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Primary Tumor Preparation
原代肿瘤细胞制备   

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Abstract

This protocol has been developed for culturing primary glioblastoma cells. We have most experience in using it on rodent preparations, but it can also be used in culturing cells from other species.

Materials and Reagents

  1. Phosphate buffered saline (PBS)
  2. Fetal bovine serum (FBS)
  3. Trypsin (Corning, Cellgro®, catalog number: 25-054-CI )
  4. Minimum essential medium (MEM)
  5. HEPES
  6. Sucrose
  7. B104 conditioned media
  8. N2 supplement (Life Technologies, Invitrogen™, catalog number: 17502-048 )
  9. T3 (Sigma-Aldrich, catalog number: T2877 )
  10. Penicillin/streptomycin/amphotericin (Life Technologies, Invitrogen™, catalog number: 15240-062 )
  11. DMEM (Life Technologies, Invitrogen™, catalog number: 11965-167 )
  12. Poly-L-lysine
  13. PDGF-AA (Sigma-Aldrich)
  14. FGFb (Life Technologies, Gibco®)
  15. Basal media (in DMEM) (see Recipes)

Equipment

  1. Mesh (BD Biosciences, Falcon®)
  2. Shaking bath
  3. Centrifuges
  4. 6-well tissue culture plates

Procedure

  1. Perform ex vivo gross total resection of the tumor, shred and mince the tissue in a small amount of PBS.
    Note: Enzymatic and mechanical dissociation followed a modified protocol (Gensert and Goldman., 2001).
  2. Treat the shredded tissue with 11 ml per sample digestive enzyme (1:1,000 dilution of Trypsin 2.5% in MEM containing 20 mM HEPES) for 30 min in a 37 °C shaking bath. After the digestion, filter the dissociated cells through a 70 µm mesh.
  3. Add 5 ml of 10% heat-inactivated FBS to inactivate the trypsin. Centrifuge cells for 10 min at 450 x g and re-suspend in MEM containing 20 mM HEPES and 30% sucrose.
  4. Centrifuge cells for 20 min at 770 g and re-suspend in media.
  5. Plate tumor cells onto 6-well tissue culture plates coated with poly-L-lysine (concentration) at a concentration of 2 million cells per well.
  6. Grow the cells in culture media containing a 2:1 mixture of basal media and B104 conditioned media (Canoll et al., 1996), further supplemented with 10 ng/ml PDGF-AA and 10 ng/ml FGFb.
  7. Basal media contained N2 supplement, 20 ng/ml T3, 0.5% FBS, and penicillin/streptomycin/amphotericin in DMEM.
  8. Collect B104 conditioned media from confluent cultures of the B104 neuroblastoma cell line maintained in basal media for 48 h.

Recipes

  1. Basal media (in DMEM)
    N2 supplement
    20 ng/ml T3
    0.5% FBS
    Penicillin/streptomycin/amphotericin

Acknowledgments

This protocol was developed in Dr. Peter Canoll’s lab at Columbia University, NY, USA. Please cite (Lei et al., 2011) if you use this protocol in your publications.

References

  1. Canoll, P. D., Musacchio, J. M., Hardy, R., Reynolds, R., Marchionni, M. A. and Salzer, J. L. (1996). GGF/neuregulin is a neuronal signal that promotes the proliferation and survival and inhibits the differentiation of oligodendrocyte progenitors. Neuron 17(2): 229-243.
  2. Gensert, J. M. and Goldman, J. E. (2001). Heterogeneity of cycling glial progenitors in the adult mammalian cortex and white matter. J Neurobiol 48(2): 75-86.
  3. Lei, L., Sonabend, A. M., Guarnieri, P., Soderquist, C., Ludwig, T., Rosenfeld, S., Bruce, J. N. and Canoll, P. (2011). Glioblastoma models reveal the connection between adult glial progenitors and the proneural phenotype. PLoS One 6(5): e20041.

简介

这个协议已经开发用于培养原发性胶质母细胞瘤细胞。 我们在啮齿动物制剂中使用它有最多的经验,但它也可以用于培养其他物种的细胞。

材料和试剂

  1. 磷酸盐缓冲盐水(PBS)
  2. 胎牛血清(FBS)
  3. 胰蛋白酶(Corning,Cellgro ,目录号:25-054-CI)
  4. 最低基本培养基(MEM)
  5. HEPES
  6. 蔗糖
  7. B104条件培养基
  8. N2补充剂(Life Technologies,Invitrogen TM,目录号:17502-048)
  9. T3(Sigma-Aldrich,目录号:T2877)
  10. 青霉素/链霉素/两性霉素(Life Technologies,Invitrogen TM,目录号:15240-062)
  11. DMEM(Life Technologies,Invitrogen TM,目录号:11965-167)
  12. 聚-L-赖氨酸
  13. PDGF-AA(Sigma-Aldrich)
  14. FGFb(Life Technologies,Gibco )
  15. 基础培养基(在DMEM中)(参见配方)

设备

  1. Mesh(BD Biosciences,Falcon )
  2. 摇匀浴
  3. 离心机
  4. 6孔组织培养板

程序

  1. 对肿瘤进行体外总切除,切碎并用少量PBS切碎组织。
    注意:酶促和机械解离遵循修改的方案(Gensert和Goldman,2001)。
  2. 用11ml /样品消化酶(1:1000稀释的胰蛋白酶2.5%,在含有20mM HEPES的MEM中)在37℃振荡浴中处理切碎的组织30分钟。消化后,通过70μm网孔过滤解离的细胞
  3. 加入5毫升10%热灭活的FBS灭活胰蛋白酶。在450×g离心细胞10分钟并重悬在含有20mM HEPES和30%蔗糖的MEM中。
  4. 以770g离心细胞20分钟,并重悬在培养基中
  5. 将肿瘤细胞以2×10 6个细胞/孔的浓度涂布在涂有聚-L-赖氨酸(浓度)的6孔组织培养板上。
  6. 在含有基础培养基和B104条件培养基(Canoll等,1996)的2:1混合物的培养基中生长细胞,进一步补充有10ng/ml PDGF-AA和10ng/ml FGFb。
  7. 基础培养基在DMEM中含有N2补充物,20ng/ml T3,0.5%FBS和青霉素/链霉素/两性霉素。
  8. 从维持在基础培养基中的B104神经母细胞瘤细胞系的汇合培养物中收集B104条件培养基48小时。

食谱

  1. 基础培养基(在DMEM中)
    N2补助
    20 ng/ml T3
    0.5%FBS
    青霉素/链霉素/两性霉素

致谢

该协议由Peter Canoll博士在美国纽约哥伦比亚大学的实验室开发。如果您在出版物中使用此协议,请引用(Lei 等人,2011)。

参考文献

  1. Canoll,P.D.,Musacchio,J.M.,Hardy,R.,Reynolds,R.,Marchionni,M.A。和Salzer,J.L。(1996)。 GGF /神经调节蛋白是促进增殖和存活并抑制少突胶质细胞祖细胞分化的神经元信号。 神经元 17(2):229-243
  2. Gensert,J.M。和Goldman,J.E。(2001)。 成年哺乳动物皮质和白质中循环的胶质祖细胞的异质性。 J Neurobiol 48(2):75-86
  3. Lei,L.,Sonabend,A.M.,Guarnieri,P.,Soderquist,C.,Ludwig,T.,Rosenfeld,S.,Bruce,J.N.and Canoll, 胶质母细胞瘤模型揭示成人胶质祖细胞与易发表型之间的关系。 PLoS One 6(5):e20041。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lei, L. (2012). Primary Tumor Preparation. Bio-protocol 2(12): e229. DOI: 10.21769/BioProtoc.229.
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