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Mouse Müller Cell Isolation and Culture
小鼠Müller细胞分离与培养

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Abstract

Müller cells are the major supportive and protective glial cells across the retina. Unlike in fish, they have lost the capacity to regenerate the retina in mammals. But, mammalian Müller cells still retain certain retinal stem cell properties with various degree of self-renewal and differentiation potentials, and thereby held a merit in cell-based therapies for treating retinal degeneration diseases. In our laboratory, we use an enzymatic procedure to isolate, purify, and culture mouse Müller cells.

Keywords: Mouse(小鼠), Müller glial cells(Müller胶质细胞), Stem cells(干细胞), Retinal cell isolation(视网膜细胞分离), Primary cell culture(原代细胞培养), Worthington Papain Kit(Worthington木瓜蛋白酶试剂盒)

Background

Müller glial cell is a major lineage in the retina that functions to maintain retinal homeostasis through synthesis of neurotrophic factors, uptake and recycle of neurotransmitters, spatial buffering of ions, and maintenance of the blood-retinal barrier (Bringmann et al., 2006; De Melo Reis et al., 2008). Müller glia serve as retinal progenitor/stem cells in fish and, to a limited extent, in birds (Vihtelic and Hyde, 2000; Fischer and Reh, 2001). But, mammalian Müller cells have lost such a capacity to regenerate the retina, though still retain certain properties of adult stem cells such as proliferation upon a retinal damage. Researches in restoring of the lost capacity of mammalian Müller cells to repair retinal damage and understanding of the underlying mechanism are undertaken in laboratories with primary cells isolated from model animal retinas. Proteolytic enzymes are widely used in Müller cell dissociation and papain is less damaging and more effective than other proteases. Sarthy and Lam developed a method for dissociation and separation of glial cells with papain digestion followed by gentle mechanical dissociation, they found that among the enzymes used for dissociating turtle retina, papain produced the least trauma (Sarthy and Lam, 1978).

Materials and Reagents

  1. Cell culture dishes: 35 x 10 mm (Corning, catalog number: 430166 )
  2. 15 ml centrifuge tubes (Corning, catalog number: 430790 )
  3. Tipone® pipette tips (USA Scientific, catalog number: 1126-7810 )
  4. 5 ml pipets (Corning, Falcon®, catalog number: 357543 )
  5. 2 ml cryopreservation vials (Corning, catalog number: 430659 )
  6. Alcohol Prep Pads (PDI, catalog number: B33905 )
  7. Animals: 2- to 4-week-old mice
  8. Worthington Papain Kit (papain dissociation system) (Worthington Biochemical, catalog number: LK003150 )
    Note: The components of kit include vial 1 (Sterile Earle’s Balanced Salt Solution, EBSS), vial 2 (Papain containing L-cysteine and EDTA), vial 3 (Deoxyribonuclease I, DNase), and vial 4 (Ovomucoid protease inhibitor with bovine serum albumin).
  9. 70% ethanol
  10. Phosphate buffered saline (PBS) (Sigma-Aldrich, catalog number: P4417-100TAB )
  11. Penicillin-streptomycin (Pen/Strep) (10,000 μg/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  12. Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Cellgro®, catalog number: 10-013-CV )
  13. Fetal bovine serum (FBS) (GE Healthcare, HyCloneTM, catalog number: SH30071.03 )
  14. Gelatin (Sigma-Aldrich, catalog number: G1890-100G )
  15. 0.25% trypsin ethylenediaminetetraacetic acid (EDTA) solution (Mediatech, Cellgro®, catalog number: 25-053-CI )
  16. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  17. Cell culture medium (see Recipes)
  18. 0.1% gelatin solution (see Recipes)
  19. Cell freezing medium (see Recipes)

Equipment

  1. Pipettes (Eppendorf)
  2. Pipet-aid (Drummond)
  3. Single Edge Blade (Sparco, catalog number: SPR11820 )
  4. Dissection forceps and scissors
    1. Iris Scissors Sharp Straight (Storz Ophthalmic Instruments, catalog number: E3344 )
    2. Castroviejo Suturing Forceps 0.12 mm (Storz Ophthalmic Instruments, catalog number: E1796 )
  5. 37 °C, 5% CO2 cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Model 3250 )
  6. 37 °C incubator (VWR, model: 1545 )
  7. Allegra bench-top centrifuge (Beckman Coulter, model: Allegra® X-15R )
  8. Lab quake rotisserie shaker (Barnstead Thermolyne LabQuake, model: 4152110 )
  9. Inverted routine microscope (Nikon Instruments, model: Eclipse TS100 )
  10. Stereo binocular microscope (Olympus, model: SZ40 )
    Note: Mice are euthanized by carbon dioxide asphyxiation, a carbon dioxide source, regulated dispenser, and euthanasia chamber. All animal manipulations are conducted in accordance with the policies and guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) were approved by the University of Louisville, Louisville, Kentucky, USA.

Procedure

  1. Preparation of papain dissociation system
    1. Prepare Worthington Papain Kit according to the manufacturer’s instructions.
    2. Add 32 ml of EBSS (vial 1) to the albumin ovomucoid inhibitor mixture (vial 4). Completely dissolve, then store in a 4 °C refrigerator and use it within 1 week.
    3. Add 5 ml of EBSS (vial 1) to a papain vial (vial 2), place vial 2 in a 37 °C incubator (or water bath) for 10 min or until the papain is completely dissolved.
    4. Add 500 µl of EBSS (vial 1) to a DNase vial (vial 3), mix gently, and then add 250 µl of the mixture to the papain vial 2 saved at step A3. The remaining 250 µl will be used in Procedure C, step C5.
    5. Transfer the above mixed vial 2 to a 15 ml conical centrifuge tube.

  2. Mouse eye dissection
    1. Disinfect dissection forceps and scissors with 70% ethanol and burn them to dry.
    2. Prepare three dishes containing 2 ml of PBS and 1% penicillin-streptomycin, and mark them with numbers as shown in Figure 1.


      Figure 1. Prepare three dishes, and mark them with numbers

    3. At least 2 mice are euthanized by CO2 asphyxiation and then placed onto a sterile surgical pad. Disinfect surface of the eyelid with 70% alcohol preparation pads.
    4. Mouse eyeballs with the optical bundles are extracted by inserting the forceps along the socket until the optical nerve bundle, and placed in the first dish for 10 min (Figure 2A).
    5. Under a stereo binocular microscope, carefully remove the muscle, fascia and conjunctiva tissues around the eyeballs in the first dish, and then transfer them to the second dish (Figure 2B, Video 1).
    6. Eyeball dissection: remove the anterior portion of the eye, including the cornea, the iris and the lens, reserve the posterior eye cup in the second dish (Figure 2C, Video 1).
    7. From the remaining posterior eyecup, the retinas are dissected free of the retinal pigment epithelium (RPE) in the third dish (Figure 2D, Video 1).


      Figure 2. Eyeball dissection. The enucleated mouse eyeballs are first immersed in PBS with the antibiotics for disinfection (A); and then transferred to the second dish (B) to remove the most anterior eye tissues (C), and to isolate the retina in the third dish (D).

      Video 1. Mouse eyeball dissection

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


  3. Isolation and culture of Müller cell
    1. Place the retina in a 15 ml conical centrifuge tube containing papain and DNase solution saved at step A5. Cut the retina into pieces as small as possible with a single edge blade (Figure 3A).
    2. Incubate the tube containing the tissue in a 37 °C incubator with gently shaking by a constant shaker for 60 min until the solution becomes clear (Figure 3B).


      Figure 3. The medium turns to be cloudy before incubation (A) and most pieces of the retina disappear after incubation (B)

    3. Triturate the mixture with a 5 ml pipette for 2-3 times. Allow any pieces of undigested tissue residues to settle to the bottom of the tube.
    4. Carefully transfer the cloudy cell suspension to a 15-ml conical centrifuge tube, and centrifuge at 300 x g for 5 min at room temperature. During this time, prepare cell culture medium to resuspend the pelleted cells.
    5. Mix medium in a sterile tube with 2.7 ml of EBSS (vial 1), 300 µl of reconstituted albumin-ovomucoid inhibitor solution (vial 4) saved at step A2, 150 µl of DNase solution (vial 3) saved at step A4.
    6. Discard the supernatant and then immediately resuspend the cell pellet by adding the medium mix at step C5.
    7. Add 5.0 ml of the albumin-inhibitor solution (vial 4) to the centrifuge tube, carefully lay the cell suspension on top, then centrifuge at 70 x g for 6 min at room temperature. The interface between the two layers of the gradients should be clearly visible. Dissociated cell pellet is at the bottom of the tube while the membrane fragments remain at the interface.
    8. Discard the supernatant and immediately resuspend the pelleted cells in the culture medium (DMEM) with 10% fetal bovine serum (FBS) and 1% Pen/strep antibiotics (see Recipes).
    9. Prepare 100 ml of 0.1% gelatin in PBS (see Recipes), add 1 ml or enough to cover the bottom of a 35-mm dish to coat the surface, leave the coating dish with the lid on in a 37 °C cell culture incubator for over 30 min, aspirate the coating solution and the coated dish is ready for use.
    10. The dissociated cells are collected and cultured in the above gelatin-coated dish at 5.5% CO2. The primary cells before passage (P0) are actually a mixture of Müller cells, photoreceptors, and other retinal neurons.
    11. After plating, adherent cells are maintained for 7 days at 37 °C. The medium is replaced every 5 days until cells are confluent and passaged at 1:1 ratio to a fresh dish coated with 0.1% gelatin (Figure 4A). Adherent Müller glial cells become relatively pure after 2 to 3 passages identified by cell morphology and immunostaining with glutamine synthetase (GS, Müller glial cell marker) (Figure 4B).


      Figure 4. Mouse Müller monolayer cells cultured for 10 days after one passage (A) and the cultured cells are stained with Müller cell-specific marker GS (B)

  4. Cell cryopreservation in liquid nitrogen
    1. Remove the culture medium from confluent monolayer-culture dish.
    2. Rinse the dish twice with PBS at room temperature.
    3. Add 0.75 ml or 2 ml of 0.25% trypsin solution to a 6-cm or 10-cm dish, respectively, or enough amount to cover the monolayer.
    4. Incubate in a 37 °C incubator for 1-2 min until cells come off the dish after gently striking the dish to a bench edge.
    5. Add the culture medium that contains 10% FBS, 2-3 times of the amount of the added trypsin, and gently mix for a minute, and transfer to a 15 ml tube.
    6. Centrifuge at 500 x g for 10 min to collect cell pellet.
    7. Discard the supernatant, and re-suspend the cell pellet in 1-3 ml of cell freezing medium (see Recipes) (usually 1 ml per 2 x 105 cells).
    8. Aliquot into 2 ml cryopreservation vial.
    9. Place the vial in a -20 °C freezer for 1 day, then transfer to -80 °C for 1 day, and finally store in a liquid nitrogen tank for long-term storage.

Data analysis

Using the above procedure, we successfully obtained large number of Müller cells isolated from 4 C57BL/6 mice (total 8 eye cups) by continuously passaging the primary cells at 1:1 ratio until passage 6 (P6) when most cells manifested a stress-induce premature senescence (SIPS) phenotype–large and flat with an obvious heterochromatin foci nucleus and positive for β-galactosidase activity. These senescent Müller cells were not proliferative, but could survive in culture for a long period of time if keeping medium timely refreshed, but eventually died in 3-4 months. Cells should be preserved in liquid nitrogen tank for a long-term storage if not used for month, and could be recovered by directly thawing the frozen cell vials in a 37 °C water bath and thereafter seeding cells in culture with little loss of viability. The percentage of the Müller cell purity differs from passage to passage because other retinal neural cells will not survive under such a culture condition. The estimation of the purity after 2 times of passage is more than 95%.

Notes

  1. All tissue and cell manipulations must be under sterile condition either in a tissue dissecting or cell culture hood with surface disinfected by 70% ethanol.
  2. We strongly recommend to collect and dissect two eyeballs together from the same mouse, and to isolate and culture Müller cells separately from other mice to minimize possible cross contamination.
  3. The primary cells are including Müller cells, photoreceptors and other neurons. Photoreceptor and other neuron cells cannot be adapted for adherent culture and will go apoptosis after passage 2 (P2).
  4. The dish for culture is specifically coated with 0.1% gelatin to allow Müller cells to better adhere.

Recipes

  1. Cell culture medium
    Dulbecco’s modified Eagle’s medium (DMEM)
    10% fetal bovine serum (FBS)
    1% penicillin-streptomycin (Pen/Strep)
  2. 0.1% gelatin solution
    100 ml of dH2O plus 0.1 g of gelatin, autoclaved
  3. Cell freezing medium
    Dulbecco’s modified Eagle’s medium (DMEM)
    20% fetal bovine serum (FBS)
    1% penicillin-streptomycin (Pen/Strep)
    10% dimethyl sulfoxide (DMSO)

Acknowledgments

This protocol was initially adopted in the laboratory for pig (Xu et al., 2017), and now modified for mouse. The work was supported by National Institute of General Medical Sciences (P20GM103453 to Y. L.), University of Louisville School of Medicine (E0819 to Y. L.), and Research to Prevent Blindness (to the Department of Ophthalmology and Visual Sciences at Louisville).

References

  1. Bringmann, A., Pannicke, T., Grosche, J., Francke, M., Wiedemann, P., Skatchkov, S. N., Osborne, N. N. and Reichenbach, A. (2006). Müller cells in the healthy and diseased retina. Prog Retin Eye Res 25: 397-424.
  2. De Melo Reis, R. A., Ventura, A. L., Schitine, C. S., de Mello, M. C. and de Mello, F. G. (2008). Müller glial as an active compartment modulating nervous activity in the vertebrate retina: neurotransmitters and trophic factors. Neurochem Res 33: 1466-1474.
  3. Fischer, A. J. and Reh, T. A. (2001). Müller glial glia are a potential source of neural regeneration in the postnatal chicken retina. Nat Neurosci 4: 247-252.
  4. Sarthy, P. V. and Lam, D. M. (1978). Biochemical studies of isolated glial (Müller) cells from the turtle retina. J Cell Biol 78(3): 675-684.
  5. Vihtelic, T. S. and Hyde, D. R. (2000). Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina. J Neurobiol 44(3): 289-307.
  6. Xu, N., Chen, Y., Dean, K. C., Lu, X., Liu, X., Wang, W., Dean, D. C., Kaplan, H. J., Gao, L., Dong, F. and Liu, Y. (2017). Sphere-induced rejuvenation of swine and human muller glia is primarily caused by telomere elongation. Stem Cells 25(6): 1579-1591.

简介

Müller细胞是视网膜上的主要支持和保护性胶质细胞。 与鱼类不同,它们已经失去了在哺乳动物中再生视网膜的能力。 但是,哺乳动物Müller细胞仍然保留了具有不同程度的自我更新和分化潜能的某些视网膜干细胞特性,从而在基于细胞的疗法中治疗视网膜变性疾病具有优点。 在我们的实验室,我们使用酶法来分离,纯化和培养小鼠Müller细胞。
【背景】Müller胶质细胞是视网膜的主要谱系,其功能是通过神经营养因子的合成,神经递质的摄取和再循环,离子的空间缓冲和血液视网膜屏障的维持来维持视网膜稳态(Bringmann等人,,2006; De Melo Reis等人,2008)。 Müller神经胶质细胞作为鱼类中的视网膜祖细胞和干细胞,在有限的范围内作为鸟类(Vihtelic和Hyde,2000; Fischer and Reh,2001)。但是,哺乳动物Müller细胞已经失去了再生视网膜的能力,尽管仍然保留成体干细胞的某些性质,例如视网膜损伤时的增殖。研究恢复哺乳动物Müller细胞丧失能力以修复视网膜损伤和理解底层机制的研究在与模型动物视网膜分离的原代细胞的实验室进行。蛋白水解酶广泛用于Müller细胞解离,木瓜蛋白酶的损伤较小,比其他蛋白酶更有效。 Sarthy和Lam开发了一种解离和分离神经胶质细胞的方法,用木瓜蛋白酶消化,然后轻柔的机械解离,发现在用于解离龟视网膜的酶中,木瓜蛋白酶产生最少的创伤(Sarthy和Lam,1978)。

关键字:小鼠, Müller胶质细胞, 干细胞, 视网膜细胞分离, 原代细胞培养, Worthington木瓜蛋白酶试剂盒

材料和试剂

  1. 细胞培养皿:35×10mm(Corning,目录号:430166)
  2. 15ml离心管(Corning,目录号:430790)
  3. Tipone ®移液器技巧(USA Scientific,目录号:1126-7810)
  4. 5 ml移液管(Corning,Falcon ®,目录号:357543)
  5. 2 ml冷冻保存瓶(Corning,目录号:430659)
  6. 酒精预处理垫(PDI,目录号:B33905)
  7. 动物:2至4周龄小鼠
  8. Worthington木瓜蛋白酶试剂盒(木瓜蛋白酶解离系统)(Worthington Biochemical,目录号:LK003150)
    注意:试剂盒的组分包括小瓶1(无菌Earle的平衡盐溶液,EBSS),小瓶2(含有L-半胱氨酸和EDTA的木瓜蛋白酶),小瓶3(脱氧核糖核酸酶I,DNA酶)和小瓶4(卵泡蛋白酶抑制剂用牛血清白蛋白)。
  9. 70%乙醇
  10. 磷酸盐缓冲盐水(PBS)(Sigma-Aldrich,目录号:P4417-100TAB)
  11. 青霉素 - 链霉素(Pen / Strep)(10,000μg/ ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  12. Dulbecco改良的Eagle's培养基(DMEM)(Mediatech,Cellgro ,目录号:10-013-CV)
  13. 胎牛血清(FBS)(GE Healthcare,HyClone TM,目录号:SH30071.03)
  14. 明胶(Sigma-Aldrich,目录号:G1890-100G)
  15. 0.25%胰蛋白酶乙二胺四乙酸(EDTA)溶液(Mediatech,Cellgro ,目录号:25-053-CI)
  16. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  17. 细胞培养基(参见食谱)
  18. 0.1%明胶溶液(见配方)
  19. 细胞冷冻培养基(见食谱)

设备

  1. 移液器(Eppendorf)
  2. 吸管(Drummond)
  3. 单刃刀片(Sparco,目录号:SPR11820)
  4. 解剖钳和剪刀
    1. 虹膜剪刀夏普直(Storz Ophthalmic Instruments,目录号:E3344)
    2. Castroviejo缝合钳0.12 mm(Storz Ophthalmic Instruments,目录号:E1796)
  5. 37℃,5%CO 2细胞培养箱(Thermo Fisher Scientific,Thermo Scientific,型号:3250型)
  6. 37℃培养箱(VWR,型号:1545)
  7. Allegra台式离心机(Beckman Coulter,型号:Allegra X-15R)
  8. 实验室地震烤架(Barnstead Thermolyne LabQuake,型号:4152110)
  9. 倒置常规显微镜(Nikon Instruments,型号:Eclipse TS100)
  10. 立体声双目显微镜(Olympus,型号:SZ40)
    注意:小鼠通过二氧化碳窒息,二氧化碳源,调节分配器和安乐死室来安乐死。所有动物操作均按照机构动物护理和使用委员会(IACUC)制定的政策和指南进行,由美国肯塔基州路易斯维尔路易斯维尔大学批准。

程序

  1. 木瓜蛋白酶解离系统的制备
    1. 根据制造商的说明准备Worthington木瓜蛋白酶包。
    2. 将32ml EBSS(小瓶1)加入到白蛋白卵类粘蛋白抑制剂混合物(小瓶4)中。完全溶解,然后储存在4°C冰箱中,并在1周内使用
    3. 向木瓜蛋白酶瓶(小瓶2)中加入5ml EBSS(小瓶1),将小瓶2置于37℃培养箱(或水浴)中10分钟,或直到木瓜蛋白酶完全溶解。
    4. 将500μlEBSS(小瓶1)加入DNase小瓶(小瓶3),轻轻混匀,然后加入250μl混合物至步骤A3保存的木瓜蛋白酶2。剩余的250μl将用于步骤C,步骤C5。
    5. 将上述混合的小瓶2转移到15ml锥形离心管中
  2. 小鼠眼睛解剖
    1. 用70%乙醇消毒解剖镊子和剪刀,并将其烧干。
    2. 准备含有2ml PBS和1%青霉素 - 链霉素的三个皿,并用数字标记,如图1所示。


      图1.准备三个菜肴,并用数字
      标记
    3. 至少2只小鼠被CO 2窒息安乐死,然后置于无菌手术垫上。用70%酒精制备垫消毒眼睑表面。
    4. 通过沿插座插入镊子直到光学神经束,并将其置于第一道中10分钟(图2A),提取具有光束的鼠标眼球。
    5. 在立体双眼显微镜下,小心地取出第一盘中眼球周围的肌肉,筋膜和结膜组织,然后将其转移到第二道菜(图2B,视频1)。
    6. 眼球解剖:去除眼睛的前部,包括角膜,虹膜和镜片,将后眼杯保留在第二道碟中(图2C,视频1)。
    7. 从剩余的后眼中,视网膜在第三个盘中被解剖为没有视网膜色素上皮(RPE)(图2D,视频1)。


      图2.眼球解剖。将摘除的小鼠眼球首先浸入PBS中,用抗生素进行消毒(A);然后转移到第二个盘(B)以除去最前面的眼组织(C),并分离第三个盘(D)中的视网膜。

      视频1

  3. Müller细胞的分离和培养
    1. 将视网膜置于15 ml锥形离心管中,包含在步骤A5保存的木瓜蛋白酶和DNA酶溶液。用单边刀片将视网膜切成小块(图3A)。
    2. 将含有组织的管在37℃的培养箱中孵育,用恒定的振荡器轻轻摇动60分钟直到溶液变澄清(图3B)。

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      图3.孵化前培养基变得混浊(A),大部分视网膜在孵育后消失(B)

    3. 用5ml移液管研磨混合物2-3次。允许任何未消化的组织残留物沉淀到管的底部。
    4. 小心地将混浊细胞悬浮液转移到15-ml锥形离心管中,并在室温下以300×g离心5分钟。在此期间,准备细胞培养基以重悬细胞沉积
    5. 将无菌管中的培养基与2.7ml EBSS(小瓶1)混合,在步骤A2保存的300μl重构白蛋白 - 卵类粘蛋白抑制剂溶液(小瓶4),在步骤A4保存的150μlDNA酶溶液(小瓶3)。
    6. 弃去上清液,然后通过在步骤C5中加入培养基混合物立即重悬细胞沉淀
    7. 将5.0ml白蛋白抑制剂溶液(小瓶4)加入到离心管中,小心地将细胞悬液置于顶部,然后在室温下以70×g离心6分钟。两层梯度之间的界面应清晰可见。分离的细胞沉淀位于管的底部,而膜片段保留在界面处
    8. 弃去上清液,立即将沉淀的细胞重悬于具有10%胎牛血清(FBS)和1%Pen / strep抗生素(见食谱)的培养基(DMEM)中。
    9. 准备100毫升0.1%的明胶在PBS中(参见食谱),加入1毫升或足以覆盖35毫米皿的底部以覆盖表面,将盖子盖在37℃的细胞培养箱超过30分钟,吸出涂层溶液,涂层盘即可使用。
    10. 将解离的细胞收集并在5.5%CO 2的上述明胶包被的培养皿中培养。传代前的原代细胞(P <0>)实际上是Müller细胞,感光细胞和其他视网膜神经元的混合物。
    11. 电镀后,贴壁细胞在37℃下保持7天。培养基每5天更换一次,直到细胞融合并以1:1的比例传播到涂有0.1%明胶的新鲜培养皿中(图4A)。粘附的Müller神经胶质细胞在通过细胞形态和用谷氨酰胺合成酶(GS,Müller胶质细胞标记物)的免疫染色鉴定的2至3代之后变得相对纯净(图4B)。


      图4.一次传代(A)培养10天后的小鼠Müller单层细胞,培养的细胞用Müller细胞特异性标志物GS(B) >
  4. 液氮中的细胞冷冻保存
    1. 从汇合单层培养皿中取出培养基
    2. 用PBS在室温下冲洗两次。
    3. 将0.75ml或2ml 0.25%胰蛋白酶溶液分别加入到6cm或10cm的培养皿中,或足够的量覆盖单层。
    4. 在37℃的培养箱中孵育1-2分钟,直至细胞从盘子上轻轻地打到台面边缘后才能脱落。
    5. 加入含有10%FBS的培养基,加入胰蛋白酶的2-3倍,轻轻混匀一分钟,转移至15 ml管中。
    6. 以500xg离心10分钟以收集细胞沉淀。
    7. 弃去上清液,并将细胞沉淀重新悬浮在1-3ml细胞冷冻培养基中(参见食谱)(通常每2×10 5个细胞1ml)。
    8. 等分成2ml冷冻保存小瓶。
    9. 将小瓶置于-20°C冷冻箱中1天,然后转移至-80°C 1天,最后储存在液氮罐中长期储存。

数据分析

使用上述方法,通过以大约1:1的比例连续传代原代细胞,直到第6代(P6),当大多数细胞表现出应激反应时,我们成功地获得了从4只C57BL / 6小鼠(共8只眼睛杯)分离的大量Müller细胞,诱导早衰(SIPS)表型大,平坦,具有明显的异染色质核心核,对β-半乳糖苷酶活性呈阳性。这些衰老的Müller细胞不是增殖的,但是如果保持中和时间更新,可以在文化中长时间存活,但最终在3-4个月内死亡。如果不使用月份,细胞应保存在液氮罐中以进行长期储存,并且可以通过在37℃水浴中直接解冻冷冻的细胞小瓶来回收,然后以较少的活力损失将细胞接种在培养物中。 Müller细胞纯度的百分比不同于通过,因为其他视网膜神经细胞在这样的培养条件下不能存活。估计2次通过后的纯度大于95%。

笔记

  1. 所有组织和细胞操作必须处于无菌状态,或者在组织切除或细胞培养罩中,用70%乙醇表面消毒。
  2. 我们强烈建议从同一只小鼠一起收集和解剖两个眼球,并分离和培养Müller细胞与其他小鼠分开,以尽可能减少交叉污染。
  3. 原代细胞包括Müller细胞,感光细胞和其他神经元。光感受器和其他神经元细胞不能适应贴壁培养,并且会在第2代后出现凋亡(P 2 )。
  4. 用培养皿特异性地涂上0.1%明胶,以使Müller细胞更好地粘附。

食谱

  1. 细胞培养基
    Dulbecco改良的Eagle's培养基(DMEM)
    10%胎牛血清(FBS)
    1%青霉素 - 链霉素(Pen / Strep)
  2. 0.1%明胶溶液
    100ml dH 2 O加0.1g明胶,高压灭菌
  3. 细胞冷冻培养基
    Dulbecco改良的Eagle's培养基(DMEM)
    20%胎牛血清(FBS)
    1%青霉素 - 链霉素(Pen / Strep)
    10%二甲基亚砜(DMSO)

致谢

该方案最初在猪实验室采用(Xu等人,2017),现在修改为小鼠。这项工作得到了国立普通医学科学研究所(P20GM103453至Y.L。),路易斯维尔医学院(E0819至Y.L。)和防止失明研究(对路易斯维尔的眼科和视觉科学系)的支持。

参考

  1. Bringmann,A.,Pannicke,T.,Grosche,J.,Francke,M.,Wiedemann,P.,Skatchkov,SN,Osborne,NN and Reichenbach,A。(2006)。&lt; a class =在健康和患病的视网膜中插入文件“href =”https://www.ncbi.nlm.nih.gov/pubmed/16839797“target =”_ blank“>Müller细胞。 Prog Retin Eye Res < / em> 25:397-424。
  2. De Melo Reis,RA,Ventura,AL,Schitine,CS,de Mello,MC and de Mello,FG(2008)。&nbsp; Müller胶质细胞作为调节脊椎动物视网膜神经活动的活性隔室:神经递质和营养因子。 Neurochem R 33:1466-1474。
  3. Fischer,AJ和Reh,TA(2001)。&nbsp; Müller胶质神经胶质是出生后视网膜神经再生的潜在来源。 Nat Neurosci 4:247-252。
  4. Sarthy,PV and Lam,DM(1978)。&nbsp; 生化研究来自乌龟视网膜的孤立胶质细胞(Müller)。细胞生物学78(3):675-684。
  5. Vihtelic,TS和Hyde,DR(2000)。&nbsp; Light诱导的杆和锥细胞在成年白血病斑马鱼(Danio rerio)视网膜中的死亡和再生。 J Neurobiol 44(3):289-307。
  6. Xu,N.,Chen,Y.,Dean,KC,Lu,X.,Liu,X.,Wang,W.,Dean,DC,Kaplan,HJ,Gao,L.,Dong,F.and Liu,Y 。(2017)。猪圈和猪圈的复活人乳头状胶质细胞主要是由端粒伸长引起的。干细胞 25(6):1579-1591。
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引用:Liu, X., Tang, L. and Liu, Y. (2017). Mouse Müller Cell Isolation and Culture. Bio-protocol 7(15): e2429. DOI: 10.21769/BioProtoc.2429.
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