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Isolation and Culture of Neurospheres for the Study of Pathogenesis of Prion Disease
分离和培养神经球用于研究朊病毒病发病机理

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Abstract

Neurosphere contains neural stem cells that are capable of self-renewal and multilineage differentiation including neurons, astrocytes, and oligodendrocytes (Gage, 2000). Cell culture model using differentiated neurosphere cultures are suggested to be a valuable tool for studying the pathogenesis of prion disease at the cellular level (Iwamaru et al., 2013). This protocol describes the procedure for a culture of whole brain-derived neurospheres from newborn mouse brains. Neurosphere formation steadily occurs within a week from the cultures of neonatal whole brains and these cells have stem cell properties.

Keywords: Neural stem cell(神经干细胞), Isolation and culture(分离和培养), Neurosphere(神经球), Neonatal mouse brain(新生小鼠大脑), Prion protein(朊病毒蛋白)

Materials and Reagents

  1. Newborn mice (age at one day after birth)
    Present culture method can be applied to any mouse strains, regardless of sex. We used prion protein overexpressing transgenic mouse and prion protein-deficient mouse for our present study (Iwamaru et al., 2013).
  2. Dulbecco's phosphate buffered saline without Ca and Mg (D-PBS) (Nacalai Tesque, catalog number: 14249-95 )
  3. Di‐sodium dihydrogen ethylenediamine tetraacetate dihydrate (EDTA) (Nacalai Tesque, catalog number: 151-11 )
  4. Hank's balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
  5. Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (DMEM/F12 Ham) (Sigma-Aldrich, catalog number: D8437 )
  6. N-2 supplement (Life Technologies, catalog number: 17502-048 )
  7. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P0781 )
  8. Fetal bovine serum (FBS) (Hyclone, catalog number: SH30070.03 )
  9. Accutase (Innovative Cell Technologies, catalog number: AT104 )
  10. Trypsin (Sigma-Aldrich, catalog number: T8003 ) (see Recipes)
  11. Deoxyribonuclease I (DNase I) (Worthington Biochemical Corporation, catalog number: LS002139 ) (see Recipes)
  12. Epidermal growth factor human (EGF) (Sigma-Aldrich, catalog number: E9644 ) (see Recipes)
  13. Fibroblast growth factor-basic human (bFGF) (Sigma-Aldrich, catalog number: F0291 ) (see Recipes)
  14. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9576 ) (see Recipes)
  15. Neurosphere basal medium (see Recipes)

Equipment

  1. Falcon 15 ml conical centrifuge tube (Corning, catalog number: 352096 )
  2. Falcon 100 mm cell culture dish (Corning, catalog number: 353003 )
  3. Nunc 60 mm dish with HydroCell surface (Thermo Fisher Scientific, catalog number: 174912 )
  4. Disposable micro homogenizer (TaKaRa Bio, TaKaRa BioMasher Standard, catalog number: 9791A )
  5. Aerosol resistant tip (ART 1000 REACH tips) (Thermo Fisher Scientific, model: 2079 )
  6. Membrane filter (EMD Millipore, Millex-GV 0.22 μm, 33 mm)
  7. Dissecting microscope (Olympus, model: SZH10 )
  8. Centrifuge (KUBOTA, model: 5220 )
  9. CO2 incubator (set at 37 °C and 5% CO2-95% air)
  10. Reciprocal shaker (TAITEC, model: NR-1 )
  11. Laminar flow hood (W800 x H1050 x D500 mm) (Panasonic Corporation, SANYO, model: MCV-710ATS )
  12. Scissors (NAPOX B-5H, 154 mm; NAPOX B-1112H, 110 mm), fine forceps (NAPOX A-5, 110 mm; NAPOX MA-45, 110 mm), spatula (Laboran 9-891-03, 180 mm)
  13. Ice bucket

Procedure

  1. Dissect the brain from newborn mice under Laminar flow hood (Figure 1). Spray 70% ethanol upon newborn mice and cut the head (Figure 2A). Make small incisions (Figure 2B, arrows) and remove the skin. Then, carefully remove the skull with forceps and expose the brain (Figure 2C).


    Figure 1. Dissecting equipments arranged in a Laminar flow hood


    Figure 2. Dissection procedures to obtain neonatal mouse brain

  2. Scoop the brain by a small spatula and place them in ice cold HBSS (10 ml in 100 mm dish, Figure 3A). By using fine forceps, remove meninges under dissecting microscope (Figure 3B-C).


    Figure 3. Removal of meninges (B: arrow) from neonatal mouse brain under dissecting microscope. After removal of meninges, the brain becomes whitish color (C).

  3. Place cortices in sterilized disposable micro homogenizer and centrifuge at 5000 x g for 30 sec at 4 °C.
  4. Pipet the minced tissue with 2 ml of D-PBS containing 1 mM EDTA, 100 μg/ml trypsin, and 400 μg/ml DNase I until evenly homogenized and place them in a 15 ml Conical Centrifuge Tube.
  5. Incubate for 15 min at 37 °C in an incubator with constant agitation using a reciprocal shaker at 50 rpm. Add 1 ml of FBS, then gently pipet up and down to help the dissociation by using aerosol resistant tip to avoid contamination. Stand tubes for 2 min at 25 °C and collect supernatant by pipetting. Avoid to suck any visible tissue fragments.
  6. Centrifuge cell suspensions at 200 × g for 5 min at 25 °C and resuspend the pellet in Neurosphere basal medium (10 ml) by pipetting.
  7. Centrifuge the cell suspension at 200 x g for 5 min at 25 °C. Discard the supernatant, resuspend in 5 ml of Neurosphere basal medium at 25 °C and transfer it into Nunc 60 mm dishes with HydroCell Surface. Culture the cells at 37 °C in humidified CO2 incubator (5% CO2-95% air).
  8. Refill 0.5 ml of Neurosphere basal medium everyday.
  9. After 5-7 days, neurospheres with variable sizes (50-100 μm in diameter) are formed in the culture (Figure 4). Centrifuge neurosphere suspensions at 200 × g for 5 min at 25 °C, discard the supernatant, and resuspend in 2 ml of Accutase at 37 °C for 10 min.


    Figure 4. Neurosphere formation in Neurosphere basal medium after 5 days of culture. Scale bar = 100 μm

  10. Gently pipet up and down (about ten times) using aerosol resistant tip to dissociate into single cells. Check the cells under microscope for successful dissociation. Add 12 ml of HBSS and centrifuge at 200 x g for 5 min at 25 °C. Resuspend the pellet in 10 ml of Neurosphere basal medium at 25 °C and subculture into new Nunc 60mm dishes with HydroCell Surface at 1:4 split ratio (i.e. 25% of the harvested cells are seeded into the new dish with same size). Refill 0.5 ml of Neurosphere basal medium everyday. The neurospheres are subcultured at 4-6 day intervals.

Recipes

  1. Deoxyribonuclease I (DNase I)
    Dissolved at 4 mg /ml in D-PBS
    Sterile filtered (0.22 μm)
    Aliquoted (4 ml) and stored at -20 °C
    Use within 6 months
  2. Epidermal growth factor human (EGF)
    Dissolved at 50 μg /ml in sterile DMEM/F12 Ham containing 1% BSA
    Aliquoted (100 μl) and stored at -20 °C
    Use within 6 months
  3. Fibroblast growth factor-basic human (bFGF)
    Dissolved at 50 μg /ml in sterile DMEM/F12 Ham containing 1% BSA
    Aliquoted (50 μl) and stored at -20 °C
    Use within 6 months
  4. Bovine serum albumin (BSA)
    Dissolved at 300 mg /ml in D-PBS
    Sterile filtered (0.22 μm)
    Stored at 4 °C
    Use within 6 months
  5. Neurosphere basal medium
    Reagents
    Volume
    Final conc.
    DMEM/F12 Ham
    487.33 ml

    EGF
    500 μl
    50 ng/ml
    bFGF
    500 μl
    50 ng/ml
    BSA
    1.67 ml ml
    0.1%
    N2 supplement
    5 ml
    1%
    Penicillin-streptomycin
    5 ml
    100 IU/ml and 100 μg/ml, respectively
  6. Trypsin
    Dissolved at 1 mg /ml in D-PBS containing 10 mM EDTA
    Sterile filtered (0.22 μm)
    Aliquoted (4 ml) and stored at -20 °C
    Use within 6 months
    For the dissociation of minced brain tissue, mix the reagents as follows:
    Reagents
    Volume
    Final conc.
    Trypsin
    1 ml
    100 μg/ml
    DNase I
    1 ml
    400 μg/ml
    D-PBS
    8 ml


Acknowledgments

This protocol was adapted from our previously published paper: Iwamaru et al. (2013). This work was supported by a Grant-in-Aid from the BSE and Other Prion Disease Control Project of the Ministry of Agriculture, Forestry, and Fisheries of Japan and by a Grant-in-Aid for Young Scientists (category B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

  1. Gage, F. H. (2000). Mammalian neural stem cells. Science 287(5457): 1433-1438.
  2. Iwamaru, Y., Takenouchi, T., Imamura, M., Shimizu, Y., Miyazawa, K., Mohri, S., Yokoyama, T. and Kitani, H. (2013). Prion replication elicits cytopathic changes in differentiated neurosphere cultures. J Virol 87(15): 8745-8755.

简介

神经球包含能够自我更新和多向分化的神经干细胞,包括神经元,星形胶质细胞和少突胶质细胞(Gage,2000)。 使用分化的神经球培养物的细胞培养模型被认为是用于研究朊病毒疾病在细胞水平的发病机制的有价值的工具(Iwamaru等人,2013)。 该协议描述了来自新生小鼠脑的全脑衍生的神经球的培养的程序。 神经球形成稳定地发生在新生儿全脑的培养物的一周内,并且这些细胞具有干细胞特性。

关键字:神经干细胞, 分离和培养, 神经球, 新生小鼠大脑, 朊病毒蛋白

材料和试剂

  1. 新生小鼠(出生后一天的年龄)
    目前的培养方法可以应用于任何小鼠品系,而不论性别。 我们使用朊病毒蛋白过表达转基因小鼠和朊病毒蛋白缺陷小鼠为我们目前的研究(Iwamaru等人,2013年)。
  2. 不含Ca和Mg的Dulbecco磷酸盐缓冲盐水(D-PBS)(Nacalai Tesque,目录号:14249-95)
  3. 二钠乙二胺四乙酸二钠二水合物(EDTA)(Nacalai Tesque,目录号:151-11)
  4. Hank's平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H8264)
  5. Dulbecco's改良Eagle培养基/营养混合物F-12 Ham(DMEM/F12 Ham)(Sigma-Aldrich,目录号:D8437)
  6. N-2补充剂(Life Technologies,目录号:17502-048)
  7. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P0781)
  8. 胎牛血清(FBS)(Hyclone,目录号:SH30070.03)
  9. Accutase(Innovative Cell Technologies,目录号:AT104)
  10. 胰蛋白酶(Sigma-Aldrich,目录号:T8003)(参见Recipes)
  11. 脱氧核糖核酸酶I(DNase I)(Worthington Biochemical Corporation,目录号:LS002139)(参见Recipes)
  12. 表皮生长因子人(EGF)(Sigma-Aldrich,目录号:E9644)(参见Recipes)
  13. 成纤维细胞生长因子 - 碱性人(bFGF)(Sigma-Aldrich,目录号:F0291)(参见配方)
  14. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9576)(参见Recipes)
  15. 神经球基础培养基(见配方)

设备

  1. Falcon 15ml锥形离心管(Corning,目录号:352096)
  2. 表皮生长因子人(EGF)(Sigma-Aldrich,目录号:E9644)(参见Recipes)
  3. 成纤维细胞生长因子 - 碱性人(bFGF)(Sigma-Aldrich,目录号:F0291)(参见配方)
  4. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9576)(参见Recipes)
  5. 神经球基础培养基(见配方)

设备

  1. Falcon 15ml锥形离心管(Corning,目录号:352096)...
  2. Centrifuge (KUBOTA, model: 5220)
  3. CO2 incubator (set at 37 °C and 5% CO2-95% air)
  4. Reciprocal shaker (TAITEC, model: NR-1)
  5. Laminar flow hood (W800 x H1050 x D500 mm) (Panasonic Corporation, SANYO, model: MCV-710ATS)
  6. Scissors (NAPOX B-5H, 154 mm; NAPOX B-1112H, 110 mm), fine forceps (NAPOX A-5, 110 mm; NAPOX MA-45, 110 mm), spatula (Laboran 9-891-03, 180 mm)
  7. Ice bucket

Procedure

  1. Dissect the brain from newborn mice under Laminar flow hood (Figure 1). Spray 70% ethanol upon newborn mice and cut the head (Figure 2A). Make small incisions (Figure 2B, arrows) and remove the skin. Then, carefully remove the skull with forceps and expose the brain (Figure 2C).


    Figure 1. Dissecting equipments arranged in a Laminar flow hood


    Figure 2. Dissection procedures to obtain neonatal mouse brain

  2. Scoop the brain by a small spatula and place them in ice cold HBSS (10 ml in 100 mm dish, Figure 3A). By using fine forceps, remove meninges under dissecting microscope (Figure 3B-C).


    图3.在解剖显微镜下从新生小鼠脑中取出脑膜(B:箭头)。去除脑膜后,大脑变成发白的颜色(C)。

  3. 将皮质置于无菌的一次性微型匀浆器中,在4℃下以5000×g离心30秒。
  4. 用2ml含有1mM EDTA,100μg/ml胰蛋白酶和400μg/ml DNA酶I的D-PBS吸取切碎的组织,直至均匀匀浆,并将其放置在15ml锥形离心管中。
  5. 在37℃下在恒温搅拌器中使用往复振荡器在50rpm下孵育15分钟。加入1毫升的FBS,然后轻轻地吸上下来帮助解离通过使用气溶胶抗性尖端,以避免污染。在25℃下放置管2分钟,通过移液收集上清液。避免吸入任何可见的组织碎片。
  6. 在25℃下以200×g离心细胞悬浮液5分钟,并通过移液将沉淀物重悬于Neurosphere基础培养基(10ml)中。
  7. 在25℃下以200×g离心细胞悬浮液5分钟。弃去上清液,在25℃下重悬在5ml Neurosphere基础培养基中,并将其转移到具有HydroCell表面的Nunc 60mm培养皿中。在37℃在潮湿的CO 2培养箱(5%CO 2 -95%空气)中培养细胞。
  8. 每天补充0.5ml Neurosphere基础培养基。
  9. 5-7天后,在培养物中形成具有可变尺寸(直径50-100μm)的神经球(图4)。在25℃下以200×g离心神经球悬浮液5分钟,弃去上清液,并在37℃下在2ml Accutase中重悬浮10分钟。


    图4.培养5天后在Neurosphere基础培养基中形成神经球。比例尺=100μmμm

  10. 轻轻吸上下(约10次)使用气溶胶抗性尖端分离成单个细胞。检查细胞在显微镜下成功解离。加入12ml HBSS并在25℃下以200×g离心5分钟。在25℃下将沉淀物重悬在10ml Neurosphere基础培养基中,并以1:4分流比(即,25%的收获细胞)接种到具有HydroCell表面的新的Nunc 60mm培养皿中盘具有相同的大小)。每天补充0.5毫升Neurosphere基础培养基。神经球以4-6天的间隔进行传代培养

食谱

  1. 脱氧核糖核酸酶I(DNase I)
    以4mg/ml溶于D-PBS中
    无菌过滤(0.22μm)
    分装(4ml)并贮存在-20℃下 在6个月内使用
  2. 表皮生长因子人(EGF)
    以50μg/ml溶于含有1%BSA的无菌DMEM/F12 Ham中溶解 分装(100μl)并贮存在-20℃下 在6个月内使用
  3. 成纤维细胞生长因子 - 碱性人(bFGF)
    以50μg/ml溶于含有1%BSA的无菌DMEM/F12 Ham中溶解 分装(50μl)并储存在-20℃下
    在6个月内使用
  4. 牛血清白蛋白(BSA)
    以300mg/ml溶于D-PBS中
    无菌过滤(0.22μm)
    储存在4°C
    在6个月内使用
  5. 神经球基础培养基
    试剂

    最终浓度。
    DMEM/F12 Ham
    487.33 ml

    EGF
    500微升
    50 ng/ml
    bFGF
    500微升
    50 ng/ml
    BSA
    1.67ml ml
    0.1%
    N2补助
    5 ml
    1%
    青霉素 - 链霉素 5 ml
    100 IU/ml和100μg/ml,分别为
  6. 胰蛋白酶
    在含有10mM EDTA的D-PBS中以1mg/ml溶解 无菌过滤(0.22μm)
    分装(4ml)并贮存在-20℃下 在6个月内使用
    对于切碎的脑组织的解离,混合试剂如下:
    试剂

    最终浓度。
    胰蛋白酶
    1 ml
    100μg/ml
    DNase I
    1 ml
    400μg/ml
    D-PBS
    8 ml


致谢

这个协议改编自我们以前发表的文章: Iwamaru (2013)。这项工作得到了日本农业,林业和渔业部BSE和其他朊病毒病防治项目的资助,以及来自该部的青年科学家资助(B类)日本教育,文化,体育,科学和技术。

参考文献

  1. Gage,F.H。(2000)。 哺乳动物神经干细胞 Science 287(5457) :1433-1438。
  2. Iwamaru,Y.,Takenouchi,T.,Imamura,M.,Shimizu,Y.,Miyazawa,K.,Mohri,S.,Yokoyama,T.and Kitani,H。 Prion复制引起分化的神经球培养物中的细胞病变。 em> 87(15):8745-8755。
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引用:Iwamaru, Y., Takenouchi, T. and Kitani, H. (2014). Isolation and Culture of Neurospheres for the Study of Pathogenesis of Prion Disease. Bio-protocol 4(6): e1081. DOI: 10.21769/BioProtoc.1081.
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