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Mar 2020
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Ex vivo Tissue Culture Protocols for Studying the Developing Neocortex
研究发育中的新皮质的体外组织培养方案   

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Abstract

The size of the neocortex and its morphology are highly divergent across mammalian species. Several approaches have been utilized for the analysis of neocortical development and comparison among different species. In the present protocol (Note: This protocol requires basic knowledge of brain anatomy), we describe three ex vivo neocortical slice/tissue culture methods: (i) organotypic slice culture (mouse, ferret, human); (ii) hemisphere rotation culture (mouse, ferret); and (iii) free-floating tissue culture (mouse, ferret, human). Each of these three culture methods offers distinct features with regard to the analyses to be performed and can be combined with genetic manipulation by electroporation and treatment with specific inhibitors. These three culture methods are therefore powerful techniques to examine the function of genes involved in neocortical development.

Keywords: Human (人类), Mouse (老鼠), Ferret (雪貂), Neocortex (大脑新皮质), Development (发育), Evolution (进化), Neural stem/Progenitor cells (神经干细胞/祖细胞)

Background

Several slice/tissue culture methods have been reported for studying brain development ex vivo (Noctor et al., 2001; Miyata et al., 2002; Tabata and Nakajima, 2003; Namba et al., 2007 and 2019; Schenk et al., 2009; Betizeau et al., 2013; Lim et al., 2018; Long et al., 2018; Nakagawa et al., 2019; Güven et al., 2020). In combination with in/ex utero and ex vivo genetic manipulation techniques [e.g., electroporation (Tabata and Nakajima, 2003; Namba et al., 2014), virus transfection (Noctor et al., 2001), and microinjection (Taverna et al., 2012)], the slice/tissue culture methods allow the investigation of cell behavior by time-lapse imaging (Miyata et al., 2001; Noctor et al., 2001; Tabata and Nakajima, 2003; Namba et al., 2011 and 2014; Taverna et al., 2012; Betizeau et al., 2013; Lim et al., 2018; Long et al., 2018; Nakagawa et al., 2019), signaling pathways by studying the effects of pharmacological reagents (Schenk et al., 2009; Long et al., 2018; Kalebic et al., 2019; Namba et al., 2020), and brain tissue development by immunohistochemistry (Schenk et al., 2009; Long et al., 2018; Güven et al., 2020; Namba et al., 2020) (Figure 1). In the present protocol, we describe three different methods to study developing neocortical tissue: (i) organotypic slice culture (Taverna et al., 2012); (ii) hemisphere rotation (HERO) culture (Schenk et al., 2009); and (iii) free-floating tissue (FFT) culture (Long et al., 2018).


Organotypic slice culture has been widely used over the past few decades (Noctor et al., 2001; Miyata et al., 2002; Tabata and Nakajima, 2003; Namba et al., 2007; Taverna et al., 2012) and readily allows time-lapse imaging of labeled cells (Miyata et al., 2001; Noctor et al., 2001; Tabata and Nakajima, 2003; Namba et al., 2011 and 2019; Taverna et al., 2012; Mora-Bermudez et al., 2014; Long et al., 2018; Nakagawa et al., 2019); however, the tissue can become damaged by slicing. The other two methods may overcome this potential problem. HERO culture was originally described by Schenk and colleagues (Schenk et al., 2009) and has been used for the pharmacological treatment of neocortical tissue (Schenk et al., 2009; Namba et al., 2020). FFT culture has recently been developed and used in our group (Long et al., 2018) to study human neocortical development following pharmacological and genetic manipulation (Long et al., 2018; Kalebic et al., 2019; Kostic et al., 2019; Namba et al., 2020).



Figure 1. Overview of ex vivo slice/tissue culture protocols and their applications. A. Summary of the three methods. B. Slice culture method. C. Hemisphere rotation (HERO) culture method. D. Free-floating tissue (FFT) culture method.


The optimal method for a given experiment depends on the species of the sample, the specific application, and the degree of tissue architecture preservation that is required. The cultured slice/hemisphere/tissue can be subjected to histological and biochemical analyses (Long et al., 2018; Kalebic et al., 2019; Kostic et al., 2019; Güven et al., 2020; Namba et al., 2020). Due to the limitation of sample availability, FFT and organotypic slice culture can lend themselves to the analysis of human neocortical development following manipulation of gene function (Long et al., 2018; Kalebic et al., 2019; Kostic et al., 2019; Namba et al., 2020). Time-lapse imaging (Taverna et al., 2012; Mora-Bermudez et al., 2014; Long et al., 2018) is generally performed in organotypic slice culture. If an experiment requires maintenance of intact tissue architecture, HERO and FFT cultures are the methods of choice.

Materials and Reagents

  1. Animals and human samples

    1. Ferret embryos (E33-E36)

    2. Human neocortical tissue [11 post-conception week (PCW)-14 PCW]

    3. Mouse embryos [embryonic day (E) 13.5-E15.5]


  2. Common materials and reagents

    1. Pasteur pipet (e.g., SARSTEDT, catalog number 86.1171.001)

    2. Fine-tip Pasteur pipet (e.g., SARSTEDT, catalog number 86.1175.001)

    3. 3.5-cm dishes (for time-lapse imaging, use glass-bottomed dishes, e.g., Thermo Fisher Scientific, Nunc, catalog number: 150680)

    4. 6-cm Petri dishes (Greiner, catalog number: 628102)

    5. 100× N2 supplement (Thermo Fisher Scientific, Invitrogen, catalog number:17502048)

    6. 100× Penicillin-Streptomycin (Merck, Gibco, catalog number: 15140122)

    7. 10 mM HEPES-NaOH (pH 7.3)

    8. 50× B27 supplement (Thermo Fisher Scientific, Invitrogen, catalog number: 17504044)

    9. Knockout Serum Replacement (KOSR, Thermo Fisher Scientific, Gibco, catalog number: 10828028)

    10. 200 mM L-glutamine (Thermo Fisher Scientific, Gibco, catalog number: 25030081)

    11. Neurobasal medium (Thermo Fisher Scientific, Gibco, catalog number: 21103049)

    12. PBS (made in-house)

    13. Rat serum (Charles River Laboratories Japan, catalog number: P00052)


  3. Organotypic slice culture

    1. DMEM-F12 (Merck, catalog number: D8900-10X1L)

    2. Low-melting agarose (Merck, catalog number: A2790)

    3. Sodium bicarbonate (NaHCO3) (Merck, catalog number: 1063290500)

    4. Type IA collagen (Nitta Gelatin, Cellmatrix, catalog number: 631-00651)

    5. Tyrode’s salt (Merck, catalog number: T2145)

    6. Tyrode's solution (see Recipes)

    7. 3% (w/v) low-melting agarose (see Recipes)

    8. Slice culture medium for mouse and ferret tissue (SCM, see Recipes)

    9. Slice culture medium for human tissue (SCM-KOSR, see Recipes)

    10. Collagen gel mixture (see Recipes)

    11. Reconstitution buffer (see Recipes)

    12. 5× DMEM-F12 solution (see Recipes)

Equipment

  1. Cell culture hood/biological safety cabinet for human samples

  2. Cell culture incubator

  3. Dissection microscope (e.g., Olympus, catalog number: SZX10)

  4. Dissection scissors (FST, catalog number: 15000-10)

  5. Heating plate (VWR, catalog number: 75838-286)

  6. Gas mixture (5% CO2 + 40% O2 + 55% N2 or 5% CO2 + 60% O2 + 35% N2)

  7. Scalpels (Surgical Disposable Scalpel, Braun, catalog number: 5518032)

  8. Spoon (for HERO culture, FST, catalog number: 10370-19)

  9. Spoon (for human tissue samples, chemical spoon)

  10. Vibratome (e.g., Leica, catalog number: VT1000S)

  11. Water bath

  12. Whole-embryo culture bottles (Nakayama, catalog number: 010-032-11)

  13. Whole-embryo culture system (Nakayama, catalog number: 10-0310)

Procedure

Part I: Organotypic slice culture

Please see Figure 2 for images of the selected procedures.


  1. Dissection and slicing

    1. Warm 2 × 50 ml Tyrode’s solution, SCM (see below) to 37°C.

    2. Melt 3% low-melting agarose in PBS and keep at 37°C.

    3. Dissect mouse or ferret embryos in Tyrode’s solution (3 embryos per experiment).

    4. Move the heads to warm Tyrode’s solution (37°C) and dissect the brains one by one in a 6-cm Petri dish.

    5. Dissect the telencephala and store on a heating plate in Tyrode’s solution.

      Note: For human tissues, start from this step.

    6. Remove the meninges after incubating the telencephala in Tyrode’s solution.

    7. Embed the telencephala in low-melting agarose (takes about 30 min).

    8. Cut the telencephala in PBS with a vibratome into 250-300-µm slices.

    9. Dissect the neocortical region of interest using a scalpel, if necessary.


  2. Collagen gel embedding and culture

    1. Prepare the collagen gel mixture (see below) on ice under a hood.

    2. Transfer the slices, pre-rinsed in collagen, to a dish containing collagen (on ice) using a Pasteur pipet and ensure that the slices are fully immersed in the collagen gel mixture by gently pipetting up and down.

    3. Transfer the slices and collagen gel mixture (200-300 µl) to a clean dish.

    4. Place the slices in the desired position.

    5. Remove the excess collagen using a fine-tip Pasteur pipet (<200 µl).

    6. Polymerize the collagen gel on a heating plate at 37°C for 5 min.

    7. Transfer the dishes to a cell culture incubator and incubate the slices for 30-40 min.

    8. Add 2 ml SCM (for mouse and ferret) or SCM-KOSR (for human) and continue slice culture for the desired time.

      Note: Start inhibitor treatment at this step.

    9. Keep the slices in the cell culture incubator at 37°C in an atmosphere of 5% CO2 + 40% O2 + 55% N2 for up to two days.


Part II: Hemisphere rotation (HERO) culture

  1. Dissection

    1. Dissect the mouse or ferret brain from the head and place into a 6-cm Petri dish containing PBS at room temperature.

    2. Remove the meninges in PBS.

      Note: You do not need to completely remove the meninges. If you are interested in the lateral neocortex, you can keep the meninges in the medial part.

    3. Remove the medulla and cerebellum.


  2. Culture

    1. Warm the SCM to 37°C.

    2. Add 1.5 ml SCM to a whole-embryo culture bottle.

    3. Transfer the hemisphere(s) to the whole-embryo culture bottle (1-3 hemispheres per bottle) using a spoon.

      Note: Start inhibitor treatment at this step.

    4. Place the hemispheres in the whole-embryo culture incubator at 37°C, in an atmosphere of 5% CO2 + 40% O2 + 55% N2 for mouse tissue and 5% CO2 + 60% O2 + 35% N2 for ferret tissue, with continuous rotation at 6 rpm for up to two days.


Part III: Free-floating tissue (FFT) culture

  1. Dissection and culture of mouse and ferret neocortex

    1. Dissect the brain from the head and place in a 6-cm Petri dish containing PBS at room temperature.

    2. Remove the meninges in PBS.

    3. Dissect the neocortical region of interest using a scalpel and dissection scissors. The size of the tissue is approximately 20-50 mm2.

    4. Warm the SCM to 37°C.

    5. Add 1.5 ml SCM to a whole-embryo culture bottle.

      Note: Start inhibitor treatment at this step.

    6. Transfer the tissue to the whole-embryo culture bottle (1-2 tissue pieces per bottle) using a spoon.

    7. Place the tissue in the whole-embryo culture incubator at 37°C, in an atmosphere of 5% CO2 + 40% O2 + 55%N2 for mouse tissue and 5% CO2 + 60% O2 + 35% N2 for ferret tissue, with continuous rotation at 6 rpm for up to three days.


  2. Dissection, pre-incubation, and culture of human neocortex

    1. Remove the meninges in PBS.

    2. Dissect the neocortical region of interest using a scalpel and dissection scissors. The size of the tissue is approximately 20-50 mm2.

    3. Warm the SCM to 37°C.

    4. Add 1.5 ml SCM-KOSR to a whole-embryo culture bottle.

    5. Transfer the tissue to the whole-embryo culture bottle (1-2 tissue pieces per bottle) using a spoon.

    6. Pre-incubate the tissues in the whole-embryo culture incubator at 37°C in an atmosphere of 5% CO2 + 60% O2 + 35% N2 with continuous rotation at 6 rpm for 3-5 h.

    7. Remove the SCM-KOSR and add 1.5 ml fresh SCM-KOSR to the bottle.

      Note: Start inhibitor treatment at this step.

    8. Place the tissues in the whole-embryo culture incubator at 37°C in an atmosphere of 5% CO2 + 60% O2 + 35% N2 with continuous rotation at 6 rpm for up to three days.



      Figure 2. Images illustrating key steps of the ex vivo slice/tissue culture protocols. Representative images of the slice, hemisphere rotation (HERO), and free-floating tissue (FFT) culture methods. The combination of letters and numbers corresponds to the steps of each protocol. Arrowheads indicate the neocortical slices/tissues/hemispheres.

Recipes

  1. Tyrode's solution

    1. Dissolve Tyrode’s salt and sodium bicarbonate (NaHCO3, 1 g for 1 L) in sterile water

    2. Add 13 ml 1 M HEPES-NaOH (pH 7.3) for 1 L

    Sterile-filter the solution

  2. 3% (w/v) low-melting agarose

    Low-melting agarose (3 g)

    Sterile PBS (100 ml)

  3. Slice culture medium for mouse and ferret (SCM) tissue, 100 ml

    Neurobasal medium (84 ml),

    Rat serum (10%, vol/vol) (10 ml)

    200 mM L-glutamine (1 ml)

    100× Pen-strep (1 ml)

    100× N2 supplement (1 ml)

    50× B27 supplement (2 ml)

    1 M HEPES-NaOH (pH 7.3) (1 ml)

    Store aliquots at -20°C

  4. Slice culture medium for human tissue (SCM-KOSR), 100 ml

    Neurobasal medium (84 ml)

    KOSR (10%, vol/vol) (10 ml)

    200 mM L-glutamine (1 ml)

    100× Pen-strep (1 ml)

    100× N2 supplement (1 ml)

    50× B27 supplement (2 ml)

    1 M HEPES-NaOH (pH 7.3) (1 ml)

    Store aliquots at -20°C

  1. Collagen gel mixture, 2.5 ml

    Type IA collagen (1.25 ml)

    Distilled water (0.5 ml)

    5× DMEM-F12 solution (0.5 ml)

    Reconstitution buffer (0.25 ml)

  2. Reconstitution buffer (100 ml)

    NaHCO3 (262 mM, 2.2 g in 100 ml)

    1 M NaOH (5 ml for 100 ml)

    1 M HEPES-NaOH (pH 7.3) (20 ml for 100 ml)

    Add distilled water to 100 ml

    Sterile-filter the solution and store at 4°C in air-tight tubes

  3. 5× DMEM-F12 solution (200 ml)

    Add 1 bottle DMEM-F12 to 200 ml distilled water

Acknowledgments

This protocol was adapted from Long et al. (2018), Güven et al. (2020), and Namba et al. (2020).

We are grateful to P. Wimberger for providing human fetal samples, and to the Services and Facilities of the Max Planck Institute of Molecular Cell Biology and Genetics for the outstanding support provided, notably J. Helppi and his Biomedical Services (BMS) team. We would like to thank all members of the Huttner group for helpful discussions. We appreciate support from the Laboratory Animal Center of the University of Helsinki. We thank V. Gkini for her technical assistance. We also thank D. Gerrelli, S. Lisgo, and their teams at the HDBR for the invaluable support from this resource. W.B.H. was supported by grants from the DFG (SFB 655, A2), ERC (250197), and ERA-NET NEURON (MicroKin).

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments (mice and ferrets) were performed in accordance with the German Animal Welfare legislation (‘‘Tierschutzgesetz’’). All procedures regarding the animal experiments were approved by the Governmental IACUC (‘‘Landesdirektion Sachsen’’) and overseen by the Institutional Animal Welfare Officer(s). The mouse embryo shown in Figure 2 was collected with approval from the University of Helsinki.

Fetal human brain tissue (PCW 10-14) was obtained from the Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe, Universitätsklinikum Carl Gustav Carus of the Technische Universität Dresden, with approval from the local University Hospital Ethical Review Committees and informed written maternal consent, and from the Human Development Biology Resource (HDBR), with the human fetal material being provided by the Joint MRC/Wellcome Trust (MR/R006237/1) Human Developmental Biology Resource (http://www.hdbr.org/).

References

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简介

[摘要]新皮层和它的形态的大小是高度在哺乳动物物种中发散的。小号everal方法已被用来为ANALY的SIS新皮层发展和COMPAR ISON之间不同的物种。在本协议中(注:该协议需要脑解剖学的基础知识),我们描述了三种离体的新皮层切片/组织培养方法:(i )器官切片培养(小鼠,雪貂,人); (ii)半球旋转培养(小鼠,雪貂);(iii)自由漂浮的组织培养物(小鼠,雪貂,人)。这三种培养方法中的每一种都针对要进行的分析提供了独特的功能,并且可以与通过电穿孔进行的遗传操作相结合,并使用特定的抑制剂进行处理。这三种培养方法,因此强大的技术,以考试系统Ë基因的功能涉及新皮层的发展。

[Backgrou ND ]几个切片/组织培养方法已被报道为研究大脑发育在体外(Noctor等,2001;宫田等,2002;田端和中岛,2003;难波等人,2007年至2019年,申克等等人,2009; Betizeau等人,2013; Lim等人,2018; Long等人,2018; Nakagawa等人,2019;Güven等人,2020)。在C ombin通货膨胀与在/前子宫内和离体内遗传操作技术[例如,电穿孔(田端和中岛,2003;难波。等人,2014) ,病毒转染(Noctor 。等人,2001) ,和显微注射(塔韦尔纳等。人,2012)] ,切片/组织培养方法允许的investigat的离子通过延时成像细胞行为(宫田等人,2001; Noctor 。等人,2001;田端和中岛,2003;难波等。(2011年和2014年; Taverna等人,2012年; Betizeau等人,2013年; Lim等人,2018年; Long等人,2018年; Nakagawa等人,2019年),通过研究药理试剂的作用来传递信号通路。(Schenk et al。,2009; Long et al。,2018; Kalebic et al。,2019; Namba et al。,2020),以及通过免疫组织化学进行脑组织发育(Schenk et al。,2009; Long et al。,2018) ;Güven等人,2020; Namba等人,2020)(图1)。在本协议中,我们描述了三种不同的方法来研究开发新皮层组织:(我)器官切片培养(塔韦尔纳等人。,2012); (ii)半球旋转(HERO)培养(Schenk等,2009);和(ⅲ)的自由浮动的组织(FFT)培养(龙等人,2018)。

器官切片培养已被广泛使用在p AST几十年(Noctor等,2001;宫田等,2002;田端和中岛,2003;难波等人。,2007;来自Taverna等,2012)和容易允许对标记的细胞进行延时成像(Miyata等人,2001; Noctor等人,2001; Tabata和Nakajima,2003; Namba等人,2011和2019 ; Taverna等人,2012; Mora-Bermudez等人。,2014;长等人,2018;中川等人。,2019); ħ H但是,所述组织可被过来损坏由切片。其他两种方法可以克服此潜在问题。HERO培养最初由Schenk及其同事描述(Schenk等,2009),并且已经用于新皮质组织的药理学治疗(Schenk等,2009; Namba等,2020)。FFT文化最近开发和使用在我们的小组(龙等,2018) ,以研究人类大脑皮层的发展以下药理和遗传操作(长等人。,2018; Kalebic等,2019;科斯蒂奇等人,2019 ; Namba等人,2020)。





图1.离体切片/组织培养方案及其应用概述。A.三种方法的总结。B.小号虱培养方法。C. ħ Emisphere公司旋转(雄)培养方法。D. ˚F REE浮动组织(FFT)培养方法。



给定实验的最佳方法取决于样品的种类,特定的应用以及所需的组织结构保存程度。可以对培养的切片/半球/组织进行组织学和生化分析(Long等人,2018; Kalebic等人,2019; Kostic等人,2019;Güven等人,2020; Namba等人,2020 )。由于该限制的样品的可用性,FFT和器官切片培养可以借给自己的ANALY SIS的人新皮层发展以下基因功能的操作(长等人,2018; Kalebic等人,2019;科斯蒂奇。等人,2019 ; Namba等人,2020)。延时成像(Taverna等,2012; Mora-Bermudez等,2014; Long等,2018)通常在器官型切片培养中进行。如果实验需要的维护完整组织架构,英雄和FFT文化是选择的方法。

关键字:人类, 老鼠, 雪貂, 大脑新皮质, 发育, 进化, 神经干细胞/祖细胞



材料和试剂


动物和人类样本
雪貂胚胎(E33-E36)
人新皮层组织[受孕后第11周(PCW)-14 PCW]
小鼠胚胎[胚胎日(E)13.5-E15.5]


联合MMON材料和试剂
巴斯德吸管(例如,SARSTEDT,目录号86.1171.001)
细头巴斯德吸管(例如,SARSTEDT,目录号86.1175.001)
3.5 cm培养皿(对于时间推移成像,使用克升屁股-底部ED菜,例如,热Fisher Scientific公司,Nunc公司,目录号:150680)
6厘米培养皿(Greiner,货号:628102)
100 × N2补充物(热费舍尔小号系统求解,Invitrogen公司,Ç atalog号:17502048)
100 ×青霉素-链霉素(默克,Gibco公司,Ç atalog号:15140122 )
10 mM HEPES - NaOH (pH 7.3)
50 × B27添加剂(热费舍尔小号系统求解,Invitrogen公司,Ç atalog号:17504044)
敲除血清替代品(KOSR,热费舍尔小号系统求解,Gibco公司,Ç atalog号:10828028)
200 mM的L-克lutamine(热费舍尔小号系统求解,Gibco公司,Ç atalog号:25030081 )
的Neurobasal米edium(热费舍尔小号系统求解,Gibco公司,Ç atalog号:21103049 )
PB S(内部制造)
血清中的R (日本Charles River Laboratories,目录号:P00052)


器官型切片培养
DMEM-F12(默克(Merck),目录号:D8900-10X1L)
低熔点琼脂糖(Merck ,目录号:A2790 )
碳酸氢钠(NaHCO 3 )(Merck,目录号:1063290500)
I型A胶原蛋白(Nitta明胶,Cellmatrix ,目录号:631-00651 )
蒂罗德盐(默克(Merck),目录号:T2145 )
泰罗德的解决方案(请参阅食谱)
3%(W / V)升流-熔化琼脂糖(见配方)
用于小鼠和雪貂组织的切片培养基(SCM ,请参见食谱)
用于人体组织的切片培养基(SCM -KOSR ,请参见食谱)
胶原蛋白凝胶混合物(请参阅食谱)
ř econstitution缓冲器(见配方)
5 × DMEM-F12解决方案(请参阅食谱)


设备


细胞培养柜/生物安全柜
细胞培养箱
解剖显微镜(例如,Olympus,目录号:SZX10)
解剖剪小号(FST,目录号:15000-10 )
加热板(VWR,目录号:75838-286)
混合气体(5%CO 2 + 40%O 2 + 55%N 2或5%CO 2 + 60%O 2 + 35%N 2 )
手术刀(外科一次性手术刀,Braun,目录号:5518032)
汤匙(用于HERO培养,FST,目录号:10370-19 )
汤匙(用于人体组织样本,化学汤匙)
振动切片机(例如,莱卡,目录号:VT1000S)
水洗澡
全胚培养瓶(中山,目录号010-032-11 )
全胚培养系统(中山,目录号10-0310 )


程序


第一部分:器官型切片培养


P租赁参见图2的所选择的程序的图像。


解剖和切片
将2 × 50 ml的Tyrode's溶液SCM(请参见下文)加热至37 °C 。
熔融3%在PBS中的琼脂糖低熔点和ķ EEP在37 ℃下。
在Tyrode溶液中解剖小鼠或雪貂胚胎(每个实验3个胚胎)。
将磁头移到预热台氏溶液(37 ℃下)和解剖大脑逐一在6厘米P ETRI菜。
解剖的telencephala上并存储一个在台氏液加热板。
注意:F或人体组织,请从此步骤开始。


孵育后删除脑膜telencephala我ñ台氏液。
嵌入的telencephala在低熔点琼脂糖(约需30分钟)。
切的telencephala用PBS中一个振动切片机成250 - 300 -微米切片。
              如有必要,用手术刀解剖感兴趣的新皮层区域。


胶原蛋白凝胶的包埋和培养
P repare与c ollagen凝胶混合物(见下文)上的冰的罩下。
转移的切片,预冲洗中的胶原蛋白,以一个培养皿含有胶原(在冰上)使用巴斯德吸移管,并确保所述片完全由向下轻轻吹打向上和浸渍在胶原凝胶混合物。
转移的切片和胶原凝胶混合物(200-300微升)到一个干净的培养皿中。
将切片放在所需的位置。
ř EMOVE的过量胶原使用细尖巴斯德吸管(<200微升)。
聚合的胶原凝胶上一个加热板在37 ℃下5分钟。
Ť转让(BOT)的菜一个细胞培养培养箱中孵育切片30 - 40分钟。
加入2 ml的SCM (小鼠和雪貂)或SCM-KOSR(人类),并继续培养切片为所述期望的时间。
注意:š在该步骤馅饼抑制剂治疗。


保持所述切片在细胞培养恒温箱中在37 ℃下在5%CO的气氛中2 + 40%氧气2 + 55%N 2为长达两天。


第二部分:^ h EM isphere旋转(HERO)文化


解剖
DISS ECT的鼠标或雪貂b雨水从头部和对花边成一个6厘米的P ETRI培养皿含有PBS在室温下。
除去PBS中的脑膜。
注:Ÿ欧不需要完全去除的脑膜。如果你有兴趣在横向大脑皮层,你可以保持在脑膜的内侧部分。


取出延髓和小脑。


文化
将SCM加热到37 °C 。
向整个胚胎培养瓶中加入1.5 ml SCM。
用勺子将半球转移到整个胚胎培养瓶中(每瓶1-3个半球)。
注意:š在该步骤馅饼抑制剂治疗。


将半球放置在37 °C的全胚培养箱中,在5%CO 2 + 40%O 2 + 55%N 2的小鼠组织和5%CO 2 + 60%O 2 + 35%N的气氛中2为雪貂组织,与在6rpm连续旋转为至多两个天内进行付运。


第三部分:自由浮动组织(FFT )培养


小鼠和雪貂新皮层的解剖和培养
解剖的从脑的头和对花边在一个6厘米P ETRI培养皿含有PBS在室温下。
除去PBS中的脑膜。
解剖用手术刀和解剖剪刀感兴趣的新皮层区域小号。的组织的尺寸为大约20 - 50毫米2 。
将SCM加热到37 °C 。
向整个胚胎培养瓶中加入1.5 ml SCM。
注意:š在该步骤馅饼抑制剂治疗。


用勺子将组织转移到整个胚胎培养瓶中(每瓶1-2个组织块)。
将组织置于37 °C的全胚培养箱中,在5%CO 2 + 40%O 2 + 55%N 2的小鼠组织和5%CO 2 + 60%O 2 + 35%N的气氛中2为雪貂组织,与在6rpm连续旋转为至多吨重稀土天。


夹层,预孵化,文化人类大脑皮层的
除去PBS中的脑膜。
解剖用手术刀和解剖剪刀感兴趣的新皮层区域小号。的组织的尺寸为大约20 - 50毫米2 。
将SCM加热到37 °C 。
将1.5 ml SCM-KOSR添加到全胚培养瓶中。
转移的组织的全胚胎培养瓶(1 -用勺子每瓶2组织片)。
预孵育的组织在在全胚胎培养培养箱中37 ℃下在5%CO的气氛中2 + 60%氧气2 + 35%N 2与连续旋转以6rpm 35 ħ 。
取下SCM-KOSR,然后向瓶中加入1.5 ml新鲜的SCM-KOSR。
注意:š在该步骤馅饼抑制剂治疗。


放置组织在全胚胎培养培养箱中于37 ℃下在5%CO的气氛中2 + 60%氧气2 + 35%N 2与连续旋转以6rpm为至多吨重稀土天。




图2 。图像说明了离体切片/组织培养方案的关键步骤。的代表性图像的切片,半球旋转(英雄),和自由浮动的组织(FFT)培养方法小号。字母和数字的组合对应于每个协议的步骤。箭头指示新皮层片/组织/他中号ispheres。


菜谱


泰罗德的解决方案
一种。将特罗德氏盐和碳酸氢钠(NaHCO 3 ,每1升1克)溶解在无菌水中     

b。加入13 ml 1 M HEPES - NaOH (pH 7.3)1 L     

小号terile-F ILTER牛逼,他的解决方案


3%(W / V)升流-熔化琼脂糖
低-熔化琼脂糖(3克)


无菌PBS(100毫升)


小鼠和雪貂(SCM)组织的切片培养基,100毫升
              的Neurobasal米edium(84毫升),


血清中的R (10%,体积/体积)(10 ml)


2 00 mM L- g谷氨酰胺(1 ml)


              100 ×笔-链球菌(1毫升)


100 × N2补充剂(1毫升)


50 × B27补充剂(2毫升)


1 M HEPES - NaOH (pH 7.3)(1毫升)


商店等分试样在- 20 ℃下


人体组织切片培养基(SCM -KOSR ),100 ml
              的Neurobasal米edium(84毫升)中


KOSR (10%,体积/体积)(10毫升)


2 00 mM L- g谷氨酰胺(1 ml)


              100 ×笔-链球菌(1毫升)


100 × N2补充剂(1毫升)


50 × B27补充剂(2毫升)


1 M HEPES - NaOH (pH 7.3)(1毫升)


将等分试样储存在-20 °C


胶原凝胶混合物,2.5米升
类型IA胶原(1.25米升)


d istilled水(0.5米升)


5 × DMEM-F12溶液(0.5毫升)


ř econstitution缓冲器(0.25米升)


ř econstitution缓冲器(100毫升)中
的NaHCO 3 (262毫,2.2克在100毫升)中


1 M NaOH (5毫升/ 100毫升)


1 M HEPES - NaOH (pH 7.3)(20毫升/ 100毫升)


加入蒸馏水至100毫升


小号terile-F ILTER吨他溶液和在气密管储存于4℃下


5 × DMEM-F12溶液(200毫升)
                                                                      将1瓶DMEM-F12加到200毫升蒸馏水中


致谢


该协议改编自Long等。(2018),Güven等。(2020年),以及Namba等人。(2020)。


我们感谢为P 。Wimberger酒店提供人类胚胎样本,并以服务及设施分子的马克斯·普朗克研究所的细胞生物学和遗传学所提供的出色的支持,特别是J. Helppi和他的生物医学服务(BMS)的球队。我们要感谢所有成员Huttner组有用d iscussions 。我们感谢支持从赫尔辛基大学的实验动物中心。我们感谢V. Gkini的技术支持。我们也感谢D. Gerrelli ,S。Lisgo及其在HDBR上的团队提供的宝贵资源支持。WBH得到了DFG(SFB 655,A2),ERC(250197)和ERA-NET NEURON (MicroKin )的资助。


利益争夺


作者宣称没有利益冲突。


伦理


所有动物实验(小鼠和雪貂)均根据德国动物福利法规(“ Tierschutzgesetz ”)进行。有关动物实验的所有程序均已获得IACUC政府的批准(“ Landesdirektion Sachsen”),并受到机构动物福利官员的监督。m个图2所示乌斯胚胎收集与批准从赫尔辛基大学。


从获得的胚胎脑组织(PCW 10-14)KLINIK UND Poliklinik献给Frauenheilkunde UND Geburtshilfe ,Universitätsklinikum卡尔·古斯塔夫·卡鲁斯的的TECHNISCHE Universität大学德累斯顿,经批准由当地的大学医院伦理审查委员会一ñ d写书面同意的产妇,由人类发展生物学资源(HDBR)提供,人类胎儿材料由MRC /惠康联合信托(MR / R006237 / 1)人类发展生物学资源(http://www.hdbr.org/)提供。


参考


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Copyright Namba et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Namba, T., Haffner, C. and Huttner, W. B. (2021). Ex vivo Tissue Culture Protocols for Studying the Developing Neocortex. Bio-protocol 11(10): e4031. DOI: 10.21769/BioProtoc.4031.
  2. Güven, A., Kalebic, N., Long, K. R., Florio, M., Vaid, S., Brandl, H., Stenzel, D. and Huttner, W. B. (2020). Extracellular matrix-inducing Sox9 promotes both basal progenitor proliferation and gliogenesis in developing neocortex. Elife 9: e49808.
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