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Primary Culture of SVZ-derived Progenitors Grown as Neurospheres
室管膜下区(SVZ)源祖细胞生长为神经球的初代培养   

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Abstract

SVZ-derived progenitors grown as neurospheres is a well-known model to study neural stem cell and progenitor functions such as proliferation, differentiation/self-renewal, and migration (Durbec and Rougon, 2001). This protocol is for preparing a culture of SVZ-derived progenitors from 8 early postnatal mouse brains (P0 to P3). One week after cell plating, we can observe round floating neurospheres, each resulting from the clonal expansion of a single EGF/FGF responsive neural progenitor.

Keywords: Nervous system(神经系统), Adult brain(成人的大脑), Stem cells(干细胞), Subventricular zone(室管膜下区), Primary culture(原代培养)

Materials and Reagents

  1. New born mice
  2. Phosphate Buffered Saline (PBS) (Life Technologies, catalog number: 14040-091 )
  3. Hank's Balanced Salt Solution (HBSS) (Life Technologies, catalog number: 14170-088 )
  4. Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, catalog number: 61965-026 )
  5. Ham’s F-12 nutrient mix (F12) (Life Technologies, catalog number: 31765-027 )
  6. Trypsin (Sigma-Aldrich, catalog number: T5266 )
  7. Insulin (Sigma-Aldrich, catalog number: I1882 )
  8. Holo-transferrin (Sigma-Aldrich, catalog number: T0665 )
  9. Putrescine (Sigma-Aldrich, catalog number: P5780 )
  10. Progesterone (Sigma-Aldrich, catalog number: P8783 )
  11. Sodium selenite (Selenium) (Sigma-Aldrich, catalog number: S5261 )
  12. Penicillin-streptomycin (Life Technologies, catalog number: 15140-130 )
  13. Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10106-169 )
  14. B27 (Life Technologies, catalog number: 17504-044 )
  15. rhFGFbasic (Peprotech, catalog number: 167 100-18B-B )
  16. rhEGF (Peprotech, catalog number: 167 AF-100-15A )
  17. Neurospheres defined medium (see Recipes)
  18. Trypsin (see Recipes)

Equipment

  1. Sterilin Universal Container (Thermo Fisher Scientific, catalog number: 128A/P )
  2. Standard TC BD Falcon 60 mm cell culture dish (BD Biosciences, Falcon®, catalog number: 353002 )
  3. Vibratom (Microm, model: HM450 )
  4. Stereoscopic Microscope
  5. Cell culture Hood and Incubator
  6. Scissors, fine forceps, spatula, micro knives
  7. Ice bucket
  8. Fire-polished glass Pasteur pipettes
  9. Water bath

Procedure

  1. Dissect the brain from new born mice.
  2. Cut 400 μm thick coronal sections of the brain in ice cold PBS with a Vibratom (Figure 1A) (from the olfactory bulb to the anterior horn of the lateral ventricle). Keep the first two sections of each brain containing the lateral wall of the lateral ventricles and place them in ice cold HBSS.


    Figure 1. Dissection of the neonatal mouse SubVentricular Zone. A. Illustration of a newborn mice sagittal section showing the cut window when using the vibratome. B. Coronal section showing parts to dissect to isolate lateral ventricules’ tissues. C. Neurospheres obtained after 1 week long culture. Scale bar, 500 μm.

  3. Under the microscope, dissect out the lateral walls of the lateral ventricles (Figure 1B). Take a forceps with one hand to handle slice and keep it on the bottom of the dish, and a micro knife with the other to cut out the zone of interest. Discard the rest of the slice after each dissection.
  4. Cut the tissues into small cubes (400 μm cubes) using sterile micro knives.
  5. Pipette the tissues within a volume of 800 μl HBSS and place them in a 30 ml Sterilin Universal Container. Add 200 μl of trypsin 12.5 mg/ml. Incubate 5 min at 37 °C in a water bath.
  6. Add 10 volumes of 10% HBSS-FBS. Using fire-polished glass Pasteur pipettes, gently pipet up and down to help the dissociation. Take of a droplet regularly and check under the microscope whether the dissociation is complete.
  7. Centrifuge at 800 x g for 7 min at room temperature (RT), discard the supernatant, and resuspend the pellet in 10 ml of fresh HBSS. Take of a sample and count the number of cells.
  8. Centrifuge the cell suspension at 800 x g for 7 min at RT, discard the supernatant, and resuspend in Standard TC BD Falcon 60 mm cell culture dishes at the concentration of 25,000 cells/ml in neurospheres defined medium, supplemented with 2% B27, bFGF (20 ng/ml) and EGF (20 ng/ml).
  9. Every 3 days, add a half-volume of doubly-supplemented [4% B27, bFGF (40 ng/ml) and EGF (40 ng/ml)] neurospheres defined medium. To avoid any sphere attachment to the bottom of the culture dish, don’t extend the culture beyond 1 week (Figure 1C).

Recipes

  1. Neurospheres defined medium
    DMEM/F12, 3:1 volumes respectively
    5 g/ml Insulin
    100 g/ml Holo-transferrin
    100 M Putrescine
    20 nM Progesterone
    30 nM Selenium
    1% Penicillin-streptomycin (100 IU/ml and 100 g/ml, respectively)
  2. Trypsin (prepare each time a new aliquot)
    Dissolve 12.5 mg of Trypsin in 1 ml of ice cold HBSS

Acknowledgments

This protocol is adapted from Durbec and Rougon (2001) and Vernerey et al. (2013).

References

  1. Durbec, P. and Rougon, G. (2001). Transplantation of mammalian olfactory progenitors into chick hosts reveals migration and differentiation potentials dependent on cell commitment. Mol Cell Neurosci 17(3): 561-576.
  2. Vernerey, J., Macchi, M., Magalon, K., Cayre, M. and Durbec, P. (2013). Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain. J Neurosci 33(7): 3240-3250.

简介

作为神经球生长的SVZ衍生的祖细胞是研究神经干细胞和祖细胞功能如增殖,分化/自我更新和迁移的众所周知的模型(Durbec和Rougon,2001)。 该方案用于从8只早期出生后的小鼠脑(P0至P3)制备SVZ衍生祖细胞的培养物。 细胞电镀后一周,我们可以观察到圆形浮游神经球,每个由单个EGF / FGF反应性神经祖细胞的克隆扩增产生。

关键字:神经系统, 成人的大脑, 干细胞, 室管膜下区, 原代培养

材料和试剂

  1. 新生小鼠
  2. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:14040-091)
  3. Hank's平衡盐溶液(HBSS)(Life Technologies,目录号:14170-088)
  4. Dulbecco改良的Eagle培养基(DMEM)(Life Technologies,目录号:61965-026)
  5. Ham's F-12营养混合物(F12)(Life Technologies,目录号:31765-027)
  6. 胰蛋白酶(Sigma-Aldrich,目录号:T5266)
  7. 胰岛素(Sigma-Aldrich,目录号:I1882)
  8. 全血运铁蛋白(Sigma-Aldrich,目录号:T0665)
  9. 腐胺(Sigma-Aldrich,目录号:P5780)
  10. 孕酮(Sigma-Aldrich,目录号:P8783)
  11. 亚硒酸钠(硒)(Sigma-Aldrich,目录号:S5261)
  12. 青霉素 - 链霉素(Life Technologies,目录号:15140-130)
  13. 胎牛血清(FBS)(Life Technologies,目录号:10106-169)
  14. B27(Life Technologies,目录号:17504-044)
  15. rhFGFbasic(Peprotech,目录号:167100-18B-B)
  16. rhEGF(Peprotech,目录号:167AF-100-15A)
  17. 神经球定义为中等(见配方)
  18. 胰蛋白酶(见配方)

设备

  1. Sterilin通用容器(Thermo Fisher Scientific,目录号:128A/P)
  2. 标准TC BD Falcon 60mm细胞培养皿(BD Biosciences,Falcon ,目录号:353002)
  3. Vibratom(Microm,型号:HM450)
  4. 立体显微镜
  5. 细胞培养罩和孵化器
  6. 剪刀,细镊子,刮刀,微刀
  7. 冰桶
  8. 火抛光玻璃巴斯德移液器
  9. 水浴

程序

  1. 从新生小鼠解剖大脑。
  2. 在冰冷PBS中,用Vibratom切割400μm厚的冠状切片(图1A)(从嗅球到侧脑室的前角)。保持每个大脑的前两个部分包含侧脑室的侧壁,并将其放在冰冷的HBSS。


    图1.新生小鼠SubVentricular区的解剖。 A。插图的新生小鼠矢状断面显示切割窗口时,使用vibratome。 B.冠状切面显示解剖隔离侧脑室组织的部分。 C.在1周长的培养后获得的神经球。比例尺,500μm。

  3. 在显微镜下,解剖侧脑室的侧壁(图1B)。用一只手拿一把镊子处理切片,并保持在菜的底部,一个微刀与另一个切出感兴趣的区域。在每次解剖后丢弃切片的其余部分。
  4. 用无菌微刀将组织切成小立方体(400μm立方体)
  5. 移取组织在800微升HBSS体积内,并将其放置在30毫升Sterilin通用容器。加入200微升胰蛋白酶12.5毫克/毫升。在37℃下在水浴中孵育5分钟
  6. 加入10体积的10%HBSS-FBS。使用火抛光的玻璃巴斯德吸管,轻轻地吸取上下帮助离解。定期取液滴,并在显微镜下检查分离是否完全
  7. 在室温(RT)下以800×g离心7分钟,弃去上清液,并将沉淀重悬于10ml新鲜HBSS中。取样品并计数细胞数。
  8. 将细胞悬液在800×g离心7分钟,弃去上清液,并将其悬浮在标准TC BD Falcon 60mm细胞培养皿中,浓度为25,000细胞/ml,在神经球定义培养基中,补充与2%B27,bFGF(20ng/ml)和EGF(20ng/ml)
  9. 每3天,加入半量的双重补充的[4%B27,bFGF(40ng/ml)和EGF(40ng/ml)]神经球定义培养基。为了避免任何球体附着到培养皿的底部,不要将培养物延伸超过1周(图1C)。

食谱

  1. 神经球定义为中等
    DMEM/F12,3:1体积,分别为
    5μg/ml胰岛素 100μg/ml全转铁蛋白 100米腐胺
    20 nM孕酮
    30nM硒
    1%青霉素 - 链霉素(分别为100IU/ml和100μg/ml)
  2. 胰蛋白酶(每次准备一份新的等分试样)
    将12.5mg胰蛋白酶溶解在1ml冰冷的HBSS中

致谢

该协议改编自Durbec和Rougon(2001)和Vernerey等人(2013)。

参考文献

  1. Durbec,P。和Rougon,G。(2001)。 将哺乳动物嗅觉祖细胞移植到雏鸡宿主中揭示了依赖于细胞承诺的迁移和分化潜能。 Mol Cell Neurosci 17(3):561-576
  2. Vernerey,J.,Macchi,M.,Magalon,K.,Cayre,M。和Durbec,P。(2013)。 睫状神经营养因子控制成年啮齿动物脑中髓鞘再生期间的祖细胞迁移。 J Neurosci 33(7):3240-3250。
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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: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Vernerey, J., Magalon, K. and Durbec, P. (2013). Primary Culture of SVZ-derived Progenitors Grown as Neurospheres. Bio-protocol 3(16): e868. DOI: 10.21769/BioProtoc.868.
  2. Vernerey, J., Macchi, M., Magalon, K., Cayre, M. and Durbec, P. (2013). Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain. J Neurosci 33(7): 3240-3250.
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