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A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from embryonic animals and electroporated or infected with viral vectors, it is also a useful approach for the study of gene function at embryonic stages. Here, we present a retinal explant culture method that we have used extensively in various publications (Kechad et al., 2012; Cayouette et al., 2003; Cayouette and Raff, 2003; Elliott et al., 2008).

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Retinal Explant Culture
视网膜外植体培养

神经科学 > 发育 > 视网膜培养
作者: Christine Jolicoeur
Christine JolicoeurAffiliation: Cellular Neurobiology, Institut de Recherches Cliniques de Montreal, Montreal, Canada
Bio-protocol author page: a1125
 and Michel Cayouette
Michel CayouetteAffiliation: Cellular Neurobiology, Institut de Recherches Cliniques de Montreal, Montreal, Canada
For correspondence: michel.cayouette@ircm.qc.ca
Bio-protocol author page: a520
Vol 4, Iss 2, 1/20/2014, 7049 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1032

[Abstract] A particularly powerful culture method for the retina is the explant assay, which consists in culturing a small piece of retina on an organotypic filter. Retinal explants can be prepared any time between embryonic day 13 (E13) and postnatal day 4 (P4). Although retinal ganglion cells tend to degenerate shortly after they are generated in explants, and photoreceptor cells do not grow extended outer segments, the explants will develop very similarly to a retina in vivo and generate all the different retinal cell types that will migrate to the appropriate layer. The retinal explant culture assay is particularly useful in cases where a mouse mutant is embryonic lethal and its retinal development cannot be studied in vivo. Because retinal explants can be prepared from embryonic animals and electroporated or infected with viral vectors, it is also a useful approach for the study of gene function at embryonic stages. Here, we present a retinal explant culture method that we have used extensively in various publications (Kechad et al., 2012; Cayouette et al., 2003; Cayouette and Raff, 2003; Elliott et al., 2008).
Keywords: Cell lineage(细胞谱系), Neural progenitor(神经祖细胞), Asymmetric division(不对称分裂), Organotypic culture(器官培养), Differentiation(区别)

[Abstract] 视网膜的特别强大的培养方法是外植体测定,其包括在器官型过滤器上培养小片视网膜。视网膜外植体可以在胚胎第13天(E13)和出生后第4天(P4)之间的任何时间进行。虽然视网膜神经节细胞在外植体中产生后不久就会退化,并且感光细胞不会扩展外延伸的外切片,外植体将在体内与视网膜非常相似,并产生将迁移到适当的所有不同的视网膜细胞类型层。视网膜外植体培养测定在小鼠突变体是胚胎致死性并且其视网膜发育不能在体内研究的情况下特别有用。因为视网膜外植体可以从胚胎动物制备并用病毒载体电穿孔或感染,因此也是胚胎期基因功能研究的有用途径。在这里,我们提出了我们在各种出版物中广泛使用的视网膜外植体培养方法(Kechad等,2012; Cayouette等,2003; Cayouette和Raff,2003; Elliott等,2008)。

Materials and Reagents

  1. Animals
    We typically use retinas from albino Sprague Dawley rats or mice. Explants can be prepared from animals aged between embryonic day 12 to post-natal day 4.
  2. DPBS (Life Technologies, catalog number: 14040 )
  3. DMEM (Life Technologies, catalog number: 10569-010 )
  4. Fetal bovine serum (FBS) (Wisent, catalog number: 080-450 )
  5. Penicillin/streptomycin (Life Technologies, catalog number: 15070 )
  6. 70% ethanol
  7. Fungizone antimycotic (Life Technologies, catalog number: 15290026 )
  8. Sucrose (Sigma-Aldrich, catalog number: S0389 )
  9. O.C.T. compound (Somagen, catalog number: 4583 )
  10. Explant medium
  11. 4% Paraformadehyde (PFA) solution (Electron Microscopy Sciences, catalog number: 15710 ) (see Recipes)
  12. 20% sucrose/OCT (1:1) (see Recipes)
  13. 20% sucrose solution (see Recipes)

Equipment

  1. Millicell organotypic insert (EMD Millipore, catalog number: PICMORG50 )
  2. 35 mm dish or 6 well plates
  3. 37 °C and 5% CO2 incubator
  4. 100 mm dish
  5. 150 mm dish
  6. Straight forcep
  7. FIne forcep (Fine Science Tools, catalog number: 11252-23 )
  8. Curved forcep (Fine Science Tools, catalog number: 91197-00 )
  9. Sharp scissor
  10. Sharp scapel (Fine Science Tools, catalog number: 10315-12 )
  11. Razor blade
  12. P1000 pipette
  13. CO2 chamber
  14. Cryomold 15 x 15 x 5 mm (VWR International, catalog number: CA60872-016 )

Procedure

Day of Experiment

  1. Prepare the explant medium (see Recipes for the preparation of the stock solutions)
    1. Explant medium
      In a 50 ml tube, add: 45 ml of DMEM
      5 ml of fetal bovine serum (FBS)
      500 µl of pen/strep 100x
      50 µl of Fungizone antimycotic
      To equilibrate the medium at the right temperature and pH, put the lid on the 50 ml tube and unscrew ¼ of a turn. Place the tube in the incubator at 37 °C and 5% CO2 until needed.
  2. Preparation of the culture plates
    1. The explants are cultured on a Millicell organotypic insert. We typically use 35 mm dish or 6 well plates to put the insert in.
    2. Fill the dish (or well) with 1.2 ml of equilibrated explants medium and place the insert gently over it with sterile forceps.
    3. Place the dish (or plate) in the incubator at 37 °C and 5% CO2 until needed.
  3. Dissection of the retinas
    Note: The dissection should be carried out under sterile conditions. Spray the working area with 70% ethanol and use a flame. Sterilize all the instruments before use.
    Prepare two 100 mm dish filled with 10 ml of sterile DPBS, one to transfer the heads and the other to dissect the eyes and two 35 mm dish filled with 2 ml of sterile DPBS to collect the retinas.
    1. Collection of the embryos
      1. Euthanize the animal in a CO2 chamber.
      2. Place the animal on its back and clean the abdomen with 70% ethanol.
      3. Pinch the skin with a straight forcep, lift the skin and muscle and make a V shape opening using scissors on both side of the abdomen, starting from the lower abdomen and cutting all the way up on the side of the animal. Lift the skin and muscle upward to reveal the internal organs and uterus.
      4. Cut out the uterus and transfer it to a 150 mm dish.
      5. Remove the embryos from the uterus and decapitate the heads with sharp scissors.
      6. Transfer the heads to a 100 mm dish filled with DPBS.
    2. Dissection of the retinas
      1. Carefully remove the skin that covers the eye.
      2. Place a curved forcep of both side of the eye and apply a gentle pressure. The eye should pup out.
      3. Pinch the back of the eye and pull.
      4. Transfer the eye in a 100 mm dish filled with DPBS.
        1. With the lens facing downward, secure the eye in position with a fine forceps and remove the optic nerve (Figure 1, steps 1-2).
        2. Introduce the tip of another forceps through the optic nerve head and between the choroid and retina to secure the sclera.
        3. Now that the sclera is secured, you need to gently tear it open with both forceps from the optic nerve head to the cornea to expose the retina and lens (Figure 1, steps 3-4).
        4. Carefully detach the lens and place the retinas in a 35 mm dish filled with DPBS (Figure 1, steps 5-6).
        5. From E18-19 onward, it is required to remove the blood vessels covering the retina to avoid endothelial cell contamination in the culture.  
        6. The blood vessels form a reddish membrane overlying the inner face of the retina. The membrane is easily removed by pinching and pulling with fine forceps.
        7. Transfer the retinas in a 35 mm dish filled with DPBS.


      Figure 1. Procedure for retina dissection. Steps 1-5 show the procedure to dissect the retina from the eyeball. Step 6 shows the dissected retina separated from the lens.

  4. Preparation of explants
    1. For retinas older than embryonic day 15, cut small pieces of around 5 mm (Figure 2, steps 1-2). For younger retinas, place the whole retina on the insert.
    2. With a razor blade, cut the edge of a P1000 pipette tip to make a lager opening. Transfer the explants from the 35 mm dish to the insert. It doesn’t matter which side of the explants is facing upward on the insert. Gently lay the explants as flat as possible (Figure 2, step 3). Try not to fold or tear the explants as this could lead to defect in the formation of the layers and make the analysis difficult.
    3. Place the dish in the incubator at 37 °C and 5% CO2. The culture should be left until the retina is fully developed (around post natal day 14 in vivo). For explants started at embryonic day 13, this would mean 20 days in culture. For explants started at post-natal day 0, this would mean 14 days in culture.
    4. The medium should be changed every 3 days by replacing 80% of the medium with fresh one.


      Figure 2. Preparation of the explants. Starting from an entire retina (Step 1), small pieces are cut (Step 2) and placed on the organotypic filter (Step 3).

  5. Fixation of the explants
    1. After the desired culture period, remove the insert from the dish and aspirate the culture medium.
    2. Replace it with freshly made 4% PFA. Place the insert back in the dish. Gently add 1 ml of 4% PFA on top of the insert. The PFA solution needs to be at room temperature to help maintain the explant morphology.
    3. Incubate 30 min. Gently rinse 3 times with PBS.

  6. Preparation of the explants for cryosectionning
    1. Replace the PBS with a solution of 20% sucrose. Make sure that the explants are well covered. Incubate at 4 °C overnight.
    2. For cryoprotection, fill a small cryomold with a mixture of 20% sucrose/OCT (1:1).
    3. Remove the 20% sucrose solution from the top of the insert, take out the insert from the dish and, using a sharp scapel, cut the membrane around each explant.
    4. Pick the piece of membrane/explant with a fine forceps and place it in the cryomold. Gently push down the explants and lay them at the bottom of the cryomold.
    5. Make sure it is covered with enough 20% sucrose/OCT solution.
    6. Snap-freeze in liquid nitrogen (do not immerse the cryomold in liquid nitrogen, just touch the surface of the nitrogen with the bottom of the mold) and store at -80 °C until ready to section.
    7. The section thickness can vary between 10 and 30 μm depending on the type of analysis needed.


      Figure 3. Micrographs of retinal explant cryosections at 10x (A) or 40x magnification after 14 days in culture. The retina was counterstained with Hoechst to visualize the nuclei. The retinal layers are identified, ONL: outer nuclear layer, INL: inner nuclear layer.

Recipes

  1. 4% paraformaldehyde
    10 ml of 16% paraformaldehyde
    26 ml of deionised water
    4 ml of 10x PBS (pH 7.4)
  2. 20% sucrose
    Dissolve 20 g of sucrose in 80 ml of 1x PBS (pH 7.4)
    Once in solution, complete the volume to 100 ml with 1x PBS (pH 7.4)
  3. 20% sucrose/O.C.T. (1:1)
    20 ml of 20% sucrose solution
    20 ml of O.C.T. compound
    Mix thoroughly

Acknowledgments

Funding for this work was provided by the Canadian Institutes of Health Research and the Foundation Fighting Blindness Canada. This protocol was adapted from procedures published in Kechad et al. (2012) and Elliott et al. (2008). The authors wish to thank members of the Cayouette lab, past and present, for continuous support and improvements on the protocol over the years.

References

  1. Cayouette, M., Barres, B. A. and Raff, M. (2003). Importance of intrinsic mechanisms in cell fate decisions in the developing rat retina. Neuron 40(5): 897-904.
  2. Cayouette, M. and Raff, M. (2003). The orientation of cell division influences cell-fate choice in the developing mammalian retina. Development 130(11): 2329-2339. 
  3. Elliott, J., Jolicoeur, C., Ramamurthy, V. and Cayouette, M. (2008). Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron 60(1): 26-39.
  4. Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W., Harris, W. A. and Cayouette, M. (2012). Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina.  J Neurosci 32(48): 17197-17210.

材料和试剂

  1. 动物
    我们通常使用来自白化Sprague Dawley大鼠或小鼠的视网膜。 外植体可以从在胚胎第12天到产后第4天之间的动物制备
  2. DPBS(Life Technologies,目录号:14040)
  3. DMEM(Life Technologies,目录号:10569-010)
  4. 胎牛血清(FBS)(Wisent,目录号:080-450)
  5. 青霉素/链霉素(Life Technologies,目录号:15070)
  6. 70%乙醇
  7. Fungizone antimycotic(Life Technologies,目录号:15290026)
  8. 蔗糖(Sigma-Aldrich,目录号:SO389)
  9. O.C.T. 化合物(Somagen,目录号:4583)
  10. 外植体介质
  11. 4%Paraformadehyde(PFA)溶液(Electron Microscopy Sciences,目录号:15710)(参见配方)
  12. 20%蔗糖/OCT(1:1)(参见配方)
  13. 20%蔗糖溶液(见配方)

设备

  1. Millicell器官插入物(EMD Millipore,目录号:PICMORG50)
  2. 35mm皿或6孔板
  3. 37℃和5%CO 2培养箱
  4. 100毫米培养皿
  5. 150 mm皿
  6. 直线力
  7. FIne forcep(Fine Science Tools,目录号:11252-23)
  8. 弯曲力(Fine Science Tools,目录号:91197-00)
  9. 锋利的剪刀
  10. Sharp scapel(Fine Science Tools,目录号:10315-12)
  11. 剃刀刀片
  12. P1000移液器
  13. CO 2室
  14. Cryomold 15×15×5mm(VWR International,目录号:CA60872-016)

程序

实验日

  1. 准备外植体培养基(参见制备储备溶液的配方)
    1. 外植体介质
      在50ml试管中,加入:45ml DMEM
      5ml胎牛血清(FBS)
      500μl的pen/strep 100x
      50μlFungizone antimycotic
      为了在适当的温度和pH下平衡培养基,将盖子放在50ml管上并旋开¼圈。 将管置于37℃和5%CO 2的培养箱中直至需要
  2. 培养板的制备
    1. 外植体在Millicell器官型插入物上培养。 我们通常使用35mm皿或6孔板将插入物放入。
    2. 填充菜(或井)与1.2毫升平衡的外植体培养基和   用无菌镊子将插入物轻轻地放置在其上。
    3. 放置菜 (或板)在37℃和5%CO 2培养箱中培养直至需要。
  3. 视网膜解剖
    注意:解剖应在无菌条件下进行。 用70%乙醇喷洒工作区域并使用火焰。 在使用前消毒所有仪器。
    准备两个100毫米的菜充满了10毫升的无菌DPBS,一个转移头和其他解剖的眼睛和两个35毫米的菜,填充2毫升无菌DPBS收集视网膜。
    1. 胚胎的集合
      1. 在CO 2室中对动物安乐死。
      2. 将动物放在其背部,用70%乙醇清洁腹部。
      3. 捏直皮肤与直的forcep,抬起皮肤和肌肉和 做一个V形开口使用剪刀在腹部的两边, 从下腹部开始并在侧面切割一直 的动物。 抬起皮肤和肌肉向上,揭示内部 器官和子宫。
      4. 切出子宫并将其转移到150mm的皿中。
      5. 从子宫中取出胚胎,用锋利的剪刀摘下头。
      6. 将头转移到填充有DPBS的100mm皿中。
    2. 视网膜的解剖
      1. 小心去除覆盖眼睛的皮肤。
      2. 放置一个弯曲的forcep的两侧的眼睛,并施加温和的压力。 眼睛应该小狗。
      3. 捏紧眼睛的后面,拉。
      4. 将眼睛转移到填充有DPBS的100mm皿中。
        1. 使镜片面朝下,用细镊子将眼睛固定在适当位置,并去除视神经(图1,步骤1-2)。
        2. 通过视神经头引入另一镊子的尖端 在脉络膜和视网膜之间以固定巩膜。
        3. 现在 巩膜是固定的,你需要轻轻地撕开它与两者打开 镊子从视神经头到角膜以暴露视网膜   透镜(图1,步骤3-4)。
        4. 小心地取下镜片,将视网膜放在装有DPBS的35 mm培养皿中(图1,步骤5-6)。
        5. 从E18-19起,需要去除血管 覆盖视网膜以避免内皮细胞污染 文化。  
        6. 血管形成上覆的微红色膜   视网膜的内表面。 膜很容易被去除 捏和拉用精细钳子。
        7. 将视网膜转移到填充有DPBS的35mm皿中。


      图1.视网膜解剖的程序。步骤1-5显示从视网膜解剖视网膜的程序。 步骤6显示从镜片分离的解剖的视网膜
  4. 外植体的制备
    1. 对于早于胚胎第15天的视网膜,切割约5mm的小块(图2,步骤1-2)。 对于年轻的视网膜,将整个视网膜放置在插入物上。
    2. 用剃刀刀片,切开P1000移液器吸头的边缘,做一个更大的开口。 转移外植体从35毫米的菜到插入。 外植体的哪一侧面向上是无关紧要的 插入。轻轻放置外植体尽可能平坦(图2,步骤3)。尽量不要折叠或撕裂外植体,因为这可能导致层的形成缺陷,使分析困难。
    3. 将培养皿置于37℃和5%CO 2的培养箱中。应该留下培养物直到视网膜完全发育(在产后14天内在体内)。对于在胚胎第13天开始的外植体,这意味着培养20天。对于在出生后第0天开始的外植体,这意味着培养14天
    4. 每3天更换一次培养基,更换80%的培养基

      图2.外植体的制备从整个视网膜(步骤1)开始,切割小块(步骤2),并置于器官型过滤器上(步骤3)。

  5. 固定外植体
    1. 在所需的培养期后,从盘中取出插入物并抽吸培养基。
    2. 更换为新鲜4%PFA。 将插入物放回盘中。 轻轻加入1毫升的4%PFA在插入物的顶部。 PFA溶液需要在室温下以帮助维持外植体形态。
    3. 孵育30分钟。 用PBS轻轻冲洗3次。

  6. 制备用于冷冻切片的外植体
    1. 用20%蔗糖溶液替换PBS。 确保外植体覆盖良好。 在4℃孵育过夜。
    2. 对于冷冻保护,用20%蔗糖/OCT(1:1)的混合物填充小的冷冻切片。
    3. 从插入物的顶部移除20%蔗糖溶液,从盘中取出插入物,并使用锋利的scapel,切割每个外植体周围的膜。
    4. 用细镊子挑取一块膜/外植体,并将其放在冷冻切片中。轻轻地推下外植体,并将它们放在冷冻切片的底部。
    5. 确保它覆盖了足够的20%蔗糖/OCT解决方案。
    6. 在液氮中快速冷冻(不要将冷冻切片浸入液氮中,只要接触模具底部的氮气表面),并储存在-80°C直到准备切片。
    7. 根据所需分析的类型,截面厚度可在10和30μm之间变化。


      图3.培养14天后在10×(A)或40×放大倍率下的视网膜外植体冷冻切片的显微照片。用Hoechst对视网膜进行复染以显现细胞核。识别视网膜层,ONL: 外核层,INL:内核层

食谱

  1. 4%多聚甲醛
    10ml 16%多聚甲醛
    26ml去离子水
    4ml 10×PBS(pH7.4)
  2. 20%蔗糖 将20g蔗糖溶于80ml 1×PBS(pH7.4)中
    一旦溶解,用1×PBS(pH7.4)将体积补至100ml,
  3. 20%蔗糖/0.C.T。 (1:1)
    20ml 20%蔗糖溶液 20ml O.C.T。 化合物
    彻底混合

致谢

这项工作的资金由加拿大卫生研究院和加拿大基金会战斗盲人提供。 该方案改编自Kechad等人(2012)和Elliott等人(2008)中公开的程序。 作者希望感谢Cayouette实验室的成员,过去和现在,多年来对协议的持续支持和改进。

参考文献

  1. Cayouette,M.,Barres,B.A。和Raff,M。(2003)。 发育中的大鼠视网膜中细胞命运决定的内在机制的重要性。 Neuron 40(5):897-904。
  2. Cayouette,M。和Raff,M。(2003)。 细胞分裂的方向影响发育中的哺乳动物视网膜中的细胞命运选择。 em> Development 130(11):2329-2339。 
  3. Elliott,J.,Jolicoeur,C.,Ramamurthy,V。和Cayouette,M。(2008)。 Ikaros赋予小鼠视网膜祖细胞早期时间能力。 Neuron em> 60(1):26-39。
  4. Kechad,A.,Jolicoeur,C.,Tufford,A.,Mattar,P.,Chow,R.W.,Harris,W.A。和Cayouette,M。(2012)。 Numb是在发育的小鼠视网膜中产生末端不对称细胞分裂所需的。 a>  J Neurosci 32(48):17197-17210。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jolicoeur, C. and Cayouette, M. (2014). Retinal Explant Culture. Bio-protocol 4(2): e1032. DOI: 10.21769/BioProtoc.1032; Full Text
  2. Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W., Harris, W. A. and Cayouette, M. (2012). Numb is required for the production of terminal asymmetric cell divisions in the developing mouse retina.  J Neurosci 32(48): 17197-17210.




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Christine Jolicoeur的其他实验方案(1)
Michel Cayouette的其他实验方案(1)