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Mouse Corneal Stroma Fibroblast Primary Cell Culture
小鼠角膜基质成纤维细胞原代培养   

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Abstract

This protocol is developed for primary cell culture of cornea stromal keratocytes isolated from neonatal mouse eyeballs. It provides an optimal condition to isolate stromal keratocytes which maintain high viability for cell culture.

Keywords: Mouse(小鼠), Corneal stroma(角膜基质), Primary culture(原代培养)

Materials and Reagents

  1. 15 ml Falcon tube (Corning, Falcon®, catalog number: 352095 )
  2. Petri dish (Thermo Fisher Scientific, Fisher Scientific, catalog number: FB0875713 )
  3. NunclonTM tissue culture dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 172931 )
  4. New born mice (postnatal day 0)
  5. 70% sterile ethanol (prepared from ethanol 190 proof) (Decon Labs, catalog number: 2801 )
  6. Distilled water (Thermo Fisher Scientific, GibcoTM, catalog number: 15230162 )
  7. PBS without Ca2+ Mg2+ (Thermo Fisher Scientific, GibcoTM, catalog number: 20012050 )
  8. Trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200072 )
  9. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 11995-065 )
  10. Fetal bovine serum (GE Healthcare, HycloneTM, catalog number: SH30396.03 )
  11. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  12. DMSO (Sigma-Aldrich, catalog number: D2650 )
  13. Liquid nitrogen
  14. Dispase II (Roche Diagnostics, catalog number: 04942078001 )
  15. Sorbitol
  16. Collagenase (Type L) (Sigma-Aldrich, catalog number: C8170 ), sterile-filtered, dissolved in PBS and stored at -20 °C. 

  17. Hyaluronidase
  18. Disapse solution (see Recipes)
  19. Digestion buffer (see Recipes)

Equipment

  1. CO2 cabinet
  2. Chemical hood
  3. Tissue culture hood
  4. Phase-contrast inverted microscope (Electron Microscopy Sciences)
  5. Spring scissor (Fine Science Tools, catalog number: 15009-08 )
  6. Forceps (Fine Science Tools, catalog number: 00108-11 )
  7. Benchtop centrifuge (Hettich Lab Technology, model: Rotina 380 )
  8. Pipettes
  9. Hemocytometer (Hausser Scientific)

  10. CO2 incubator

Procedure

Note: All materials used in this experiment must be sterile or autoclaved to prevent contamination.

  1. Isolation of mouse corneal stroma keratocytes
    1. Euthanize five newborn pups using CO2 cabinet in a chemical hood. Briefly rinse the bodies in 70% (v/v) ethanol and PBS successively. Then place the eyeballs into a Petri dish and transfer it to a tissue culture hood.

    2. Dissect out eyeballs from each pup. Carefully cut corneas along the sclera rim using a surgical scissor and place all corneas in 15 ml Falcon tube with 10 ml of dispase solution at 4 °C in a horizontal orientation (Video 1).

      Video 1. Isolation of mouse corneas from newborn pups under a stereo microscope. Mouse corneas are carefully cut along the sclera rim using a surgical scissor.

    3. Transfer the tissues together with the dispase solution into a Petri dish (Video 1).
    4. Peel off and collect the loosened corneal epithelial sheets with forceps which can be cultured separately (Video 2).

      Video 2. Dissection of corneal stoma tissues from dispase-treated cornea. Under a stereo microscope, corneal epithelial sheets are gradually stripped off from dispase digested mouse corneas and then collect corneal stroma tissues for cell culture.

    5. Rinse the tissues in 10 ml PBS for two times to wash away dispase. Transfer tissues to a new 15 ml tube with 2 ml digestion buffer and incubate the tube at 37 °C with shaking (125 rpm) for 30 min.
    6. Spin for 5 min at 300 x g in a centrifuge at room temperature; carefully decant and discard supernatant. Wash once with 5 ml PBS by mixing thoroughly and centrifuge as above, discarding the supernatant. 

    7. Add 0.5 ml trypsin-EDTA, mix thoroughly, and incubate at 37 °C for 20 min. Then centrifuge and discard supernatant as above (step A6), resuspend pellet in 0.5 ml DMEM medium containing 10% fetal bovine serum. Using 1 ml pipette, pipet up and down several times to break up cell aggregates.

  2. Primary culture
    1. Plate the suspension from step A7 onto one 6 cm tissue culture dish, avoiding tissue mass.
    2. Add 3 ml DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and grow the cells for 48 h at 37 °C in a CO2 incubator with 5% CO2. Next, replace the old growth medium with 3 ml DMEM containing 10% FBS without antibiotics every 3 days. Within 2 weeks from the time of establishment of the culture, confluent monolayers will form, displaying the typical fibroblast morphology (Figure 1).


      Figure 1. The morphology of mouse corneal fibroblast cultured for one (A) and two (B) weeks. Scale bar = 100 μm.

  3. Subculture
    1. At 80-90% confluency, aspirate the medium and rinse the cells with PBS. Add 0.5 ml trypsin-EDTA to trypsinize the cells in the culture plate and incubate it at 37 °C for 5 min to release cells from the culture plate. Next, add 5 ml DMEM + 10% FBS to stop the reaction.
    2. Collect the cells with medium into a 15 ml tube. Spin down the cells at slow speed (300 x g for 5 min). Discard the supernatant, resuspend the cells in DMEM culture medium and count the cells using a hemocytometer. Next, plate the cells at a density of 1 x 104 cells/cm2 onto a plate containing DMEM + 10% FBS medium without antibiotics. Incubate the culture in a CO2 incubator at 37 °C and 5% CO2. 

    3. Change culture medium every 2-3 days and subculture the cells when 80-90% confluence.

  4. Cryopreservation


    Cryopreservation is necessary to maintain large quantities of cells from the same tissue samples for future experiments.
    1. Release cells using trypsin-EDTA and centrifuge as for the subculture (step C1). 

    2. Collect the cells with medium into a 15 ml tube. Spin down the cell at slow speed (300 x g for 5 min). Discard the supernatant, resuspend cells in DMEM culture medium and count the cells using a hemocytometer. 

    3. Dispense aliquots of 2 x 106 cells/ml in culture medium with 10% DMSO. 

    4. Store cells at -80 °C freezer for 24 h and then transfer cells to liquid nitrogen for long-term storage.
    5. To recover cells: thaw cells quickly in a 37 °C water bath and dilute cells tenfold with DMEM medium without antibiotics. Then subculture cells as above (step C).

Recipes

  1. Dispase solution
    Prepare 15 mg/ml dispase II, 100 mM sorbitol and 1% penicillin-streptomycin in DMEM basal medium.
    Store at -20 °C.
  2. Digestion buffer
    Prepare 2.0 mg/ml collagenase and 0.5 mg/ml hyaluronidase in DMEM basal medium.
    Store at -20 °C.

Acknowledgments

This work was supported by grant from the National Institute of Health/National Eye Institute (NIH/NEI) [RO1 EY21501, RO1 EY23086].

References

  1. Seluanov, A., Vaidya, A. and Gorbunova, V. (2010). Establishing primary adult fibroblast cultures from rodents. J Vis Exp (44).
  2. Zhang, Y., Yeh, L. K., Zhang, S., Call, M., Yuan, Y., Yasunaga, M., Kao, W. W. and Liu, C. Y. (2015). Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development. Development 142(19): 3383-3393.

简介

该协议是为从新生小鼠眼球分离的角膜基质角膜细胞的原代细胞培养而开发的。 它提供了一个最佳条件,以隔离基质角膜细胞,保持细胞培养的高生存力。

关键字:小鼠, 角膜基质, 原代培养

材料和试剂

  1. 15ml Falcon管(Corning,Falcon ,目录号:352095)
  2. 培养皿(Thermo Fisher Scientific,Fisher Scientific,目录号:FB0875713)
  3. Nunclon TM组织培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:172931)
  4. 新生小鼠(出生后第0天)
  5. 70%无菌乙醇(由乙醇190制备)(Decon Labs,目录号:2801)
  6. 蒸馏水(Thermo Fisher Scientific,Gibco TM ,目录号:15230162)
  7. 没有Ca 2+的PBS(Thermo Fisher Scientific,Gibco< sup> TM,目录号:20012050)的PBS。</sup>
  8. 胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM ,目录号:25200072)
  9. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:11995-065)
  10. 胎牛血清(GE Healthcare,Hyclone ,目录号:SH30396.03)
  11. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM ,目录号:15140122)
  12. DMSO(Sigma-Aldrich,目录号:D2650)
  13. 液氮
  14. Dispase II(Roche Diagnostics,目录号:04942078001)
  15. 山梨醇
  16. 胶原酶(类型L)(Sigma-Aldrich,目录号:C8170),无菌过滤,溶于PBS中并储存在-20℃。
  17. 透明质酸酶
  18. Disapse解决方案(请参阅配方)
  19. 消化缓冲液(参见配方)

设备

  1. CO 2 机柜
  2. 化学发动机罩
  3. 组织培养罩
  4. 相差倒置显微镜(Electron Microscopy Sciences)
  5. 弹簧剪(Fine Science Tools,目录号:15009-08)
  6. 镊子(Fine Science Tools,目录号:00108-11)
  7. 台式离心机(Hettich Lab Technology,型号:Rotina 380)
  8. 移液器
  9. 血细胞计数器(Hausser Scientific)
  10. CO <2>孵化器

程序

注意:本实验中使用的所有材料必须无菌或高压灭菌,以防止污染。

  1. 小鼠角膜基质角膜细胞的分离
    1. 使用CO 2 sub橱柜在化学罩安乐死五个新生小狗。简单地在70%(v/v)乙醇和PBS中冲洗身体连续。然后将眼球放入培养皿中,转移到组织培养罩。
    2. 从每只小狗解剖眼球。使用手术剪刀小心沿着巩膜边缘切割角膜,并将所有角膜放置在15 ml Falcon管中,在4°C水平方向(视频1)与10 ml分散酶溶液。

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      视频1.在立体显微镜下分离新生小鼠的小鼠角膜。 使用手术剪刀沿着巩膜边缘小心地切割小鼠角膜。
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    3. 将组织与分散酶溶液一起转移到培养皿中(视频1)。
    4. 剥去并收集松开的角膜上皮板与镊子,可以单独培养(视频2)。

      <! - flashid1960v99开始 - >
      视频2.来自分散治疗角膜的角膜造口组织的解剖。 在立体显微镜下,角膜上皮片层逐渐从分散酶消化的小鼠角膜剥离,然后收集角膜基质组织用于细胞培养。
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    5. 冲洗组织在10ml PBS两次,以洗去分散。转移组织到一个新的15毫升管与2毫升消化缓冲液,并孵育管在37℃下摇动(125 rpm)30分钟。
    6. 在室温下在离心机中以300×g离心5分钟;小心倾析并弃去上清液。用5ml PBS通过充分混合并如上离心,洗涤一次,弃去上清液。
    7. 加入0.5ml胰蛋白酶-EDTA,充分混合,并在37℃下孵育20分钟。然后如上所述离心并弃去上清液(步骤A6),将沉淀重悬于含有10%胎牛血清的0.5ml DMEM培养基中。使用1毫升吸管,吸上下数次,打破细胞聚集。

  2. 小学文化
    1. 将来自步骤A7的悬浮液铺在一个6cm组织培养皿上,避免组织块
    2. 加入3ml补充有10%胎牛血清和1%青霉素 - 链霉素的DMEM,并在37℃下在具有5%CO 2的CO 2培养箱中培养细胞48小时, sub>。接下来,每3天更换旧生长培养基与3毫升含有10%FBS的DMEM无抗生素。在建立培养物的2周内,将形成汇合的单层,显示典型的成纤维细胞形态(图1)。


      图1.在(A)和(B)周培养的小鼠角膜成纤维细胞的形态。比例尺=100μm。
  3. 亚文化
    1. 在80-90%汇合时,吸出培养基并用PBS冲洗细胞。加入0.5ml胰蛋白酶-EDTA,胰蛋白酶处理培养板中的细胞,并在37℃孵育5分钟从培养板中释放细胞。接下来,加入5ml DMEM + 10%FBS以终止反应
    2. 收集细胞用培养基到15毫升管。以低速(300×g)旋转细胞5分钟。弃去上清液,将细胞重悬于DMEM培养基中,并使用血细胞计数器计数细胞。接下来,以1×10 4个细胞/cm 2的密度将细胞平板于含有不含抗生素的DMEM + 10%FBS培养基的平板上。在CO 2培养箱中在37℃和5%CO 2下培养该培养物。
    3. 每2-3天更换培养基,80-90%汇合时传代培养细胞
  4. 冷冻保存
    冷冻保存对于维持来自相同组织样品的大量细胞用于将来实验是必要的
    1. 使用胰蛋白酶-EDTA释放细胞并离心用于传代培养(步骤C1)。
    2. 收集细胞用培养基到15毫升管。以低速(300×g)旋转细胞5分钟。弃去上清液,将细胞重悬于DMEM培养基中,并使用血细胞计数器计数细胞。
    3. 在具有10%DMSO的培养基中分配2×10 6个细胞/ml的等分试样。
    4. 存储细胞在-80°C冰箱24小时,然后将细胞转移到液氮长期储存。
    5. 回收细胞:在37℃水浴中迅速解冻细胞,用不含抗生素的DMEM培养基将细胞稀释十倍。然后如上所述传代培养细胞(步骤C)

食谱

  1. 分散溶液
    在DMEM基础培养基中制备15mg/ml分散酶II,100mM山梨醇和1%青霉素 - 链霉素。
    储存于-20°C。
  2. 消化缓冲区
    在DMEM基础培养基中制备2.0mg/ml胶原酶和0.5mg/ml透明质酸酶。
    储存于-20°C。

致谢

这项工作得到国家卫生研究所/国家眼科研究所(NIH/NEI)[RO1 EY21501,RO1 EY23086]的资助。

参考文献

  1. Seluanov,A.,Vaidya,A.和Gorbunova,V。(2010)。  从啮齿动物建立初级成年成纤维细胞培养物。(44)。
  2. Zhang,Y.,Yeh,LK,Zhang,S.,Call,M.,Yuan,Y.,Yasunaga,M.,Kao,WW and Liu,CY(2015)。  Wnt /β-catenin信号通过在小鼠发育期间抑制Bmp4来调节角膜上皮分层。 142(19):3383-3393
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, Y., Wang, Y., Yuka, O., Zhang, L. and Liu, C. (2016). Mouse Corneal Stroma Fibroblast Primary Cell Culture. Bio-protocol 6(19): e1960. DOI: 10.21769/BioProtoc.1960.
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