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Dissociated Retinal Cell Culture
视网膜细胞的分离培养   

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Abstract

The retina is a relatively simple and accessible part of the central nervous system, making it a powerful model to study cell fate specification mechanisms. Multipotent retinal progenitor cells (RPCs) give rise to seven major classes of retinal cell types. Mechanisms regulating cell fate choice in the retina depend on both cell intrinsic and environmental factors, but their relative contribution to specific cell fate decisions remains unclear. Dissociated retinal cell cultures provide a great assay to study this problem. RPCs are cultured in serum-free and extract-free medium, providing the investigator with a control over the environment to address questions related to the effects of a particular molecule on the development of retinal neurons. In addition, dissociated cell cultures can be used to study the importance of cell intrinsic mechanisms by isolating RPCs from their normal environment (Cayouette et al., 2003; Jensen and Raff, 1997). The method described below is suitable for the clonal-density culture of RPCs. In such cultures, RPCs are isolated from each other and from the postmitotic neurons. They divide and differentiate into different retinal cell types to form small colonies, or “clones”. In a recent study, we found that these clones are indistinguishable from the clones that develop in situ in the retina, both in terms of cell number and cell type composition, suggesting that intracellular mechanisms play a key role in retinal development (Cayouette et al., 2003).

Keywords: Retina(视网膜), Primary culture(原代培养), Retinal neuron(视网膜神经元), Retinal progenitor cells(视网膜祖细胞), Clonal density(克隆密度)

Materials and Reagents

  1. Animals
    We typically use retinas from albino Sprague Dawley rats. This method works for culturing rat retinas aged between embryonic day 17 to post-natal day 1. Younger and older retinal cells or retinal cells from mouse retinas do not seem to survive as well under these conditions, but can also be cultured.
  2. DPBS (Life Technologies, catalog number: 14040 )
  3. Neurobasal medium (Life Technologies, catalog number: 21103-049 )
  4. DMEM/F12 medium (Life Technologies, catalog number: 10565-018 )
  5. B27 supplement (Life Technologies, catalog number: 17504-044 )
  6. Pen/Strep (P/S) (Life Technologies, catalog number: 15140-122 )
  7. Poly L-lysine (PLL) (Sigma-Aldrich, catalog number: P-4707 )
  8. Mouse Laminin (Life Technologies, catalog number: 23017-015 )
  9. Papain (Worthington Biochemical, catalog number: LS003126 )
  10. L-cystein crystal (Sigma-Aldrich, catalog number: C-7352 )
  11. Trypsin Inhibitor (Roche Diagnostics, catalog number: 109878 )
  12. 0.45 µm and 0.22 µm filters (Fisher Scientific, catalog number: SLHV033RS and SLGP033RS )
  13. Hoechst 33342 (Life Technologies, Molecular Probes®, catalog number : H3570 )
  14. “Incomplete” RGM medium
  15. Growth factors (see Recipes)
    1.  bFGF (Pepro Tech, catalog number: 100-18B )
    2. EGF (Pepro Tech, catalog number: 315-09 )
    3. NT-3 (Pepro Tech, catalog number: 450-03 )
    4. BDNF (Pepro Tech, catalog number: 450-02 )
  16. 10x Lo Ovomucoid (see Recipes)
  17. 20x 4% BSA (Sigma-Aldrich, catalog number: A-4161 ) (see Recipes)
  18. 0.4% DNase (Worthington Biochemical, catalog number: LS002007 ) (see Recipes)
  19. Insulin (2.5 mg/ml) (Sigma-Aldrich, catalog number: I-6634 ) (see Recipes)
  20. cpt-cAMP (Sigma-Aldrich, catalog number: C-3912 ) (see Recipes)
  21. N-acetyl cysteine (NAC) (Sigma-Aldrich, catalog number: A-9165 ) (see Recipes)
  22. Forskolin (Sigma-Aldrich, catalog number: D-2438 ) (see Recipes)
  23. N2 Supplement (see Recipes)
  24. 4% Paraformaldehyde (Electron Microscopy Science, catalog number: 15710 ) (see Recipes)
  25. Apo-Transferrin (Sigma-Aldrich, catalog number: T-1147 ) (see Recipes)
  26. Progesterone (Sigma-Aldrich, catalog number: P-8783 ) (see Recipes)
  27. Putrescine (Sigma-Aldrich, catalog number: P-5780 ) (see Recipes)
  28. Sodium Selinite (Sigma-Aldrich, catalog number: S-5261 ) (see Recipes)

Equipment

  1. 35 mm petri dish (BD Biosciences, Falcon®, catalog number: 353001 )
  2. 50 ml conical tube
  3. CO2 incubator
  4. 15 ml conical tube
  5. 100 mm petri dish
  6. CO2 chamber
  7. Straight forcep
  8. Fine forcep (Fine Science Tools, catalog number: 11252-23 )
  9. Curved forcep (Fine Science Tools, catalog number: 91197-00 )
  10. Sharp scissor
  11. Laminar flow hood
  12. Water bath
  13. P1000 pipette
  14. Microscope
  15. Hemacytometer
  16. Centrifuge

Procedure

Day before experiment

  1. Coat culture dishes with poly-L-lysine and laminin
    1. Prepare a 50% working solution of Poly L-lysine (PLL) by diluting the stock solution in sterile water. Add 1.5 ml of PLL solution to each 35 mm petri dish. Leave at RT for 60 min. Remove PLL and let dry in the hood. Wash three times with sterile water.
    2. Prepare the mouse laminin solution at a concentration of 5 µg/ml in cold Neurobasal medium. Add 1.5 ml of mouse laminin solution to the dish.
    3. Incubate for at least overnight in an incubator equilibrated at 37 °C and 8% CO2. 5% CO2 incubation is ok, but not optimal for the Neurobasal/DMEM/F12 medium mix. Longer incubation can be done and were found to improve quality of the prep.
  2. Prepare the “incomplete” retina growth medium (RGM)
    1. In a 50 ml conical tube, under sterile condition, add to following:
      10 ml of Neurobasal medium
      10 ml of DMEM/F12 medium
      100 µl of N2 supplement
      200 µl of B27 supplement
      200 µl of 100x Pen/Strep (P/S)
      200 µl of 6 mg/ml NAC
      40 µl of 50 mM cpt-cAMP  
      20 µl of 10 mM Forskolin
      200 µl of 2.5 mg/ml Insulin
      Filter sterilize the solution using a 0.22 µm filter previously rinsed with Neurobasal medium  and stored at 4 °C until needed.

Day of Experiment

  1. Prepare the solution needed (see Recipes for the preparation of the stock solutions)
    1. “Incomplete” RGM medium add the growth factors
      4 µl of EGF 250 µg/ml
      2 µl of b-FGF 100 µg/ml
      20 µl of BDNF 50 µg/ml
      5 µl of NT-3 100 µg/ml
      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 8% CO2 until needed.
    2. Papain solution
      In a 15 ml conical tube add the following
      10 ml of DPBS
      5 µl of 1 N NaOH
      100 U of papain solution
      100 µl of 0.4% DNase
      2 mg of L-cystein crystal (to be added only prior to using the solution for dissociation)
      Keep at 37 °C in a water bath
    3. LO-OVO solution
      9 ml of DPBS
      1 ml of 10x LO-OVO
      100 µl of 0.4% DNase
      Keep at 37 °C in a water bath
  2. Culture procedure
    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 it’s back and clean the belly skin 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 to 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). From E18-19 onward, it is required to remove the blood vessel covering the retina to avoid endothelial cell contamination in the culture.
        5. The blood vessels form a reddish membrane overlying the inner face of the retina. The membrane is easily removed by pinching and pulling with a fine forceps.
        6. Transfer the retinas in a 35 mm dish filled with DPBS. Using a fine scalpel, slice the retinas in small pieces (2 mm) to help the dissociation. The number of retinas to dissect depends on your need. Typically, the yield is about 0.5 x 106 cells for one E17 retina and 1.6 x 106 cells for one P0 retina. It is optimal to dissect at least 6 to 8 retina as it improve cell recovery.


      Figure 1. Dissection of the retina. Steps 1 to 5 show the procedure to dissect the retina from the eyeball. Step 6 shows the dissected retina and lens.

    3. Dissociation of the retinas (all these steps are done in the laminar flow hood)
      1. Digestion of the tissue
        Take out the papain tube from the water bath and add 2 mg of L-cystein.
        Transfer the pieces of retina in this tube.
        Return the tube in the 37 °C water bath and incubate 5 min if you are working with E17 retinas or 8 min if you are working with P0 retinas.
      2. Neutralisation of the enzyme activity
        Take out the tube from the water bath, let the pieces of retina settle in the bottom of the tube and remove the supernatant with gentle aspiration.  
        Slowly add 4 ml of LO-OVO solution over the pieces of retina.
        Let the pieces of retina settle in the bottom of the tube and remove the supernatant with gentle aspiration. Add 1 ml of LO-OVO solution.
      3. Trituration
        Using the P1000, gently triturate up and down the pieces of retina to break them apart until the solution appears cloudy.
        Make sure that the cell suspension is well dissociated and that no clumps of cells remain by observing a small aliquot of the suspension under the microscope.
        If clumping is observed, triturate the suspension with a little more force until unicellular suspension is obtained. (If the cells are difficult to dissociate, the incubation time in papain can be increased). Be careful not to over triturate the cells, this could lead to decreased survival.
        Add the remaining 5 ml of LO-OVO solution to the cell suspension and centrifuge 11 min in a benchtop centrifuge at 200 x g at room temperature.
    4. Plating
      1. Take out the RGM medium from the incubator.
      2. Aspirate the supernatant and gently resuspend the cell pellet in 1 ml of equilibrated RGM medium.
      3. Using the hemacytometer, count the number of total cells. For clonal density culture, add 5,000 cells per 35 mm dish, for low density culture, add 20,000 cells per 35 mm dish and for high density culture, add 100,000 cells per 35 mm dish.
      4. Dilute to right amount of cells in 1 ml of RGM medium and add it to the 35 mm dish.
      5. Place in the incubator at 37 °C with 8% CO2 for 2 h.
      6. Once the cells have adhered to the bottom of the dish, gently add another 1.5 ml of medium and replace in the incubator.
    5. Feeding
      Feed the cells every 3 to 4 days by removing 50% of the culture medium and adding the same amount of equilibrated RGM medium. In optimal conditions, the cells can be cultured for 10 to 14 days.

  3. Immunostaining of cultured cells
    Note: In these culture conditions, retinal progenitor cells will divide and differentiate into the different retinal cell types, normally produced from E17 (amacrines, bipolars, rod photoreceptors, and Müller cells). Various cell-specific markers can be used to identify the different retinal cell types. Nestin for retinal progenitor cells, Brn3b and neurofilamment 165 for ganglion cells, Rhodopsin and recoverin for rod photoreceptors, Islet-1 for bipolar cells and a subtype of amacrines and ganglion cells, whereas Pax-6 is a marker of retinal progenitor cells, amacrines and ganglion cells. Here, we provide a general protocol for immunostaining.
    1. Aspirate the culture medium and fix the cells by adding cold 4% PFA  for 15 min on ice.
    2. Rinse three times with 1x PBS.
    3. Block/permeabilize for 1 h at room temperature (RT) with block/permeabilization solution (20% goat serum + 0.4% Triton in 1x PBS).
    4. Prepare the primary antibody in 1 ml of block/permeabilization solution. Dilution to be used depend on the antibody.
    5. Incubate overnight at 4 °C.
    6. Discard primary antibody.
    7. Rinse three times with 1x PBS.
    8. Prepare the secondary antibody in 1 ml of block/permeabilization solution. The secondary antibody is usually used at a 1:1,000 dilution.
    9. Incubate 1 h at RT.
    10. Discard secondary antibody.
    11. Rinse three times with 1x PBS.
    12. To counterstain the nucleus, incubate in Hoechst solution (1 ml of Hoechst in 10 ml of 1x PBS) for 5 min at RT.
    13. Observe under inverted microscope.


      Figure 2. Phase contrast and immunostaining micrographs of dissociated cultures. A. Two retinal progenitor cells 18 h after plating. Retinal progenitors have a typical bipolar morphology. B. A dissociated retinal cell culture seen after 7 days in culture showing many differentiated neurons. C. Retinal progenitor cells stained for Nestin (red) 24 h after plating.

Recipes

  1. Growth factors
    1. b-FGF:  Reconstitute in 10 mM Tris pH 8.5 to a concentration of 100 µg/ml
    2. EGF:  Reconstitute in water to a concentration of 250 µg/ml
    3. NT-3:  Reconstitute in water to a concentration of 100 µg/ml
    4. BDNF:  Dilute 1mg/ml stock solution to a concentration of 50 µg/ml in water
  2. 10x Lo Ovomucoid
    To 40 ml DPBS add 600 mg BSA, Mix well
    Add 600 mg Trypsin inhibitor and mix to dissolve
    Adjust pH to 7.4; requires the addition of approx. 400 µl of 1 N NaOH
    When completely dissolved, filter through a 0.22 µm filter pre-rinsed with PBS
    Make 1.0 ml aliquots and store at -20 °C
  3. 20x 4% BSA
    Add 2 g BSA in 50 ml DPBS
    Dissolve at 37 °C.  Adjust pH to 7.4 (with approximately 200 µl 1 N NaOH)  
    Filter sequentially through 0.45 µm and 0.22 µm filters, pre-rinsed with PBS
    Make 1.0 ml aliquots
    Stored at -20 °C
  4. 0.4% DNase
    Dissolve 40 mg in 10 ml of sterile water
    Mix by gentle inversion
    Aliquots in 200 μl and stored at -20 °C
  5. Insulin (2.5 mg/ml)
    To 10 ml sterile water add
    25 mg insulin
    50 µl 1.0 N HCl  
    Mix well
    Stored at 4 °C for no more than 4 to 6 weeks
  6. cpt-cAMP (50 mM)
    Dissolve the cpt-cAMP in sterile water at 25 mg/ml
    Aliquots in 200 µl and stored at -20 °C
  7. NAC (6 mg/ml)
    Dissolve 6 mg N-acetyl cysteine in 1 ml of Neurobasal medium
    Aliquots in 200 µl
    Stored at -20 °C
  8. Forskolin (10 mM)
    Add 2.4 ml DMSO to a 10 mg bottle (4.2 mg/ml or 10 mM)
    Make 50 µl aliquots
    Stored -20 °C
  9. N2 Supplement
    1.  Prepare N2 stock solutions
      1.  Apo-Transferrin: Dissolve 500 mg in 5 ml of water (100 mg/ml stock solution), filter (0.22 µM), aliquot and stored at -20 °C
      2. Progesterone: Dissolve 6 mg in 10 ml of ethanol to make 0.6 mg/ml stock solution, filter (0.22 µM), aliquot and stored at -20 °C
      3. Putrescine: Dissolve 1.6 g in 10 ml of water to make 1 M (160 mg/ml) stock solution, filter  (0.22 µM), aliquot and stored at -20 °C
      4. Sodium Selenite: Dissolve 2.59 mg in 5 ml of water to make 3mM stock solution, filter  (0.22 µM), aliquot and stored at -20 °C
      5. BSA: Dissolve 500 mg in 10 ml of water to make 50 mg/ml stock solution, filter  (0.22 µM), aliquot and stored at -20 °C
    2. Prepare 10 ml of 100x N2 solution
      To 7.857ml of DMEM-F12, add the following:
      1 ml  of Apo-Transferrin 100 mg/ml
      33 µl of  progesterone 0.6 mg/ml
      100 µl of putrescine 1 M
      10 µl of sodium selenite 3 mM
      1 ml of BSA 50 mg/ml
      Aliquot in 200 µl and stored at -20 °C 
  10. 4% Paraformaldehyde
    Dilute a vial of 16% PFA (10 ml) in 26 ml of water + 4 ml of 10x PBS; This solution can be kept at 4 ˚C for no longer than 1 month.

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 Cayouette et al. (2003). 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. Jensen, A. M. and Raff, M. C. (1997). Continuous observation of multipotential retinal progenitor cells in clonal density culture. Dev Biol 188(2): 267-279.

简介

视网膜是中枢神经系统相对简单易用的部分,是研究细胞命运规范机制的有力模型。多能视网膜祖细胞(RPC)产生七大类视网膜细胞类型。调节视网膜细胞命运选择的机制取决于细胞内在和环境因素,但它们对特定细胞命运决定的相对贡献仍不清楚。分离的视网膜细胞培养物提供了一个很好的分析来研究这个问题。 RPC在无血清和无提取物培养基中培养,为研究者提供对环境的控制,以解决与特定分子对视网膜神经元发育的影响有关的问题。此外,分离的细胞培养物可用于通过从其正常环境中分离RPC来研究细胞内在机制的重要性(Cayouette等人,2003; Jensen和Raff,1997)。下述方法适用于RPC的克隆密度培养。在这样的培养物中,RPC彼此分离并且从后神经元分离。它们分化和分化成不同的视网膜细胞类型以形成小菌落或“克隆”。在最近的一项研究中,我们发现这些克隆与细胞数量和细胞类型组成方面在视网膜原位发生的克隆是无法区分的,这表明细胞内机制在视网膜发育中起关键作用(Cayouette et al。 ,2003)。

关键字:视网膜, 原代培养, 视网膜神经元, 视网膜祖细胞, 克隆密度

材料和试剂

  1. 动物
    我们通常使用来自白化Sprague Dawley大鼠的视网膜。 该方法用于培养在胚胎第17天至出生后第1天之间的大鼠视网膜。来自小鼠视网膜的年轻和较老的视网膜细胞或视网膜细胞在这些条件下似乎不能存活,但也可以培养。
  2. DPBS(Life Technologies,目录号:14040)
  3. Neurobasal培养基(Life Technologies,目录号:21103-049)
  4. DMEM/F12培养基(Life Technologies,目录号:10565-018)
  5. B27补充剂(Life Technologies,目录号:17504-044)
  6. Pen/Strep(P/S)(Life Technologies,目录号:15140-122)
  7. 聚L-赖氨酸(PLL)(Sigma-Aldrich,目录号:P-4707)
  8. 小鼠层粘连蛋白(Life Technologies,目录号:23017-015)
  9. 木瓜蛋白酶(Worthington Biochemical,目录号:LS003126)
  10. L-半胱氨酸晶体(Sigma-Aldrich,目录号:C-7352)
  11. 胰蛋白酶抑制剂(Roche Diagnostics,目录号:109878)
  12. 0.45μm和0.22μm过滤器(Fisher Scientific,目录号:SLHV033RS和SLGP033RS)
  13. Hoechst 33342(Life Technologies,Molecular Probes ,目录号:H3570)
  14. "未完成"RGM介质
  15. 生长因子(参见食谱)
    1.   bFGF(Pepro Tech,目录号:100-18B)
    2. EGF(Pepro Tech,目录号:315-09)
    3. NT-3(Pepro Tech,目录号:450-03)
    4. BDNF(Pepro Tech,目录号:450-02)
  16. 10x Lo Ovomucoid(参见配方)
  17. 20x 4%BSA(Sigma-Aldrich,目录号:A-4161)(参见Recipes)
  18. 0.4%DNase(Worthington Biochemical,目录号:LS002007)(参见Recipes)
  19. 胰岛素(2.5mg/ml)(Sigma-Aldrich,目录号:I-6634)(参见Recipes)
  20. cpt-cAMP(Sigma-Aldrich,目录号:C-3912)(参见Recipes)
  21. N-乙酰半胱氨酸(NAC)(Sigma-Aldrich,目录号:A-9165)(参见Recipes)
  22. 福斯克林(Sigma-Aldrich,目录号:D-2438)(参见Recipes)
  23. N2补充(见配方)
  24. 4%多聚甲醛(Electron Microscopy Science,目录号:15710)(参见配方)
  25. Apo-Transferrin(Sigma-Aldrich,目录号:T-1147)(参见Recipes)
  26. 孕酮(Sigma-Aldrich,目录号:P-8783)(参见Recipes)
  27. 腐胺(Sigma-Aldrich,目录号:P-5780)(参见Recipes)
  28. 钠Selinite(Sigma-Aldrich,目录号:S-5261)(参见Recipes)

设备

  1. 35mm培养皿(BD Biosciences,Falcon ,目录号:353001)
  2. 50ml锥形管
  3. CO <2>孵化器
  4. 15 ml锥形管
  5. 100 mm培养皿
  6. CO 2室
  7. 直线力
  8. Fine forcep(Fine Science Tools,目录号:11252-23)
  9. 弯曲力(Fine Science Tools,目录号:91197-00)
  10. 锋利的剪刀
  11. 层流罩
  12. 水浴
  13. P1000移液器
  14. 显微镜
  15. 血细胞计数器
  16. 离心机

程序

实验前一天

  1. 用聚-L-赖氨酸和层粘连蛋白包被培养皿
    1. 通过在无菌水中稀释储备溶液制备聚L-赖氨酸(PLL)的50%工作溶液。 向每个35mm培养皿中加入1.5ml PLL溶液。 在室温下放置60分钟。 取下PLL,在机罩内干燥。 用无菌水洗三次。
    2. 在冷的Neurobasal培养基中制备浓度为5μg/ml的小鼠层粘连蛋白溶液。 加入1.5毫升小鼠层粘连蛋白溶液到盘中。
    3. 在37℃和8%CO 2平衡的培养箱中孵育至少过夜。 5%CO 2孵育确定,但对于Neurobasal/DMEM/F12培养基混合物不是最佳的。 可以进行更长时间的孵育,并且发现可以提高制备的质量
  2. 准备"不完全"视网膜生长培养基(RGM)
    1. 在50ml锥形管中,在无菌条件下,添加以下:
      10ml神经基础培养基
      10ml DMEM/F12培养基
      100μlN2补助剂
      200微升B27补充剂
      200μl100x Pen/Strep(P/S)
      200微升6毫克/毫升NAC
      40μl50mM cpt-cAMP
      20μl10mM Forskolin
      200μl2.5mg/ml胰岛素
      使用先前用Neurobasal培养基漂洗的0.22μm过滤器对溶液进行过滤灭菌。 并在4℃储存直至需要

实验日

  1. 准备所需的解决方案(见配方的储备溶液的制备)
    1. "不完全"RGM培养基添加生长因子
      4μlEGF250μg/ml
      2μlb-FGF100μg/ml
      20微升BDNF 50微克/毫升
      5μlNT-3100μg/ml
      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8%CO 2的培养箱中直至需要
    2. 木瓜溶液
      在15ml锥形管中加入以下
      10ml DPBS
      5μl1N NaOH
      100 U木瓜蛋白酶溶液
      100μl0.4%DNase
      2mg L-半胱氨酸晶体(仅在使用溶液解离之前加入)
      在37℃水浴中保持
    3. LO-OVO解决方案
      9 ml DPBS
      1ml 10×LO-OVO / 100μl0.4%DNase
      在37℃水浴中保持
  2. 文化程序
    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. 将动物放在它的背部,用70%乙醇清洁腹部皮肤。
      3. 捏直皮肤与直的forcep,抬起皮肤和肌肉和 使用剪刀在腹部的两侧做一个V形开口 从下腹部开始,一直切到侧面 这个动物。 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. 小心取下镜头 将视网膜放置在填充有DPBS的35mm培养皿中(图1,步骤 5-6)。 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。
        5. 血管形成上覆的微红色膜 视网膜的内表面。 膜通过夹紧容易地移除 并用细镊子拉扯
        6. 转移视网膜在35 mm皿中装有DPBS。 使用精细的手术刀,切片的视网膜 小片(2mm)以帮助解离。 视网膜的数量 解剖取决于你的需要。 通常,对于一个E17视网膜,产量为约0.5×10 6个细胞,对于一个P0视网膜,产量为1.6×10 6个细胞。 它是 最佳解剖至少6至8视网膜,因为它提高细胞恢复

      图1.视网膜解剖。步骤1至5显示了从视网膜解剖视网膜的程序。 步骤6显示解剖的视网膜和晶状体
    3. 视网膜的解离(所有这些步骤在层流罩中进行)
      1. 组织消化
        从水浴中取出木瓜蛋白酶管,加入2mg L-半胱氨酸。
        转移在这个管中的视网膜的片段。
        将管在37°C水浴中,孵育5分钟,如果你是 使用E17视网膜或8分钟,如果你正在使用P0视网膜。
      2. 酶活性的中和
        从水浴中取出管,让视网膜片块沉降 在管的底部并用温和的去除上清液 吸气。  
        在视网膜上慢慢加入4 ml LO-OVO溶液。
        让视网膜的片段在管的底部沉降并移除 轻柔吸出上清液。加入1ml LO-OVO溶液
      3. 研磨
        使用P1000,轻轻地研磨上下视网膜碎片,将它们分开,直到溶液显示混浊。
        确保细胞悬浮液很好地解离,没有 通过观察悬浮液的小等分试样残留细胞团块 在显微镜下 如果观察到结块,则研磨 悬浮液用更多的力直到单细胞悬浮液 获得。 (如果细胞难以解离,孵育时间  在木瓜蛋白酶中可以增加)。小心不要过度磨损 细胞,这可能会导致生存率下降。
        添加剩余  将5ml LO-OVO溶液加入细胞悬浮液中并离心11分钟  室温下在200×g下进行台式离心机。
    4. 电镀
      1. 从培养箱中取出RGM培养基。
      2. 吸出上清液,轻轻地重悬细胞沉淀在1ml平衡的RGM培养基中
      3. 使用血细胞计数器,计数总细胞数。 对于克隆 密度培养,每35mm培养皿加入5,000个细胞,用于低密度培养 培养,每35mm培养皿添加20,000个细胞并用于高密度培养, 每35mm培养皿加入100,000个细胞。
      4. 在1ml RGM培养基中稀释至正确量的细胞,并将其加至35mm培养皿中。
      5. 置于37℃,8%CO 2的培养箱中2小时。
      6. 一旦细胞粘附在培养皿的底部,轻轻加入另外1.5毫升培养基并在培养箱中更换。
    5. 喂食
      通过去除50%的培养基并加入相同量的平衡的RGM培养基,每3至4天饲喂细胞。 在最佳条件下,细胞可培养10至14天

  3. 培养细胞的免疫染色
    注意:在这些培养条件下,视网膜祖细胞将分裂并分化成不同的视网膜细胞类型,通常由E17产生(阿米库林,双极杆,杆状光感受器和穆勒细胞)。各种细胞特异性标记可用于鉴定不同的视网膜细胞类型。视网膜祖细胞的巢蛋白,神经节细胞的Brn3b和神经膜165,杆感光细胞的视紫红质和恢复蛋白,双极细胞的Islet-1和半胱氨酸和神经节细胞的亚型,而Pax-6是视网膜祖细胞,神经节细胞。在这里,我们提供了免疫染色的一般方案。
    1. 吸出培养基并通过加入冷的4%PFA固定细胞在冰上15分钟
    2. 用1x PBS冲洗三次。
    3. 在室温(RT)下用封闭/透化溶液(20%山羊血清+ 0.4%Triton在1x PBS中)封闭/透化1小时。
    4. 准备一抗在1毫升的块/透化溶液。稀释度取决于抗体。
    5. 在4℃孵育过夜。
    6. 丢弃一抗。
    7. 用1x PBS冲洗三次。
    8. 准备第二抗体在1ml的阻断/透化溶液中。 二抗通常以1:1000稀释度使用。
    9. 在室温下孵育1小时。
    10. 丢弃二抗。
    11. 用1x PBS冲洗三次。
    12. 为了复染核,在室温下在Hoechst溶液(1ml Hoechst在10ml 1×PBS中)孵育5分钟。
    13. 在倒置显微镜下观察。


      图2.解离培养物的相差显微镜和免疫染色显微照片。A.两个视网膜祖细胞在铺板后18小时。 视网膜祖细胞具有典型的双极形态。 B.在培养7天后观察到的解离的视网膜细胞培养物,显示许多分化的神经元。 C.电镀后24小时,视网膜祖细胞对巢蛋白(红色)染色

食谱

  1. 生长因子
    1. b-FGF: 在10mM Tris pH8.5中重构至100μg/ml的浓度
    2. EGF: 在水中重构至250μg/ml的浓度
    3. NT-3: 在水中重构至浓度为100μg/ml
    4. BDNF: 稀释1mg/ml储备溶液至50μg/ml的浓度在水中
  2. 10x Lo类粘蛋白
    向40ml DPBS中加入600mg BSA,混匀
    加入600mg胰蛋白酶抑制剂并混合溶解
    调节pH至7.4; 需要添加约。 400μl1N NaOH
    当完全溶解时,通过用PBS预漂洗的0.22μm过滤器过滤 制成1.0毫升等分试样并储存在-20℃下
  3. 20x 4%BSA
    在50ml DPBS中加入2μgBSA
    在37℃溶解  将pH调节至7.4(使用约200μl1N NaOH)
    依次通过0.45μm和0.22μm过滤器,用PBS预漂洗
    使1.0 ml等分
    储存于-20°C
  4. 0.4%DNase
    将40mg溶于10ml无菌水中
    通过轻微反转混合
    等分于200μl,储存于-20°C
  5. 胰岛素(2.5mg/ml)
    向10ml无菌水中加入
    25 mg胰岛素
    50μl1.0 N HCl
    混合良好
    在4°C下储存不超过4至6周
  6. cpt-cAMP(50mM) 将cpt-cAMP溶解在无菌水中,浓度为25mg/ml 等分于200μl,储存于-20°C
  7. NAC(6mg/ml) 将6mg N-乙酰半胱氨酸溶解在1ml Neurobasal培养基中
    等分于200μl
    储存于-20°C
  8. 毛喉素(10mM)
    将2.4ml DMSO加入到10mg瓶(4.2mg/ml或10mM)中 使50微升等分
    储存-20°C
  9. N2补充
    1.  准备N2储备溶液
      1. 载脂转铁蛋白:将500mg溶于5ml水(100mg/ml储备溶液)中,过滤(0.22μM),等分并保存在-20℃下。
      2. 孕酮:将6mg溶于10ml乙醇中,制成0.6mg/ml 储备溶液,过滤器(0.22μM),等分并保存在-20℃下
      3. 腐胺:将1.6g溶于10ml水中,制成1M(160mg/ml) 储液,过滤器 (0.22μM),等分并保存在-20℃下
      4. 亚硒酸钠:将2.59mg溶于5ml水中,制成3mM原液 解决方案,过滤器  (0.22μM),等分并保存在-20℃下
      5. BSA:将500mg溶解在10ml水中以制备50mg/ml储备溶液,过滤& (0.22μM),等分并保存在-20℃
    2. 准备10ml的100×N 2溶液
      向7.857ml DMEM-F12中加入以下物质:
      1 ml  的转铁蛋白100mg/ml
      33μl的  孕酮0.6mg/ml
      100μl腐胺1MW / 10μl亚硒酸钠3mM
      1ml BSA 50mg/ml
      在200μl等分试样并储存在-20℃&
  10. 4%多聚甲醛
    稀释一小瓶16%PFA(10ml)在26ml水+ 4ml 10x PBS中; 此溶液可在4℃保存不超过1个月。

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Cayouette等人(2003)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. Jensen,A.M。和Raff,M.C。(1997)。 在克隆密度培养中连续观察多潜能视网膜祖细胞。 188(2):267-279。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 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. Jolicoeur, C. and Cayouette, M. (2014). Dissociated Retinal Cell Culture. Bio-protocol 4(2): e1033. DOI: 10.21769/BioProtoc.1033.
  2. Kechad, A., Jolicoeur, C., Tufford, A., Mattar, P., Chow, R. W. Y., 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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