glass petri dish
  3. 37 °C incubator
  4. Centrifuge
  5. 24-well plate (VWR, catalog number: 29442-044 )
  6. G24 Environmental Incubator Shaker (New Brunswick Scientific)
  7. Miltenyi AutoMacs machine

Procedure

I.   Before procedure

  1. Autoclave all surgery tools.
  2. Collagen-coated dishes are used for better attachment of MCs. Mix 50 μl of the collagen solution with 950 μl sterile water and coat the 24-well plate (200 μl per well).
  3. Put the coated dish in 37 °C incubator for at least 30 min, remove the collagen solution, and then wash with PBS 3 times. Make sure each well is completely dry before plating the cell.
  4. Prepare 1 mg/ml Pronase in DMEM/F-12 medium and incubate in 37 °C incubator with gentle stirring for 20 min; Filter the solution with 0.22 μm filter into 50 ml tube before using.
  5. Prepare 5% low glucose DMEM medium.
  6. Prepare MC medium.
  7. Prepare Ketamine solution.
  8. Use 3-5 C57BL/6 male or female mice (20-30 g/each) for MC isolation. Age preference at least 8 weeks old.


II.  Isolation and culture of liver mesothelial cells

  1. Inject Ketamine solution intraperitoneally to mice with 1 ml syringe 23-25 gauge 5/8 inch needle for anesthesia, 0.1 ml per 10 mg of mouse body weight.
  2. Shave the hair and clean up with 70% ethanol, cut the skin, muscle and expose the abdomen cavity.
  3. Remove the gallbladder, cut off the ligaments connected to the liver lobes and diaphragm carefully, take out the liver lobes gently without disturbing the liver surface, and put the liver in 10 cm petri dish washing with sterile PBS.
  4. Transfer the livers in 50 ml tube, add 30 ml DMEM/F-12 and shake at 130 rpm in 37 °C shaker for 5-10 min washing.
  5. Transfer the livers to the tube with 30 ml Pronase/DMEM/F-12 medium and shake at 130 rpm in 37 °C shaker for 20 min. No need to cut the liver into small pieces.
  6. Filter the supernatant containing MCs by BD Falcon cell strainer.
  7. Collect the flow through and add 30 ml 5% low glucose DMEM.
  8. Spin down at 1,700 x g for 5 min at RT.
  9. Discard the supernatant, add 30 ml 5% low glucose DMEM, and spin down at 1,700 x g for 5 min at RT.
  10. Repeat step 9.
  11. Resuspend the pellet with 1.5 ml 5% low glucose DMEM and add 1 μl rat anti-mouse GPM6A antibodies at 1:1,500 dilutions.
  12. Incubate on ice for 15-30 min.
  13. Add 5% low glucose DMEM to 30 ml, spin down at 1,700 x g for 5 min at RT and discard supernatant.
  14. Add 1 ml 5% low glucose DMEM to gently suspend the pellet.
  15. Add 10 μl goat anti-rat IgG microbeads (1 μl/5 x 105 cells) and incubate on ice for 20 min.
  16. Add 5% low glucose DMEM to 30 ml, spin down at 1,700 x g for 5 min at RT and discard supernatant.
  17. Suspend pellet in 1 ml 5% low glucose DMEM and filter with BD Falcon cell strainer.
  18. Use Miltenyi AutoMACS machine to separate the MCs according to their instruction (https://www.miltenyibiotec.com). In brief, the machine will load the cell suspension to the magnetic column and the magnetically labeled GPM6A+ cells will be retained in the column under the magnetic field. After washing, GPM6A+ MCs will be eluted to a new tube.
    Note: Or use Miltenyi magnetic separator to separate MC.
  19. Add 30 ml low glucose DMEM medium and spin down at 1,700 x g for 5 min at RT, discard supernatant.
  20. Add 1 ml MC medium to gently suspend the pellet, count the cell number (yield: about 3 x 104 MCs from 1 mouse) and plate the cells to the wells of the plate (2 x 104 cells per well).
  21. Culture MCs in 5% CO2 at 37 °C incubator, change with MC medium every 3 days. Primary MCs form epithelial colonies and become confluent within 1 week. From 1 week after plating, some MCs start to lose epithelial cell polarity and become fibroblastic cells. Following two passages, neither epithelial nor fibroblastic MCs attach to the dish and survive.

Recipes

  1. 5% low glucose DMEM medium
    Low glucose DMEM
    5% FBS
    1% Antibiotic-Antimycotic
  2. MC medium
    Low glucose DMEM
    5% FBS
    1% Antibiotic-Antimycotic
    Hydrocortisone solution (1:1,000 dilution)
    Insulin-Transferrin-Selenium-X (1:100 dilution)
  3. Ketamine solution
    4.5 ml Ketamine (conc. 100 mg/ml)
    0.75 ml Xylazine (conc. 20 mg/ml)
    19.5 normal saline 0.9%

References

  1. Wu, D. F., Koch, T., Liang, Y. J., Stumm, R., Schulz, S., Schroder, H. and Hollt, V. (2007). Membrane glycoprotein M6a interacts with the micro-opioid receptor and facilitates receptor endocytosis and recycling. J Biol Chem 282(30): 22239-22247.
  2. Bochmann, L., Sarathchandra, P., Mori, F., Lara-Pezzi, E., Lazzaro, D. and Rosenthal, N. (2010). Revealing new mouse epicardial cell markers through transcriptomics. PLoS One 5(6): e11429.
  3. Li, Y., Wang, J. and Asahina, K. (2013). Mesothelial cells give rise to hepatic stellate cells and myofibroblasts via mesothelial-mesenchymal transition in liver injury. Proc Natl Acad Sci U S A 110(6): 2324-2329.

简介

间皮细胞(MCs)形成单个鳞状上皮细胞层,并覆盖内部器官的表面,以及腔壁。 MC的分离对于研究它们的功能和特征对于理解肝脏的生理学和病理生理学是非常重要的。 糖蛋白M6a(GPM6A)最初被鉴定为在神经元中表达的细胞表面蛋白,并且最近在心外膜和肝脏MC中报道了其表达(Wu等人,2001; Bochmann等人, ,2010; Li等人,2012)。 在这里我们描述了一种方法从成年小鼠肝脏用抗GPM6A抗体分离MCs。 在低葡萄糖和血清浓度下,原代MC生长并形成上皮集落(图1)。

关键字:抗体, 上皮间质转化, 纤维化, gpm6a, 链霉蛋白酶

图1.肝脏MCs培养2天(20倍目标)

材料和试剂

  1. Dulbecco改良的Eagle培养基(DMEM)具有稳定的L-谷氨酰胺的低葡萄糖(Thermo Fisher Scientific,目录号:SH30021.01)
  2. DMEM/F-12(Thermo Fisher Scientific,目录号:SH30023.FS)
  3. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F9665)
  4. PBS,pH7.4(Sigma-Aldrich,目录号:P3813-10PAK)
  5. 牛胶原溶液,I型(Advanced BioMatrix,目录号:5005-B)
  6. Pronase(Roche,目录号:11459643001)
  7. 抗生素 - 抗真菌剂(100x)(Life Technologies,目录号:15240-062)
  8. BD Falcon聚丙烯锥形管(BD Biosciences,目录号:352070)
  9. 具有70μm尼龙网(BD Biosciences,目录号:352350)的BD Falcon细胞过滤器
  10. 大鼠抗小鼠糖蛋白M6a(GPM6A)抗体(MBL International,目录号:D0553)
  11. 山羊抗大鼠IgG微珠(Miltenyl Biotec,目录号:130-048-501)
  12. 氢化可的松溶液(Sigma-Aldrich,目录号:H6909-10ML)
  13. 胰岛素 - 转铁蛋白-Selenium-X(Life Technologies,目录号:41400-045)
  14. 氯胺酮(Clipper分销公司,目录号:NDC57319-542-02)
  15. 5%低葡萄糖DMEM培养基(参见Recipes)
  16. MC介质(参见配方)
  17. 氯胺酮溶液(见配方)

设备

  1. 37℃摇床
  2. 手术工具:钳子,剪刀和玻璃培养皿
  3. 37℃孵育器
  4. 离心机
  5. 24孔板(VWR,目录号:29442-044)
  6. G24环境孵化器(新不伦瑞克科学)
  7. Miltenyi AutoMacs机器

程序

I.   手术前

  1. 高压灭菌所有手术工具。
  2. 胶原包被   菜肴用于更好地附着MC。 混合50微升的胶原蛋白   溶液与950微升无菌水和涂布24孔板(200微升 每孔)。
  3. 将包被的培养皿在37°C孵育器至少30分钟,删除胶原溶液,然后用PBS洗3次。 确保每个井完全干燥后再铺设电解池。
  4. 准备   1mg/ml链霉蛋白酶在DMEM/F-12培养基中并在37℃培养箱中孵育 轻轻搅拌20分钟; 用0.22μm过滤器过滤溶液   进入50ml管中。
  5. 制备5%低葡萄糖DMEM培养基
  6. 准备MC培养基。
  7. 准备氯胺酮溶液。
  8. 使用3-5 C57BL/6雄性或雌性小鼠(20-30克/每)进行MC隔离。 年龄偏好至少8周龄。


II。  肝间皮细胞的分离和培养

  1. 注射氯胺酮溶液腹腔注射1毫升注射器小鼠23-25规格5/8英寸针麻醉,0.1毫升每10毫克小鼠体重。
  2. 剃毛,用70%乙醇清理,切皮,肌肉和暴露腹腔。
  3. 取出胆囊,切除与肝叶和隔膜连接的韧带,小心地取出肝叶,轻轻地取出肝叶,而不干扰肝脏表面,并将肝脏置于用无菌PBS洗涤的10cm培养皿中。
  4. 转移肝脏在50ml管,加入30毫升DMEM/F-12,并在130 rpm在37℃振荡器摇动5-10分钟洗涤。
  5. 用30ml链霉蛋白酶/DMEM/F-12培养基将肝脏转移到管中,并在37℃振荡器中以130rpm摇动20分钟。 无需将肝脏切成小块。
  6. 用BD Falcon细胞过滤器过滤含有MC的上清液
  7. 收集流过并加入30 ml 5%低葡萄糖DMEM
  8. 在室温下以1,700英寸×g 旋转5分钟。
  9. 弃去上清液,加入30ml 5%低葡萄糖DMEM,并在室温下以1,700×g离心5分钟。
  10. 重复步骤9.
  11. 用1.5 ml 5%低葡萄糖DMEM重悬沉淀,加入1μl大鼠抗小鼠GPM6A抗体,稀释度为1:1,500。
  12. 在冰上孵育15-30分钟。
  13. 加入5%低葡萄糖DMEM至30ml,在室温下以1,700×g离心5分钟并弃去上清液。
  14. 加入1ml 5%低葡萄糖DMEM,轻轻悬浮沉淀
  15. 加入10μl山羊抗大鼠IgG微珠(1μl/5×10 5个细胞),并在冰上孵育20分钟。
  16. 加入5%低葡萄糖DMEM至30ml,在室温下以1,700×g离心5分钟并弃去上清液。
  17. 将沉淀悬浮在1ml 5%低葡萄糖DMEM中,并用BD Falcon细胞滤器过滤
  18. 使用Miltenyi AutoMACS机器根据其说明分离MC( https://www.miltenyibiotec.com )。简而言之,机器将负载细胞悬浮液到磁性柱,并且磁性标记的GPM6A +细胞将在磁场下保留在柱中。洗涤后,将GPM6A + MCs洗脱到新管中。
    注意:或使用Miltenyi磁性分离器分离MC。
  19. 加入30ml低葡萄糖DMEM培养基,并在室温下以1,700×g离心5分钟,弃去上清液。
  20. 加入1ml MC培养基以轻轻悬浮沉淀,计数细胞数(产量:约1×10 6个MCs),并将细胞平板接种到板的孔中(2×10 6个细胞/sup> 4 细胞/孔)
  21. 在37℃培养箱中培养MCs在5%CO 2中,每3天用MC培养基更换。 原发性MC形成上皮集落,并在1周内变得汇合。 从铺板后1周,一些MC开始丧失上皮细胞极性并成为成纤维细胞。 两次传代后,既没有上皮也没有成纤维细胞MC附着在培养皿上并存活

食谱

  1. 5%低葡萄糖DMEM培养基 低葡萄糖DMEM
    5%FBS
    1%抗生素 - 抗真菌剂
  2. MC介质
    低葡萄糖DMEM
    5%FBS
    1%抗生素 - 抗真菌剂 氢化可的松溶液(1:1,000稀释)
    胰岛素 - 转铁蛋白 - 硒-X(1:100稀释)
  3. 氯胺酮溶液
    4.5ml氯胺酮(浓度100mg/ml) 0.75ml甲苯噻嗪(浓度20mg/ml) 19.5生理盐水0.9%

参考文献

  1. Wu,D.F.,Koch,T.,Liang,Y.J.,Stumm,R.,Schulz,S.,Schroder,H。和Hollt,V。(2007)。 膜糖蛋白M6a与微阿片受体相互作用,促进受体内吞作用和回收。 J Biol Chem 282(30):22239-22247
  2. Bochmann,L.,Sarathchandra,P.,Mori,F.,Lara-Pezzi,E.,Lazzaro,D。和Rosenthal,N。(2010)。 透过转录组学揭示新的小鼠心外膜细胞标记。 PLoS One 5(6):e11429。
  3. Li,Y.,Wang,J.and Asahina,K。(2013)。 间皮细胞通过肝损伤中的间皮 - 间质转化产生肝星状细胞和肌成纤维细胞。 a> Proc Natl Acad Sci USA 110(6):2324-2329。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, Y., Lua, I. and Asahina, K. (2013). MACS Isolation and Culture of Mouse Liver Mesothelial Cells. Bio-protocol 3(13): e815. DOI: 10.21769/BioProtoc.815.
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