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Culture of Rat Olfactory Ensheathing Cells Using EasySep® Magnetic Nanoparticle Separation
使用EasySep®磁性纳米粒分离法培养大鼠嗅鞘细胞   

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Abstract

Olfactory ensheathing cells (OECs) can be isolated and purified from a range of postnatal day 7-day to 10-day rat olfactory bulbs. Rat OECs express the CD271/p75NTR receptor and using the “Do-It-Yourself” magnetic nanoparticle EasySep kit from STEMCELL technologies this protocol allows the selective purification of these cells in less than 50 min. Similar procedure can be used for mouse cultures.

Keywords: Olfactory(嗅), Glia(神经胶质细胞), Purification(净化), P75NTR(p75NTR), Magnetic beads(磁珠)

Materials and Reagents

  1. EasySep® "Do-It-Yourself" Selection Kit (STEMCELL Technologies, catalog number: 18098 )
  2. Mouse IgG1 P75NTR antibody (Abcam, catalog number: ab8877 )
  3. 2% Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F4135 ) in PBS (2 ml in 98 ml  of PBS,10 mM)
  4. Phosphate buffered saline (PBS) (Life Technologies, catalog number: 00-3002 )
  5. Dissolve tablets in 100 ml of distilled H2O , The buffer contains 10 mM phosphate, 150 mM sodium chloride (pH 7.3-7.5 )
  6. 10x Trypsin solution (Life Technologies, InvitrogenTM , catalog number: 15090046 )
  7. Low-glucose DMEM (Life Technologies, InvitrogenTM , catalog number: 21885025 )
  8. 5% v/v FBS (Sigma-Aldrich, catalog number: F4135)
  9. 2 mM L-glutamine (Life Technologies , InvitrogenTM, catalog number: 25030024 )
  10. Bovine serum albumin Pathocyte (MP Biomedicals, catalog number: 810111 )
  11. Bovine pancreatic insulin (Sigma-Aldrich, catalog number: I-5500 )
  12. Human transferrin (Sigma-Aldrich, catalog number: T-2252 )
  13. Progesterone (Sigma-Aldrich, catalog number: P-0130 )
  14. Putrescine (Sigma-Aldrich, catalog number: P7505 )
  15. l-thyroxine (Sigma-Aldrich, catalog number: T-2501 )
  16. Selenium (Sigma-Aldrich, catalog number: S-1382 )
  17. 3,3’,5-triiodo-l-thyronine (Sigma-Aldrich, catalog number: T-2752 )
  18. FGF2 (25 ng/ml ) (Peprotech , catalog number: 100-18B )
  19. Heregulin β-1 (50 ng/ml ) (R&D Systems, catalog number: 396-HB-050 )
  20. Forskolin (5 x 10-7 M) (Sigma-Aldrich , catalog number: F6886 )
  21. Gentamicin solution (50 mg/ml use at 1 ml/l) (Sigma-Aldrich , catalog number: G1397 )
  22. L15 media (Leibovitz medium) (Sigma-Aldrich, catalog number: L1518 )
  23. 1.33% collagenase (MP Biomedicals UK, catalog number: 195109 Type I )
  24. Plastic bijou bottle (Sterilin ) (Thermo Fisher Scientific , catalog number: 129A )
  25. Bovine pancreas DNAse (Sigma-Aldrich , catalog number: D4263 )
  26. Bovine serum albumin fraction v (Sigma-Aldrich , catalog number: A2153
  27. Astrocyte-conditioned media (ACM; use at 1:5) ACM is fresh serum-free media (DMEM-BS) collected from a confluent astrocyte monolayer for 48 h (noble  and Murray, 1984; Alexander et al., 2002 )
  28. Poly-L-Lysine (Sigma-Aldrich, catalog number: P1274 )
  29. 70% ethanol
  30. 10% v/v serum free DMEM-Bottenstein and Sato (DMEM-BS) (Bottenstein et al., 1979) (see Recipes)

Equipment

  1. EasySep® Magnet Catalog (STEMCELL technologies, catalog number: 18000 )
  2. FACS tubes 5 ml Polystyrene Round-Bottom Tubes (BD Biosciences, catalog number: 352058 )
  3. 50 ml centrifuge tubes (BD Biosciences, catalog number: 352070 )
  4. 40 μm cell strainers (BD Biosciences, catalog number: 352340 )
  5. 1.5 ml polypropylene microcentrifuge tube (Griener Bio-One GmbH, catalog number: 616201 )
  6. Poly-l-lysine coated (13.3 μg/ml, MW-100,000) (Sigma-Aldrich, catalog number: P1274), T25 cm2 tissue culture flasks (Greiner Bio-One GmbH, catalog number: 690175 )
  7. Nunc sterile centrifuge tube (Thermo Fisher Scientific, catalog number: 339651 )
  8. 25 cm2 flask (Greiner Bio-One GmbH, catalog number: 690160 )
  9. 40 μm cell strainer (BD Biosciences, Falcon®, catalog number: 352340)
  10. 37 °C incubator
  11. Plastic bijou bottle (Thermo Fisher Scientific, Sterilin®, catalog number: 129A)
  12. 21 gauge needle (BD Biosciences , catalog number: 305167
  13. 23 G needle (BD Biosciences , catalog number: 305143

Procedure

  1. Coating tissue culture flask with poly-L-Lysine (PLL, MW ~100,000)
    Make up 4 mg/ml of pLL in ddH2O (e.g. dilute 25 mg in 6.25 ml), filter sterilise using 0.22 μm filter and store at -20 °C. For use dilute 1:300 in dH2O (final concentration 13.3 μg/ml).
  2. Follow the protocol provided with the EasySep® "Do-It-Yourself" Selection Kit to make up the positive selection antibody cocktail. Briefly, add 15 μg (15 μl) of mouse IgG1 P75NTR to a 1.5 ml microcentrifuge tube. Add 100 μl of component A (supplied in the kit) to the vial and mix well. Add 100 μl of component B (supplied in the kit) to the vial and mix well. Tightly cap the vial and place it into a humidified 37 °C incubator in 7% CO2 overnight. The following day bring the vial to a final volume of 1.0 ml by adding 985 ml PBS. The positive selection antibody cocktail is now ready for use.
  3. Dissect out the olfactory bulbs from postnatal rat pups by first decapitating the pups under Home Office License. Pin the head dorsal-side up onto a dissecting board and spray with 70% ethanol. Using sterile instruments remove the skin from the head using curved scissors and make a large circular cut to remove the skull to reveal the brain and the two olfactory bulbs at the nose tip. Using curved forceps gently remove the olfactory bulbs. Enzymatically digest the bulbs in 1.33% collagenase (MP Biomedicals UK, 195109 Type I) in 500 μl of L15 media containing 50 μg/ml gentamicin in a 7 ml plastic bijou bottle. The tissue mix is placed in a humidified 37 °C incubator in 7% CO2 for 15 min to aid dissociation. After dissociation DNase (500 μl) of stock containing 0.04 mg/ml bovine pancreas DNAse and 3.0 mg/ml bovine serum albumin fraction v diluted in L15 is added to prevent cell clumping and the tissue is dissociated by passing gently and slowly through syringes carrying a 21 gauge needle first followed by a 23 G needle. The cell suspension is transferred to a 15 ml Nunc sterile centrifuge tube and spin at 1,200 rpm (~480 x g) for 5 min and plate cells in OEC media in a 25 cm2 flask, and replace half the medium twice a week. After 1 week in culture remove cells off the flask using trypsin. First wash the monolayer with 2.0 ml of PBS, remove and then add 1 ml of PBS containing 100 μl of trypsin (10x) and allow the cells to detach for 1-2 min in the incubator. Add 1 ml of 2% FBS to neutralise the trypsin, spin down cells to generate an unpurified mixed of olfactory bulb cells (Higginson and Barnett, 2011).
  4. Using 5 ml 2% FBS, wash detached cells through a 40 μm cell strainer and centrifuge at 1,200 rpm for 5 min.
  5. Resuspend cell pellet in 100 μl 2% FBS and transfer them to a 5 ml FACS tube.
  6. Add 10 μl of the positive selection cocktail that has been assembled in step 1 to the cell suspension. Mix well and incubate at room temperature for 15 min.
  7. Mix EasySep® Magnetic Nanoparticles to ensure that they are in a uniform suspension by pipetting up and down at least 5 times. Vortexing is not recommended. Add 5 μl of the magnetic nanoparticles and mix well. Incubate at room temperature for 10 min.
  8. Bring cell suspension to a total volume of 2.5 ml by adding 2% FBS. Mix the cells in the tube by gently pipetting up and down 2-3 times. Place the tube (without cap) into the EasySep® magnet. Set aside for 5 min.
  9. Pick up the magnet and in one continuous motion, invert the magnet and tube, pouring off the supernatant fraction. The magnetically labelled cells will remain inside the tube, held by the magnetic field. Leave the magnet and tube inverted for 2-3 sec then return to upright position. Do not shake or blot off any drops that may remain hanging from the mouth of the tube.
  10. Remove the tube from the magnet and add 2.5 ml of 2% FBS. Mix the cell suspension by gently pipetting up and down 2-3 times. Place the tube back in the magnet and set aside for a further 5 min.
  11. Repeat steps 7 to 9 once more, for a total of four 5 min separations in the magnet.
  12. Remove tube from magnet and resuspend cells in 3 ml OEC medium.
  13. Centrifuge the FACS tube containing the cells for 1,200 rpm (~480 x g) for 5 min to pellet the now purified OECs.
  14. Resuspend pellet in 50 μl of fresh OEC media and plate in a strip in a PLL coated T25 cm2 tissue culture flask. Allow cells to attach for 15 min at 37 °C. Cells are plated in a small strip to allow cells to attach to the flask in a high density which promotes viability.
  15. Flood flask with 3 ml OEC media and incubate at 37 °C, 7% CO2. We use 7% CO2 as this was a general protocol for all glial cells when the original purification of OECs was carried out in Prof Mark Noble’s lab (Barnett et al., 1993).
  16. After 7 days the strip of cells will be confluent and OECs can be harvested by standard trypsination and bulked up for further use.
  17. Using this system OECs will be at least 98-99% pure of any contaminating fibroblasts or other cells (see below image, Figure 1).


    Figure 1. Image of P75NTR (green) positive OECs (blue DAPI to visualise nuclei).

Recipes

  1. 10% v/v serum free DMEM-Bottenstein and Sato (DMEM-BS) (Bottenstein et al., 1979)
    Made by combining DMEM-45 g/L glucose, and supplemented with:
    25 μg/ml gentamicin
    0.0286% bovine serum albumin Pathocyte
    0.5 μg/ml bovine pancreatic insulin
    100 μg/ml human transferrin
    0.2 μM progesterone
    0.10 μM putrescine
    0.45 μM l-thyroxine
    0.224 μM selenium
    0.49 μM 3,3’,5' -triiodo-l-thyronine

Acknowledgments

The protocol was adapted from Franceschini and Barnett, (1996) and Higginson and Barnett, (2011). The work was funded by the MRC.

References

  1. Alexander, C. L., Fitzgerald, U. F. and Barnett, S. C. (2002). Identification of growth factors that promote long-term proliferation of olfactory ensheathing cells and modulate their antigenic phenotype. Glia 37(4): 349-364. 
  2. Barnett, S. C., Hutchins, A. M. and Noble, M. (1993). Purification of olfactory nerve ensheathing cells from the olfactory bulb. Dev Biol 155(2): 337-350.
  3. Bottenstein, J. E. and Sato, G. H. (1979). Growth of a rat neuroblastoma cell line in serum-free supplemented medium. Proc Natl Acad Sci U S A 76(1): 514-517. 
  4. Franceschini, I. A. and Barnett, S. C. (1996). Low-affinity NGF-receptor and E-N-CAM expression define two types of olfactory nerve ensheathing cells that share a common lineage. Dev Biol 173(1): 327-343.
  5. Higginson, J. R. and Barnett, S. C. (2011). The culture of olfactory ensheathing cells (OECs)--a distinct glial cell type. Exp Neurol 229(1): 2-9. 
  6. Noble, M. and Murray, K. (1984). Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J 3(10): 2243-2247. 

简介

嗅鞘细胞(OECs)可以从出生后第7天到10天的大鼠嗅球分离和纯化。 大鼠OEC表达CD271 / p75NTR受体,并使用STEMCELL技术的“Do-It-Yourself”磁性纳米颗粒EasySep试剂盒,该方案允许在不到50分钟内对这些细胞进行选择性纯化。 类似的方法可用于小鼠培养。

关键字:嗅, 神经胶质细胞, 净化, p75NTR, 磁珠

材料和试剂

  1. EasySep ®"自己动手"选择工具包(STEMCELL Technologies,目录号:18098)
  2. 小鼠IgG1 P75 NTR 抗体(Abcam,目录号:ab8877)
  3. 在PBS(2ml,98ml PBS,10mM)中的2%胎牛血清(FBS)(Sigma-Aldrich,目录号:F4135)
  4. 磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:00-3002)
  5. 将片剂溶解在100ml蒸馏的H 2 O中。缓冲液含有10mM磷酸盐,150mM氯化钠(pH 7.3-7.5)
  6. 10x胰蛋白酶溶液(Life Technologies,Invitrogen TM ,目录号:15090046)
  7. 低葡萄糖DMEM(Life Technologies,Invitrogen TM ,目录号:21885025)
  8. 5%v/v FBS(Sigma-Aldrich,目录号:F4135)
  9. 2mM L-谷氨酰胺(Life Technologies,Invitrogen TM ,目录号:25030024)
  10. 牛血清白蛋白Pathocyte(MP Biomedicals,目录号:810111)
  11. 牛胰胰岛素(Sigma-Aldrich,目录号:I-5500)
  12. 人转铁蛋白(Sigma-Aldrich,目录号:T-2252)
  13. 孕酮(Sigma-Aldrich,目录号:P-0130)
  14. 腐胺(Sigma-Aldrich,目录号:P7505)
  15. (Sigma-Aldrich,目录号:T-2501)
  16. 硒(Sigma-Aldrich,目录号:S-1382)
  17. 3,3',5-三碘-1-甲腺原氨酸(Sigma-Aldrich,目录号:T-2752)
  18. FGF2(25ng/ml)(Peprotech,目录号:100-18B)
  19. 调蛋白β-1(50ng/ml)(R& D Systems,目录号:396-HB-050)
  20. 福斯克林(5×10 -7 M)(Sigma-Aldrich,目录号:F6886)
  21. 庆大霉素溶液(50mg/ml,以1ml/l使用)(Sigma-Aldrich,目录号:G1397)
  22. L15培养基(Leibovitz培养基)(Sigma-Aldrich,目录号:L1518)
  23. 1.33%胶原酶(MP Biomedicals UK,目录号:195109 I型)
  24. 塑料瓶(Sterilin)(Thermo Fisher Scientific,目录号:129A)
  25. 牛胰腺DNAse(Sigma-Aldrich,目录号:D4263)
  26. 牛血清白蛋白级分v(Sigma-Aldrich,目录号:A2153)
  27. 星形胶质细胞条件培养基(ACM;以1:5使用)ACM是从汇合的星形胶质细胞单层收集的新鲜无血清培养基(DMEM-BS)48小时(noble& Murray,1984; Alexander等人,/em>,2002)
  28. 聚-L-赖氨酸(Sigma-Aldrich,目录号:P1274)
  29. 70%乙醇
  30. 10%v/v无血清DMEM-Bottenstein和Sato(DMEM-BS)(Bottenstein等人,1979)(参见Recipes)

设备

  1. EasySep ®磁铁目录(STEMCELL technologies,目录号:18000)
  2. FACS管5ml聚苯乙烯圆底管(BD Biosciences,目录号:352058)
  3. 50ml离心管(BD Biosciences,目录号:352070)
  4. 40μm细胞过滤器(BD Biosciences,目录号:352340)
  5. 1.5ml聚丙烯微量离心管(Griener Bio-One GmbH,目录号:616201)
  6. 将聚-L-赖氨酸包被的(13.3μg/ml,MW-100,000)(Sigma-Aldrich,目录号:P1274),T25cm 2组织培养烧瓶(Greiner Bio-One GmbH,目录号: 690175)
  7. Nunc无菌离心管(Thermo Fisher Scientific,目录号:339651)
  8. 25cm 2烧瓶(Greiner Bio-One GmbH,目录号:690160)
  9. 40μm细胞滤器(BD Biosciences,Falcon ,目录号:352340)
  10. 37℃孵育器
  11. 塑料瓶(Thermo Fisher Scientific,Sterilin ,目录号:129A)
  12. 21号针(BD Biosciences,目录号:305167)<
  13. 23 G针(BD Biosciences,目录号:305143)<

程序

  1. 用聚-L-赖氨酸(PLL,MW〜100,000)涂覆组织培养瓶
    在ddH 2 O中(例如在25℃下在6.25ml中稀释25mg)制备4mg/ml的pLL,使用0.22μm过滤器进行过滤灭菌并在-20℃下储存。使用时在dH 2 O中稀释1:300(终浓度为13.3μg/ml)。
  2. 按照EasySep ®"Do-It-Yourself"选择试剂盒提供的方案,构成阳性选择抗体混合物。简单地说,将15μg(15μl)小鼠IgG1 P75 NTR 加入到1.5ml微量离心管中。将100μl组分A(试剂盒中提供)加入到小瓶中,混匀。加入100微升组分B(试剂盒中提供)到小瓶中,混匀。将小瓶盖紧并将其置于加湿的37℃培养箱中,在7%CO 2中过夜。第二天通过加入985ml PBS使小瓶达到1.0ml的最终体积。阳性选择抗体混合物现在可以使用。
  3. 通过首先在家庭办公室许可下斩首幼仔,从出生后的幼崽中解剖出嗅球。将头部背面朝上放在解剖板上,并用70%乙醇喷雾。使用无菌仪器使用弯曲剪刀从头部清除皮肤,并做一个大的圆形切割,以去除头骨,显露大脑和两个嗅球在鼻尖。使用弯曲的镊子轻轻地删除嗅球。在500ml含有50μg/ml庆大霉素的L15培养基中的7ml塑料瓶中,在1.33%胶原酶(MP Biomedicals UK,195109 I型)中酶解消化球茎。将组织混合物置于加湿的37℃培养箱中,在7%CO 2中15分钟以帮助解离。在解离后,加入稀释在L15中的含有0.04mg/ml牛胰腺DNAse和3.0mg/ml牛血清白蛋白级分v的DNA酶(500μl)储备液,以防止细胞结块,并且通过轻轻地和缓慢地通过携带21号针头,随后是23G针头。将细胞悬浮液转移到15ml Nunc无菌离心管中,并以1,200rpm(〜480x/min)旋转5分钟,并将平板细胞在OEC培养基中在25℃ cm 2烧瓶中,并且每周更换一半培养基两次。在培养1周后,使用胰蛋白酶从烧瓶中除去细胞。首先用2.0ml PBS洗涤单层,除去,然后加入1ml含有100μl胰蛋白酶(10x)的PBS,并允许细胞在培养箱中分离1-2分钟。加入1ml 2%FBS以中和胰蛋白酶,旋转细胞以产生未纯化的嗅球细胞混合物(Higginson和Barnett,2011)。
  4. 使用5ml 2%FBS,通过40μm细胞过滤器洗涤分离的细胞并在1,200rpm离心5分钟。
  5. 重悬细胞沉淀在100μl2%FBS,并将其转移到5ml FACS管
  6. 加入10微升的阳性选择鸡尾酒在步骤1中组装到细胞悬液。充分混合并在室温下孵育15分钟
  7. 混合EasySep ®磁性纳米颗粒,确保它们在均匀悬浮液中,通过上下吹吸至少5次。不推荐涡旋。加入5微升的磁性纳米粒子并混匀。在室温下孵育10分钟。
  8. 通过加入2%FBS使细胞悬浮液达到2.5ml的总体积。通过轻轻吹打上下2-3次将细胞在试管中混合。将管(无帽)放入EasySep ®磁体。搁置5分钟。
  9. 拿起磁铁,在一个连续的运动,倒转磁铁和管,倾吐上清部分。磁性标记的细胞将保留在管内,由磁场保持。使磁铁和管倒置2-3秒,然后返回到直立位置。不要摇动或弄脏可能留在管口上的任何液滴。
  10. 从磁铁中取出管,加入2.5 ml的2%FBS。混合细胞悬浮液,轻轻吹打上下2-3次。将管放回磁铁,再放置5分钟。
  11. 重复步骤7到9一次,在磁体中总共有4个5分钟的分离。
  12. 从磁铁取出管,并重悬在3毫升OEC培养基中的细胞
  13. 离心含有细胞的FACS管1,200rpm(〜480×g/g)5分钟,以沉淀现在纯化的OEC。
  14. 重悬在50μl新鲜的OEC培养基中的沉淀,并在PLL包被的T25cm 2组织培养瓶中的条中平板。允许细胞在37°C附加15分钟。将细胞铺在小条中,使细胞以高密度附着到烧瓶上,从而提高活力
  15. 用3ml OEC培养基进行扩增烧瓶,并在37℃,7%CO 2下孵育。我们使用7%CO 2,因为这是所有胶质细胞的一般方案,当在蛋白质的原始纯化在Mark Noble的教授的实验室中进行时(Barnett等人 1993)。
  16. 7天后,条带的细胞将汇合,并且可以通过标准胰蛋白酶消化收获OEC,并且填充以供进一步使用。
  17. 使用该系统OECs将是至少98-99%纯的任何污染的成纤维细胞或其他细胞(见下图,图1)。


    图1. P75 NTR (绿色)阳性OEC(蓝色DAPI可视化核)的图像。

食谱

  1. 10%v/v无血清DMEM-Bottenstein和Sato(DMEM-BS)(Bottenstein等人,1979)
    通过组合DMEM-45g/L葡萄糖并补充:
    25μg/ml庆大霉素
    0.0286%牛血清白蛋白Pathocyte
    0.5μg/ml牛胰岛素
    100μg/ml人转铁蛋白
    0.2μM孕酮
    0.10μM腐胺
    0.45μMl-甲状腺素
    0.224μM硒
    0.49μM3,3',5'-三碘-1-甲腺氨酸

致谢

该方案改编自Franceschini和Barnett(1996)和Higginson和Barnett,(2011)。 这项工作由MRC资助。

参考文献

  1. Alexander,C.L.,Fitzgerald,U.F.and Barnett,S.C。(2002)。 鉴别促进嗅鞘细胞长期增殖和调节其抗原表型的生长因子< a>。 Glia 37(4):349-364。
  2. Barnett,S.C.,Hutchins,A.M.and Noble,M。(1993)。 从嗅球上纯化嗅神经鞘细胞。 /em> 155(2):337-350
  3. Bottenstein,J.E。和Sato,G.H。(1979)。 大鼠神经母细胞瘤细胞系在无血清补充培养基中的生长。 Proc Natl Acad Sci U S A 76(1):514-517。
  4. Franceschini,I.A。和Barnett,S.C。(1996)。 低亲和力NGF受体和EN-CAM表达定义两种类型的嗅神经包裹共享共同谱系的细胞。 Dev Biol 173(1):327-343。
  5. Higginson,J.R。和Barnett,S.C。(2011)。 嗅鞘细胞(OEC)的文化 - 一种独特的胶质细胞类型。 Exp Neurol 229(1):2-9。 
  6. Noble,M。和Murray,K。(1984)。 纯化星形胶质细胞促进双潜能胶质祖细胞的体外分裂。 EMBO J 3(10):2243-2247。
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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. Lindsay, S. L. and Barnett, S. C. (2013). Culture of Rat Olfactory Ensheathing Cells Using EasySep® Magnetic Nanoparticle Separation. Bio-protocol 3(8): e682. DOI: 10.21769/BioProtoc.682.
  2. Higginson, J. R., Thompson, S. M., Santos-Silva, A., Guimond, S. E., Turnbull, J. E. and Barnett, S. C. (2012). Differential sulfation remodelling of heparan sulfate by extracellular 6-O-sulfatases regulates fibroblast growth factor-induced boundary formation by glial cells: implications for glial cell transplantation. J Neurosci 32(45): 15902-15912.
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