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In vitro Migration Assays for Neural Stem Cells, Intermediate Neurogenic Progenitors and Immature Neurons
神经干细胞、中间神经性前体细胞和未成熟神经元的体外迁移分析   

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Abstract

In the vertebrate central nervous system (CNS), different neural precursor populations such as neural stem cells (NSCs), intermediate neurogenic progenitors (INPs) and immature neurons have to migrate from their places of birth to their location of function. Coordinated migration is mediated by direct cell-cell interactions and by extracellular matrix components, chemoattractants as well as repellents. The migration potential of such populations as well as the responsiveness to chemoattractive compounds can be addressed in isolated cells using in vitro migration assays. Here we describe two migration assays, a matrigel migration assay and a Boyden chamber migration assay, which allow the in vitro assessment of neural migration under defined conditions (Ladewig, Koch and Brüstle, 2014). A matrigel matrix is a soluble basement membrane extract. The major components of matrigel matrix are collagens, laminin and proteoglycans, which provide the substrate for migrating cells. In the matrigel assay migration can be analyzed using a phase contrast microscope. The Boyden chamber assay (Richards and McCullough, 1984) is based on microchemotaxis chambers, which consist of two compartments separated by a membrane with a defined pore size. Cells can be plated in the upper compartment and allowed to migrate through the pores towards the lower compartment, in which a potential chemotactic agent is loaded. Cell migration can be analyzed following fixing and immunohistochemical staining. In principle, the described protocols should be applicable to other cell populations such as endothelial cells or cancer cells using conditions adapted to the individual needs of the specific cell type.

Materials and Reagents

  1. Neural cell populations: e.g. pluripotent stem cell (PSC) derived neuroepithelial stem cells (lt-NES) and neurons derived thereof (Ladewig et al., 2008; Koch et al., 2009)
  2. DMEM/F12 (high glucose) (Life Technologies, catalog number: 11320074 )
  3. Neurobasal medium (Life Technologies, catalog number: 21103049 )
  4. B27 supplement (Life Technologies, catalog number: 17504044 )
  5. N2 supplement (GE Healthcare, catalog number: T1129,2205 )
  6. Glucose (Roche Diagnostics, catalog number: HN06.3 )
  7. Trypsin/EDTA (Life Technologies, catalog number: 15400054 )
  8. Trypsin inhibitor (Life Technologies, catalog number: 17075029 )
  9. DNAse (CellSystems Biotechnologie Vertrieb GmbH, catalog number: LS002140 )
  10. Specific for matrigel migration assay
    1. MatrigelTM Basment Membrane Matrix (BD Bioscience, catalog number: 354230 ) or equivalent such as Geltrex® Matrix (Life Technologies, catalog number: A1413202 )
    2. 4 well tissue culture dish (VWR International, catalog number: 176740 )
    3. Rock inhibitor (Cell guidance systems, catalog number: SM02-100 )
  11. Specific for Boyden chamber migration assay
    1. Millicell culture plate inserts 8-μm pore size (Millipore, catalog number: PI8P01250 )
    2. Nylon mesh (Pall, http://www.pall.com)
    3. 24 well plate (VWR International, catalog number: 734-0056 )
    4. Poly-l-ornithine (Sigma-Aldrich, catalog number: P3655-1G )
    5. Laminin (Life Technologies, catalog number: 23017015 )
    6. PBS (Life Technologies, catalog number: 14190094 )
    7. Chemoattractant such as FGF2 (R&D Systems, catalog number: 233-FB/CF ) or VEGF (R&D Systems, catalog number: 236-EG-01M )
    8. Potential inhibitors for used chem¬oattractant such as VEGF receptor 1 and VEGF receptor 2 blocking antibodies (both R&D Systems, catalog numbers: AF321 and AF357 ) and the FGF2 neutralizing antibody (Millipore, clone bFM-1)
    9. Cotton bud (drug store, e.g. Q-tip®)
    10. Solutions and antibodies for standard immunohistochemical staining
  12. Neural stem cell media (see Recipes)
  13. Neuronal differentiation media (see Recipes)

Equipment

  1. Cell culture centrifuge (e.g., Megafuge 1.0 R, Kendro Laboratory)
  2. 37 °C, 5% CO2 cell culture incubator (e.g., Thermo Fisher Scientific, model: Heracell 240 )
  3. Counting chamber (e.g., Fuchs-Rosenthal, Marienfeld)
  4. Glass object slide (e.g., Thermo Fisher Scientific, Superfrost Plus)
  5. Microscope (e.g., ZEISS, model: Axiovert 200M )

Procedure

  1. Matrigel migration assay
    1. Thaw matrigel matrix at 4 °C on ice overnight.
    2. Dilute matrigel matrix at a ratio of 1:2 in cold DMEM/F12.
      Note: 1 ml of matrigel will be sufficient to prepare 12 wells of a 4 well dish.
    3. Add 250 μl of the matrigel matrix mixture per well of a 4-well tissue culture dish and incubate in the cell culture incubator (at 37 °C) for at least 30 min for hardening.
      Note: Store pipet tips at -20 °C before using.
    4. Trypsinize cells and count them using a counting chamber.
      Note: At this step cell suspension can be incubated with 0.1% DNAse to avoid cell clumping.
    5. Pellet cells by centrifugation at 300 x g for 4 min.
    6. Remove supernatant and resuspend 100,000 cells/µl in cell culture media (e.g. Neuronal media containing DMEM/F12 with one vol% N2 supplement and Neurobasal with two vol% B27 supplement mixed at a 1:1 ratio).
      Optional: Add 5 mM Rock inhibitor for cell survival.
    7. Spot 1 µl of the cell suspension in the middle of the dish on the gel surface.
      Note: Specified 4 well dishes have a very slight round bottom which supports precise spotting.
    8. Incubate dishes for 10 min by carefully transferring them to the cell culture incubator until cells are attached.
    9. Carefully cover the cells with respective cell culture media and cultivate them in the cell culture incubator until analysis.
    10. Radial migration from the center of the cell clumps can be analyzed at different time points using static or live cell microscopy (see Figure 1).

  2. Boyden chamber migration assay
    1. Place Millicell culture plate inserts into 24 well plates and coat them from both sides with poly-l-ornithine. Incubate the culture plate for at least 2 h at 37 °C (e.g. in the cell culture incubator).
    2. Wash the 24 well dish with the millicell culture plate inserts 3 times with PBS followed by coating with laminin (1:1,000 diluted in PBS).
    3. Wrap the dish with parafilm and store at least over night at 4 °C.
      Note: Experiments should always be performed in triplicates.
    4. Trypsinize cells and count them using a counting chamber.
      Note: At this step cell suspension can be incubated with DNAse to avoid cell clumping and filtered through a nylon mesh (before counting).
    5. Pellet the cells at 300 x g for 4 min.
    6. Remove the supernatant and resuspend 1 x 106 cells/ml in neural differentiation media.
      Note: The media used should not contain any growth factors or cytokines.
    7. Remove the coating suspension and load the bottom well of the chamber with 400-450 µl media with or without the chemoattractant of interest (e.g. 10 ng/ml VEGF or 10 ng/ml FGF2).
      Notes:
      1. It is crucial that the media in the bottom well is covering the bottom side of the membrane but should not rise to the upper side.
      2. To avoid bubbles, carfully tip the pipette to the fringe of the chamber.
      3. Use respective chemoattractive-blocking agents as control (e.g. VEGF receptor 1 and VEGF receptor 2 blocking antibodies and FGF2 neutralizing antibody).
    8. Plate 100 µl of the cell suspension on the poly-l-ornithine/laminin coated membrane that separates the upper and the lower well.
      Note: Do not touch the filter and avoid creating bubbles.
    9. Place the chamber in the 37 °C, 5% CO2 incubator for 6-20 h. The incubation time varies considerably depending on the cell type and chemotactic factor.
    10. Fix the dish (with the millicell culture plate insert) with 4% PFA and wash twice with PBS.
    11. Stain the upper and lower side of the membrane with DAPI.
      Note: Check under the fluorecence microscope whether cell destribution is uniform (on both the upper and lower side of the membrane) and proceed only with those membranes. Non-uniform distribution of cells is most likely due to incomplete coating of the membrane. Take care that the membrane is not getting dry during the coating (point 1-3).
    12. Wipe off cells on the upper side of the membrane (non-migrated cells) with a cotton bud. Repeat this step at least twice.
    13. Stain the millicell culture plate inserts (in the 24 well dish) for appropiate markers (e.g. ßIII tubulin to identify neurons, nestin for neural progenitors).
    14. Cells reaching the bottom side of the membrane can be quantified under the flurecence microscope (Figure 2).
      Note: The millicell culture plate inserts can be placed on a glass object slide, use a drop of PBS to keep the membrane wet.

Representative data



Figure 1. (A-B) Matrigel migration assay addressing the migration capacity of lt-NES cell derived purified neurons in comparison to neurons within non-purified population. Equal numbers of cells were plated on matrigel matrix and analyzed 48 h later. Purified neurons showed a radial symmetric distribution across a large area with migration of individual neurons from the plating site A. In contrast, the non-purified population (containing neurons as well as progenitor cells) formed spherical clusters with hardly any neurons leaving these aggregates. Instead, radial axonal outgrowth was observed B. Scale bar: A-B. 200 µm

A

B

Figure 2. Cell migration studied by using millicell culture plate inserts. A. Cells are plated on the membrane of the upper well. Chemoattractants can be added to the lower well. Migration of cells from the upper well through the membrane can be measured by wiping off the remaining cells from the upper side of the membrane using a cotton bud and counting the cells that reached the bottom side. Adapted from Erlandsson (2003). B. Assessing the migration of neurons within a cell mixture (containing neurons and progenitor cells) and of purified neurons. Bars represent the percentage of neurons reaching the lower side of the membrane after 20 h. Cell numbers were normalized to the number of neurons plated and shown as mean+SD (*P < 0.05).

Notes

The quality of the Millicell culture plate inserts can vary, thus it is crucial to ensure that the cell destribution is uniform on both the upper and lower side of the membrane as described in point 11 of the Boyden chamber migration protocol.

Recipes

  1. Neural stem cell media
    500 ml DMEM/F12, high glucose
    5 ml N2 supplement
    1.68 mg glucose
  2. Neuronal differentiation media
    50% neural stem cell media
    48% neurobasal media
    2% B27 supplement

Acknowledgments

This work was supported by the European Union (grant 222943 Neurostemcell; LSHG-CT-2006-018739, ESTOOLS), the German Research Foundation (DFG; SFB-TR3), the Hertie Foundation and the Ministry of Innovation Science and Research of North Rhine-Westphalia (Junior Research Group, L-072.0081).

References

  1. Erlandsson, A. (2003). Neural Stem Cell Differentiation and Migration.
  2. Koch, P., Opitz, T., Steinbeck, J. A., Ladewig, J. and Brüstle, O. (2009). A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci U S A 106(9): 3225-3230.
  3. Ladewig, J., Koch, P. and Brüstle, O. (2014). Auto-attraction of neural precursors and their neuronal progeny impairs neuronal migration. Nat Neurosci 17(1): 24-26.
  4. Ladewig, J., Koch, P., Endl, E., Meiners, B., Opitz, T., Couillard-Despres, S., Aigner, L. and Brüstle, O. (2008). Lineage selection of functional and cryopreservable human embryonic stem cell-derived neurons. Stem Cells 26(7): 1705-1712.
  5. Richards, K. L. and McCullough, J. (1984). A modified microchamber method for chemotaxis and chemokinesis. Immunol Commun 13(1): 49-62.

简介

在脊椎动物中枢神经系统(CNS)中,不同的神经前体细胞如神经干细胞(NSCs),中间神经源性祖细胞(INPs)和不成熟的神经元必须从他们的出生地迁移到它们的功能位置。配位迁移通过直接的细胞 - 细胞相互作用和细胞外基质组分,化学引诱物以及驱避剂介导。这些群体的迁移潜力以及对化学吸引化合物的反应性可以使用体外迁移测定在分离的细胞中解决。在这里我们描述两个迁移测定,基质胶迁移测定和博伊登室迁移测定,其允许在确定的条件下的神经迁移的体外评估(Ladewig,Koch和Brüstle,2014)。基质胶基质是可溶性基底膜提取物。基质胶基质的主要成分是胶原,层粘连蛋白和蛋白聚糖,它们为迁移细胞提供了底物。在基质胶试验中,可以使用相差显微镜分析迁移。 Boyden室测定法(Richards和McCullough,1984)基于微化学趋化室,其由通过具有限定孔径的膜分开的两个室组成。细胞可以接种在上室中并允许通过孔迁移到下室,其中加载了潜在的趋化剂。可以在固定和免疫组织化学染色后分析细胞迁移。原则上,所描述的方案应该适用于其它细胞群体,例如内皮细胞或癌细胞,使用适合于特定细胞类型的个体需要的条件。

材料和试剂

  1. 神经细胞群体:例如多能干细胞(PSC)衍生的神经上皮干细胞(lt-NES)及其衍生的神经元(Ladewig等人,2008; Koch& et al。,2009)
  2. DMEM/F12(高葡萄糖)(Life Technologies,目录号:11320074)
  3. Neurobasal培养基(Life Technologies,目录号:21103049)
  4. B27补充剂(Life Technologies,目录号:17504044)
  5. N2补充剂(GE Healthcare,目录号:T1129,2205)
  6. 葡萄糖(Roche Diagnostics,目录号:HN06.3)
  7. 胰蛋白酶/EDTA(Life Technologies,目录号:15400054)
  8. 胰蛋白酶抑制剂(Life Technologies,目录号:17075029)
  9. DNAse(CellSystems Biotechnologie Vertrieb GmbH,目录号:LS002140)
  10. 特异于matrigel迁移测定
    1. Matrigel TM基底膜基质(BD Bioscience,目录号: 354230)或等同物例如Geltrex Matrix(Life Technologies, 目录号:A1413202)
    2. 4孔组织培养皿(VWR International,目录号:176740)
    3. 岩石抑制剂(细胞引导系统,目录号:SM02-100)
  11. 特定于Boyden室迁移测定
    1. Millicell培养板插入8-μm孔径(Millipore,目录号:PI8P01250)
    2. 尼龙网(Pall, http://www.pall.com
    3. 24孔板(VWR International,目录号:734-0056)
    4. 聚-1-鸟氨酸(Sigma-Aldrich,目录号:P3655-1G)
    5. 层粘连蛋白(Life Technologies,目录号:23017015)
    6. PBS(Life Technologies,目录号:14190094)
    7. 化学吸引剂如FGF2(R& D Systems,目录号: 233-FB/CF)或VEGF(R& D Systems,目录号:236-EG-01M)
    8. 使用的化学引诱物例如VEGF受体1的潜在抑制剂 和VEGF受体2阻断抗体(R& D Systems,目录 编号:AF321和AF357)和FGF2中和抗体(Millipore, 克隆bFM-1)
    9. 棉花芽(药店,例如 Q-tip ®
    10. 用于标准免疫组织化学染色的溶液和抗体
  12. 神经干细胞培养基(见配方)
  13. 神经元分化培养基(参见配方)

设备

  1. 细胞培养离心机(例如, Megafuge 1.0 R,Kendro Laboratory)
  2. 37℃,5%CO 2细胞培养箱(例如Thermo Fisher Scientific,型号:Heracell 240)中。
  3. 计数室(,例如,Fuchs-Rosenthal,Marienfeld)
  4. 玻璃物体幻灯片(例如 Thermo Fisher Scientific,Superfrost Plus)
  5. 显微镜(例如,ZEISS,型号:Axiovert 200M)

程序

  1. Matrigel迁移测定
    1. 在4℃下在冰上解冻基质胶基质过夜
    2. 在冷DMEM/F12中以1:2的比例稀释基质胶基质 注意:1ml matrigel将足以准备4孔培养皿的12个孔。
    3. 每孔加入250微升的Matrigel基质混合物的4孔 并在细胞培养箱中孵育(37℃) ℃)至少30分钟进行硬化 注意:使用之前,请将移液器吸头置于-20°C。
    4. 胰酶消化细胞并使用计数室计数 注意:在这一步,细胞悬浮液可以与0.1%DNA酶孵育以避免细胞聚集。
    5. 通过在300×g离心4分钟来沉淀细胞
    6. 取出上清液并在细胞培养物中重悬100,000细胞/μl 培养基(例如含有具有一个体积%N 2补充物的DMEM/F12的神经元培养基和具有以1:1混合的两个体积%B27补充物的神经原 比率)。
      可选:添加5 mM岩石抑制剂用于细胞存活。
    7. 点1在细胞悬液的中间的凝胶表面上的菜。
      注意:指定的4孔培养皿有一个非常轻微的圆底,支持精确的点样。
    8. 孵育10分钟,仔细将它们转移到细胞培养箱,直到细胞附着
    9. 用相应的细胞培养基和小心地覆盖细胞 在细胞培养箱中培养,直到分析
    10. 从细胞团块的中心的径向迁移可以在 不同的时间点使用静态或活细胞显微镜(见图 1)。

  2. Boyden室迁移试验
    1. 将Millicell培养板插入24孔板并涂层 从两侧用聚-1-鸟氨酸。 孵育培养板 在37℃下至少2小时(例如在细胞培养箱中)
    2. 用millicell培养板插入物洗涤24孔皿3次 PBS,然后用层粘连蛋白(1:1000稀释于PBS中)包被
    3. 包装用石蜡膜的菜,并存储至少过夜,在4°C。
      注意:实验应一律执行三次。
    4. 胰酶消化细胞并使用计数室计数 注意:在这一步骤中,细胞悬浮液可以与DNAse温育以避免   细胞团块并通过尼龙网过滤(计数前)。
    5. 在300×g下将细胞沉淀4分钟。
    6. 除去上清液,并在神经分化培养基中重悬1×10 6个细胞/ml。
      注意:使用的培养基不应含有任何生长因子或细胞因子。
    7. 取下涂层悬浮液,并加载底部的孔 室与400-450微升培养基有或没有化学吸引剂 兴趣(例如10ng/ml VEGF或10ng/ml FGF2)。
      注意:
      1. 它 至关重要的是底部井中的介质覆盖底部   的膜,但不应上升到上侧。
      2. 为了避免气泡,将移液管牢牢地吸到腔室的边缘。
      3. 使用各自的化学吸引阻断剂作为对照(例如VEGF 受体1和VEGF受体2阻断抗体和FGF2 中和抗体)。
    8. 平板100微升的细胞悬液 分离上部的聚-1-鸟氨酸/层粘连蛋白包被的膜 和下井。
      注意:请勿触摸过滤器,避免产生气泡。
    9. 将室在37℃,5%CO 2培养箱中放置6-20小时。 的 孵育时间显着取决于细胞类型和 趋化因子
    10. 用4%PFA固定培养皿(用millicell培养板插入物),并用PBS洗涤两次
    11. 用DAPI染色膜的上下侧。
      注意:在荧光显微镜下检查细胞是否死亡 是均匀的(在膜的上侧和下侧)和 只能使用这些膜。细胞的不均匀分布 最可能是由于膜的不完全涂覆。小心 膜在涂覆期间不变干(点1-3)。
    12. 用棉芽擦去膜上侧的细胞(未迁移的细胞)。重复此步骤至少两次。
    13. 染色毫细胞培养板插入物(在24孔培养皿中)  适当标记(例如ßIII微管蛋白识别神经元,巢蛋白) 神经祖细胞)
    14. 到达膜底部的细胞可以在荧光显微镜下定量(图2) 注意:毫细胞培养板插入物可以放置在玻璃载玻片上,使用一滴PBS保持膜湿润。

代表数据



图1.(A-B)Matrigel迁移测定,解决与非纯化群体中的神经元相比的来自lt-NES细胞的纯化神经元的迁移能力。 将等量的细胞接种在基质胶基质上,48小时后分析。纯化的神经元显示跨越大面积的径向对称分布,其中个体神经元从铺板位点A迁移。相比之下,非纯化群体(含有神经元以及祖细胞)形成几乎没有任何神经元离开这些聚集体的球形簇。相反,观察到径向轴突生长B.比例尺:A-B。 200μm

A

B

图2.通过使用millicell培养板插入物研究的细胞迁移。 A.将细胞接种在上部孔的膜上。可以将化学引诱物加入到下部孔中。细胞从上部孔迁移 通过膜可以通过使用棉芽从膜的上侧擦拭剩余的细胞并计数到达底侧的细胞来测量。 摘自Erlandsson(2003)。 B.评估细胞混合物(含有神经元和祖细胞)和纯化的神经元内神经元的迁移。 条表示在20小时后达到膜下侧的神经元的百分比。 将细胞数量相对于接种的神经元的数量标准化,并显示为平均值±SD(* P <0.05)。

笔记

Millicell培养板插入物的质量可以变化,因此,如Boyden腔室迁移方案的第11点所述,确保细胞分泌在膜的上侧和下侧均匀是至关重要的。

食谱

  1. 神经干细胞培养基
    500 ml DMEM/F12,高葡萄糖
    5ml N <2>补充剂
    通过膜可以通过使用棉芽从膜的上侧擦拭剩余的细胞并计数到达底侧的细胞来测量。 摘自Erlandsson(2003)。 B.评估细胞混合物(含有神经元和祖细胞)和纯化的神经元内神经元的迁移。 条表示在20小时后达到膜下侧的神经元的百分比。 将细胞数量相对于接种的神经元的数量标准化,并显示为平均值±SD(* P <0.05)。

    笔记

    Millicell培养板插入物的质量可以变化,因此,如Boyden腔室迁移方案的第11点所述,确保细胞分泌在膜的上侧和下侧均匀是至关重要的。

    食谱

    1. 神经干细胞培养基
      500 ml DMEM/F12,高葡萄糖
      5ml N <2>补充剂
      ... 通过膜可以通过使用棉芽从膜的上侧擦拭剩余的细胞并计数到达底侧的细胞来测量。 摘自Erlandsson(2003)。 B.评估细胞混合物(含有神经元和祖细胞)和纯化的神经元内神经元的迁移。 条表示在20小时后达到膜下侧的神经元的百分比。 将细胞数量相对于接种的神经元的数量标准化,并显示为平均值±SD(* P <0.05)。

      笔记

      Millicell培养板插入物的质量可以变化,因此,如Boyden腔室迁移方案的第11点所述,确保细胞分泌在膜的上侧和下侧均匀是至关重要的。

      食谱

      1. 神经干细胞培养基
        500 ml DMEM/F12,高葡萄糖
        5ml N <2>补充剂
        ...... 通过膜可以通过使用棉芽从膜的上侧擦拭剩余的细胞并计数到达底侧的细胞来测量。 摘自Erlandsson(2003)。 B.评估细胞混合物(含有神经元和祖细胞)和纯化的神经元内神经元的迁移。 条表示在20小时后达到膜下侧的神经元的百分比。 将细胞数量相对于接种的神经元的数量标准化,并显示为平均值±SD(* P <0.05)。

        笔记

        Millicell培养板插入物的质量可以变化,因此,如Boyden腔室迁移方案的第11点所述,确保细胞分泌在膜的上侧和下侧均匀是至关重要的。

        食谱

        1. 神经干细胞培养基
          500 ml DMEM/F12,高葡萄糖
          5ml N <2>补充剂
          .........
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引用:Ladewig, J., Koch, P. and Brüstle, O. (2015). In vitro Migration Assays for Neural Stem Cells, Intermediate Neurogenic Progenitors and Immature Neurons. Bio-protocol 5(1): e1371. DOI: 10.21769/BioProtoc.1371.
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