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Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors
小鼠乳腺肿瘤上皮细胞的分离   

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Abstract

The isolation of cancer epithelial cells from mouse mammary tumor is accomplished by digestion of the solid tumor. Red blood cells and other contaminates are removed using several washing techniques such that primary epithelial cells can further enriched. This procedure yields primary tumor cells that can be used for in vitro tissue culture, fluorescence-activated cell sorting (FACS) and a wide variety of other experiments (Lo et al., 2012).

Materials and Reagents

  1. Bovine serum albumin (Sigma-Aldrich, catalog number: A9576 )
  2. Sterile Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: D8537 )
  3. NH4Cl solution (StemCell Technologies, catalog number: 0 7850 )
  4. Scalpel or Razor Blade
  5. EasySep Negative Selection Kit (StemCell Technologies, catalog number: 19757 )
  6. Accutase (ITC, catalog number: AT104 )
  7. Collagenase (StemCell Technologies, catalog number: 0 7912 )
  8. Penicillin/Streptomycin (Life Technologies, InvitrogenTM, catalog number: 10378-016 )
  9. Dulbecco’s Modified Eagle Medium/F12 (Sigma-Aldrich, catalog number: D8437 )

Equipments

  1. Swing bucket centrifuge
  2. Incubated shaker
  3. EasySep Magnet (StemCell Technologies, catalog number: 18000 )
  4. Sterile Nylon Mesh 40 micrometer cell strainer (BD Biosciences, Falcon®, catalog number: 352340 )
  5. 50 ml conical tube

Procedure

  1. Sacrifice or anesthetize the animal and restrain the animal by pinning its feet into a foam surface using pushpins. Make a parallel incision from the base of the tail up to the neck along the mouse’s abdomen, careful not to puncture the peritoneum. Gently pull back the skin and pin it to the foam surface to reveal the tumor(s). Cut the tumor free from the skin and the body, carefully removing as much excess tissue surrounding the tumor as possible.
  2. Store the harvested tumors in serum-free DMEM/F12 medium on ice until the digestion procedure.
    Notes: Tumors should be digested as soon as possible after removal. We would not recommend more than 1 h on ice.
  3. In a 10 cm Petri dish, mince the tumor (0.5-1.0 mg or more dependent on experimental needs) into 1-2 mm pieces using a razor or scalpel and place into a 50 ml conical tube. This should be performed in a biological cabinet at room temperature. A small amount of digestion medium can be added to the tumor to make mincing easier. It is not essential to mince the cells on ice. The tumors should be minced efficiently but not in a rush so as to leave pieces that are too large.
  4. Add the digestion medium to the 50 ml conical tube in a 10:1 ratio to the volume of the tumor such that there is approximately ten times more digestion medium in the tube than tumor.
  5. Place the tube securely onto an incubated shaker in an inclined position such that the medium and tumor are constantly mixing. Digest the minced tumors for 3-5 h at 37 °C. Though some pieces of tumor may remain, check that most of the tumor has been dissolved for thorough digestion.
  6. Dilute the digested mixture with DMEM/F12 containing 0.2% BSA at a 1:1 ratio with the digested tumor. This will lead to a doubling of the volume.
  7. Filter the mixture through a 40 micrometer sterile nylon mesh cell strainer into another sterile 50 ml conical tube. From this point forward, keep cells on ice at all times except during centrifugation and digestion with Accutase.
  8. Spin down the filtered cells at 200 x g for 5 min and remove supernatant.
  9. Resuspend the cells in an appropriate volume of DMEM/F12-0.2% BSA to wash (~1 ml per ml cell pellet).
  10. Spin down the cells again at 200 x g for 5 min and remove supernatant.
  11. In order to lyse red blood cells contaminating the pelleted cells, mix 4 ml NH4Cl and 1 ml PBS-1.0% BSA (4:1 ratio) first and use this mixture to resuspend the pellet (these calculations are appropriate for a pellet that occupies a volume of approximately 5 ml. The amount of NH4Cl and PBS-1.0% BSA should be adjusted for the size of the pellet with the ratio remaining 4:1). Leave the cells in the solution for 2-3 min at room temperature depending on the amount of RBC contamination, which can be determined by the presence of red color. If the tumor is well-vascularized and the pellet is very red in color, repeat the procedure until the red color diminishes.
  12. Spin down the cells at 200 x g for 5 min and remove supernatant.
  13. Wash the cell pellet in DMEM/F12-0.2% BSA and spin down cells. Repeat this step several times until the supernatant becomes clear.
  14. If cells are clumped together during medium washing, digest the 1-2 x 106 cells with 1 ml Accutase for 10 min at 37 °C to dissociate aggregated cells.
  15. Add 2 ml DMEM/F12-0.2% BSA per 1 ml Accutase and mix by pipetting to neutralize the reaction of the enzyme.
  16. Spin down cells at 200 x g for 5 min and wash cells using PBS.
  17. Remove supernatant and suspend in an appropriate volume of PBS for the pellet size (~1 ml per ml of pellet present). Store cells on ice until isolation.
  18. Lineage marker negative (Lin) epithelial cells from mammary glands can then be isolated using the EasySep negative selection kit according to the manufacturer’s instructions. This will remove unwanted cells by targeting these cells with antibodies against non-epithelial cells. The Lineage marker positive (Lin+), non-epithelial cells are attracted to the magnet required for use with the EasySep negative selection kit and desired cells remain free to be collected. This yields a purified epithelial sample for use in other experiments.
  19. Isolated cells can be kept on ice for at least 1 h until they are used for experimental analyses. Isolate cells can also be kept in appropriate cell freezing medium and then stored at -80 °C or in liquid nitrogen for long-term storage.

Recipes

  1. Tumor Digestion Medium (50 ml)
    25 ml Accutase
    50 mg Collagenase
    0.5 ml 100x Penicillin/Streptomycin
    24.5 ml Dulbecco’s Modified Eagle Medium/F12

Acknowledgments

This protocol was first described in our manuscript Lo et al. (2012). This work was supported by the Elsa U. Pardee Cancer Foundation grant (B94AFFAA), the American Cancer Society Research Award (RSG-10-067-01-TBE) to HC and NIH grant (3P20RR017698-08) to HC and QW.

References

  1. Lo, P. K., D. Kanojia, X. Liu, U. P. Singh, F. G. Berger, Q. Wang and H. Chen (2012). CD49f and CD61 identify Her2/neu-induced mammary tumor-initiating cells that are potentially derived from luminal progenitors and maintained by the integrin-TGFbeta signaling. Oncogene 31(21): 2614-2626.

简介

通过消化实体瘤完成从小鼠乳腺肿瘤中分离癌上皮细胞。 使用几种洗涤技术除去红细胞和其它污染物,使得原代上皮细胞可以进一步富集。 该程序产生可用于体外组织培养,荧光激活细胞分选(FACS)和各种其它实验的原发性肿瘤细胞(Lo等人, ,2012)。

材料和试剂

  1. 牛血清白蛋白(Sigma-Aldrich,目录号:A9576)
  2. 无菌Dulbecco's磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:D8537)
  3. NH 4 Cl溶液(StemCell Technologies,目录号:07850)
  4. 手术刀或剃刀刀片
  5. EasySep Negative Selection Kit(StemCell Technologies,目录号:19757)
  6. Accutase(ITC,目录号:AT104)
  7. 胶原酶(StemCell Technologies,目录号:07912)
  8. 青霉素/链霉素(Life Technologies,Invitrogen TM ,目录号:10378-016)
  9. Dulbecco's Modified Eagle Medium/F12(Sigma-Aldrich,目录号:D8437)

设备

  1. 回转桶离心机
  2. 孵育摇床
  3. EasySep磁体(StemCell Technologies,目录号:18000)
  4. 无菌尼龙网40微米细胞滤器(BD Biosciences,Falcon ,目录号:352340)
  5. 50ml锥形管

程序

  1. 牺牲或麻醉动物,并通过使用图钉将其脚钉入泡沫表面来约束动物。沿着小鼠的腹部从尾根部到颈部做一个平行的切口,小心不要刺破腹膜。轻轻拉回皮肤,并将其固定到泡沫表面以显露肿瘤。切除肿瘤免于皮肤和身体,仔细除去尽可能多的肿瘤周围的多余组织
  2. 将收获的肿瘤存储在无血清DMEM/F12培养基中的冰上,直到消化程序。
    注意:移除肿瘤后应尽快消化。我们不会在冰上推荐超过1小时。
  3. 在10cm培养皿中,使用剃刀或解剖刀将肿瘤(0.5-1.0mg或更多取决于实验需要)切成1-2mm片,并放入50ml锥形管中。这应在室温下在生物柜中进行。可以向肿瘤中加入少量消化介质以使切碎更容易。在冰上剁碎细胞不是必要的。肿瘤应该有效地切碎,但不要急于取出,以留下太大的碎片
  4. 将消化介质以50:1的比例与肿瘤体积比例加入到50 ml锥形管中,使得管中的消化介质比肿瘤大约多十倍。
  5. 将管牢固地放置在倾斜位置的培养摇床上,使培养基和肿瘤不断混合。在37℃下消化切碎的肿瘤3-5小时。虽然一些肿瘤可能残留,但检查大多数肿瘤已经溶解完全消化
  6. 用消化的肿瘤以1:1的比例用含有0.2%BSA的DMEM/F12稀释消化的混合物。这将导致音量翻倍。
  7. 通过40微米无菌尼龙网格细胞过滤器将混合物过滤到另一个无菌的50ml锥形管中。从这一点开始,除了在离心和用Accutase消化之前,始终将细胞保持在冰上
  8. 在200 x 下旋转过滤的细胞5分钟,除去上清液。
  9. 将细胞重悬在适当体积的DMEM/F12-0.2%BSA中以洗涤(〜1ml/ml细胞沉淀)。
  10. 再次以200 x em/g 再次旋转细胞5分钟,除去上清液。
  11. 为了裂解污染沉淀的细胞的红细胞,首先混合4ml NH 4 Cl和1ml PBS-1.0%BSA(4:1比率),并使用该混合物重悬沉淀物(这些计算对于占据大约5ml体积的沉淀是合适的。应当针对颗粒的尺寸调整NH 4 Cl和PBS-1.0%BSA的量,其中比例保持4:1 )。离开细胞在溶液中2-3分钟在室内 温度取决于RBC污染的量,其可以通过存在红色来确定。 如果肿瘤血管充分,血小板颜色非常红,则重复该步骤,直到红色消失。
  12. 将细胞以200×em/g 离心5分钟,除去上清液。
  13. 在DMEM/F12-0.2%BSA中洗涤细胞沉淀并离心细胞。 重复该步骤几次,直到上清液变得澄清。
  14. 如果在培养基洗涤期间细胞团聚在一起,在37℃下用1ml Accutase消化1-2×10 6个细胞10分钟以解离聚集的细胞。
  15. 每1ml Accutase加入2ml DMEM/F12-0.2%BSA,并通过吸移混合以中和酶的反应。
  16. 将细胞以200 x em/g 离心5分钟,然后用PBS洗涤细胞。
  17. 除去上清液并悬浮在适当体积的PBS中用于颗粒大小(每ml存在的沉淀约1ml)。将细胞储存在冰上直到隔离。
  18. 然后可以使用EasySep阴性选择试剂盒根据制造商的说明书分离来自乳腺的谱系标记物阴性(Lin - )上皮细胞。这将通过用针对非上皮细胞的抗体靶向这些细胞来去除不想要的细胞。谱系标记物阳性(Lin +),非上皮细胞被吸引到与EasySep阴性选择试剂盒一起使用所需的磁体,并且期望的细胞保持游离以被收集。这产生用于其他实验的纯化上皮样品
  19. 分离的细胞可以在冰上保持至少1小时,直到它们用于实验分析。分离细胞也可以保存在适当的细胞冷冻培养基中,然后储存在-80℃或液氮中长期储存。

食谱

  1. 肿瘤消化培养基(50ml)
    25 ml Accutase
    50 mg胶原酶
    0.5ml 100x青霉素/链霉素 24.5ml Dulbecco's Modified Eagle Medium/F12

致谢

该方案首先在我们的手稿Lo 等人(2012)中描述。 这项工作得到了Elsa U. Pardee癌症基金会资助(B94AFFAA),美国癌症协会研究奖(RSG-10-067-01-TBE)到HC和NIH资助(3P20RR017698-08)到HC和QW的支持。

参考文献

  1. Lo,P.K.,D.Kanojia,X.Liu,U.P.Singh,F.G.Berger,Q.Wang和H.Chen(2012)。 CD49f和CD61可识别Her2/neu诱导的乳腺肿瘤发生 细胞,其潜在地来源于内腔祖细胞并通过整联蛋白-TGFβ信号传导维持。 Oncogene 31(21):2614-2626
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Johnson, S., Chen, H. and Lo, P. (2013). Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors. Bio-protocol 3(3): e326. DOI: 10.21769/BioProtoc.326.
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