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Apr 2019

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Isolation, Culture, and Identification of Primary Müller Cells from Human Retina
人视网膜原代 Müller 细胞的分离、培养和鉴定   

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Abstract

Müller cells, the major glial cells of the retina, play vital roles in maintaining redox homeostasis and retinal metabolism. An immortalized human Müller cell line (MIO-M1) is widely used as an in vitro model to study Müller cells’ function, but they may not be exactly the same as primarily cultured human Müller cells. The use of human primary Müller cells (huPMCs) in culture has been limited by the requirement for complicated culture systems or particular age ranges of donors. We have successfully grown huPMCs using our established protocol. The cell type was pure, and cultured cells expressed Müller cell-specific markers strongly. The cultured huPMCs were used for morphologic, metabolic, transcriptomic, and functional studies.


Graphic abstract:


Timeline for human primary Müller cell (huPMC) culture


Keywords: Müller cell (Müller细胞), Retina (视网膜), Human (人类), Primary culture (初代培养)

Background

Müller cells, the principal macroglial cells of the vertebrate retina, are responsible for retinal redox homeostasis and provide metabolic support for retinal neurons (Reichenbach and Bringmann, 2013). Müller cells are also a promising target for therapeutic regeneration (Ahmad et al., 2011). An immortalized human Müller cell line (MIO-M1) was characterized in 2002 and has been widely used as an in vitro model to study human Müller cells (Limb et al., 2002). Primary cultures of human cells may offer a more appropriate in vitro model than immortalized cell lines for single-cell studies (Schnichels, 2020). The use of human primary Müller cells (huPMCs) in culture has been limited by the requirement for complicated culture systems (Giannelli et al., 2011) or particular age ranges of donors (Lupien and Salesse, 2007). Human retina tissue from donors cultured using our modified protocol was refrigerated overnight in the dark before digestion. The tissue was then cut into small pieces and firmly pressed to the base of the cell culture flask. These pieces were cultured with a minimum amount of medium for a week then provided with a regular amount of medium. Cell colonies emerged after culturing for two to three weeks in donors from almost all age groups. Our culture system does not require growth factors or Matrigel. Firmly pressing retina tissue to the base of the flask greatly increased the success rate, even in donor samples where the post-mortem time exceeded 24 h. We have successfully detected the following Müller cell markers: CRALBP, VIMENTIN, SOX2, GS, and AQP4.

Materials and Reagents

  1. Pipette tips

  2. Corning® CellBIND® 25 cm2 (T25) rectangular canted neck cell culture flask with vent cap (Corning, catalog number: 3289)

  3. 5 ml flat bottom tube (screw cap) (Techno Plas, catalog number: P5016SU)

  4. Sterile gauze swab (LIVINGSTONE, catalog number: GSS075X5PL)

  5. 1 ml disposable syringe (LIVINGSTONE, catalog number: DSL001MLS)

  6. 18 G sterile hypodermic needle (LIVINGSTONE, catalog number: DN18GX1.5LV)

  7. Falcon® 60 mm TC-treated Easy-Grip style cell culture dish (Corning, catalog number: 353004)

  8. FisherbrandTM Pasteur Pipets (Fisher Scientific, catalog number: 22-209361)

  9. Foil

  10. Serological pipet (Corning, Costar, catalog number: 4487)

  11. 15 ml centrifuge tube (Corning, catalog number: 430791)

  12. 50 ml centrifuge tube (Corning, catalog number: 430291)

  13. DMEM (Dulbecco's modified Eagle medium), high glucose, GlutaMAXTM supplement, pyruvate (ThermoFisher, Gibco, catalog number: 10569-010)

  14. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F9423)

  15. Penicillin-streptomycin (Sigma-Aldrich, catalog number: P4333)

  16. TrypLETM select enzyme (1×) (ThermoFisher, Gibco, catalog number: 12563-029)

  17. PBS tablets (Medicago, catalog number: 09-2051-100)

  18. CO2 independent medium (ThermoFisher, Gibco, catalog number: 18045-088)

  19. 2% PFA in PBS

  20. EZ Slides (Millicell, catalog number: PEZGS0816)

  21. Microscope cover glass, rectangular (Knittel, catalog number: G417)

  22. Triton X-100 (Merck, catalog number: 30632.4N)

  23. Donkey serum (Millipore, Sigma-Aldrich, catalog number: S30-100ML)

  24. Primary antibodies:

    CRALBP (Abcam, catalog number: ab15051)

    VIMENTIN (abcam, catalog number: ab92547)

    SOX2 (Millipore, catalog number: AB5603)

  25. Secondary antibodies:

    Donkey anti-Rabbit IgG (H+L), Alexa Fluor 488 (Invitrogen, ThermoFisher, catalog number: A-21206)

    Donkey anti-Mouse IgG (H+L), Alexa Fluor 594 (ThermoFisher, Invitrogen, catalog number: A-21203)

  26. Hoechst 33342 nucleic acid stain (ThermoFisher, catalog number: H3570)

  27. Vectashield Antifade Mounting Medium (Vector Laboratories, catalog number: H-1000)

  28. DMSO (Dimethyl sulfoxide) (Sigma-Aldrich, catalog number: D2650)

  29. Cryopreservation tubes (Nunc CryoTubes) (Nunc, catalog number: V7634)

  30. Isopropyl alcohol (Sigma-Aldrich, catalog number: W292907)

  31. Sterile specimen jar (Livingstone, catalog number: TP5744SLN)

  32. Complete medium (see Recipes)

  33. Sterile PBS (see Recipes)

  34. Ethanol (80%, v/v) (see Recipes)

  35. Freezing medium (see Recipes)

  36. Blocking solution (see Recipes)

  37. Dilution solution (see Recipes)

Equipment

  1. Forceps (blink medical, katena, model: HR142, Forcep Jewellers No.5)

  2. Scissors (Eyeline, ACROfine, model number: AB-9921D)

  3. Kelly forceps (MediTools, catalog number: SHF-2718)

  4. Autoclave

  5. 37°C water bath

  6. CO2 incubator (37°C, 5% CO2)

  7. Dissection hood

  8. Dissection microscope

  9. Fume cupboard

  10. Inverted microscope

  11. Confocal microscope

  12. Shaking bed

  13. Pipette

  14. 4°C fridge

  15. Centrifuge compatible with 15 ml conical tubes

  16. Freezing container (Thermo Scientific, catalog number: 5100-0001)

  17. -80°C freezer

  18. Liquid nitrogen storage

Procedure

  1. Collect and pre-treat retina tissue (DAY 0)

    Eyes are transported from the eye bank after the cornea is removed for transplantation. Use CO2 Independent Medium to transporting the tissue under atmospheric conditions. Autoclave the dissection tools are beforehand. Use 80% ethanol (v/v) for general sterilizing in the tissue culture room. Dissect donors’ eyes and detach the retina from the eyecup. Starve the retina in DMEM without FBS or growth factors in the fridge and keep in the dark overnight. Rod photoreceptors consume more energy to maintain resting membrane potential in the dark (Okawa et al., 2008); thus, most of them are assumed to have died after being kept in the dark overnight.

    1. Transport human donor eyes (cornea already removed) with a sterile specimen jar in a CO2-independent medium.

    2. Using scissors and forceps, dissect and remove iris, lens, and vitreous from the eyecup and separate neural retina gently from the underlying retinal pigment epithelium (Video 1).


      Video 1. Dissect the neural retina


    3. Collect 1 cm2 of human neural retina in a sterile flat bottom tube containing 5 ml DMEM (for 1-2 T25 flasks).

    4. Wrap the tube with foil to avoid light and keep it at 4°C overnight.


  2. Digest and seed cells (DAY 1)

    Digestion destroys the tissue structure. The small pieces of retinal tissue, needle fixation to the flask, and CellBIND® surface facilitate cell attachment.

    1. Pre-warm the TrypLE digestion solution and complete medium (see Recipes) in a 37°C water bath before the experiment.

    2. Transfer the tissue to a new sterile flat bottom tube containing 5 ml pre-warmed TrypLE to digest the retina. Incubate in a CO2 incubator at 37°C for 60 min. Invert the tube every 20 min during digestion.

    3. Use sterile forceps to transfer the digested retina into a 60 mm cell culture dish containing 10 ml complete medium.

    4. Cut the tissue into small pieces (around 1 × 1 mm) with scissors and forceps in the medium under a dissection microscope in the dissection hood.

    5. Transfer the retinal pieces into T25-cell culture flasks using sterile Pasteur glass pipettes with a minimum volume of complete medium (Figure 1A and 1B; Video 2).


      Video 2. Transfer, distribute and fix the retinal pieces


    6. Bend the sterile 18 G needle to a 90° angle using hemostatic forceps. Separate the dissected retinal pieces and distribute them around the bottom of the flask using the angled needle to prevent clumping (Video 2).

    7. Use an 18 G angled needle to firmly press the retinal pieces onto the bottom of the T25 flasks to enhance the success rate of primary human Müller cell culture; lay down the flask under the microscope, find the retina pieces and firmly press down the syringe so that the sharp needle can fix the tiny retina pieces onto the base. (Figure 1C and 1D; Video 2). Place the flask vertically and add 2 ml complete medium into each flask. Avoid washing off the attached retinal pieces.



      Figure 1. Transfer the retinal pieces to the T25 flask (A and B) and firmly press the retinal pieces to the bottom (C and D)


    8. Place the flask vertically in the cell culture incubator (CO2, 37°C) for 15 min to allow better attachment of retinal pieces to the bottom of T-25 flasks.

    9. Place the T25 flasks down horizontally. The medium (2 ml) is enough to cover the bottom of the flask and will minimize the chance of tissue detachment during culture.

    10. Minimize the disturbance of the flasks.


  3. Increase medium volume (DAY 7)

    1. Add another 2 ml complete medium in each flask.

    2. Minimize the disturbance of the flasks.


  4. Routine culture (from DAY 10)

    It takes approximately 2-3 weeks before huPMCs colonies emerge from the tissue and another 2-3 weeks before reaching 90-100% confluency (Figure 2).

    1. Change the medium with 4 ml fresh complete medium twice a week.



      Figure 2. Morphology of cultured colonies . (A) Colony after 2 weeks of culture. (B) Colony after 6 weeks of culture, showing 90-100% confluency. Scale bars: 200 μm.


  5. Passage

    Digestion of huPMCs usually takes longer than for most cell lines (including MIO, ARPE19, or HEK293T). Passage 1:1 to begin. Apply 1:2 to 1:3 passaging after P1. Our lab uses P3 huPMCs for experiments, although passaging to P10 is generally possible (depending on donors).

    1. Remove the medium and rinse with 3 ml sterile PBS.

    2. Remove the PBS and add 2 ml TrypLE in each T25 flask to digest.

    3. Incubate the flask with TrypLE in a cell culture incubator 6-8 min (when more than half of the cells detach from the bottom under the microscope).

    4. Terminate the digestion by adding 2 ml complete medium in the flask.

    5. Detach all the cells by pipetting and transfer all the liquid to a 15 ml centrifuge tube.

    6. Pellet the huPMCs by centrifuging (200 × g, 5 min, and 20°C).

    7. Resuspend the pellet in 1 ml complete medium and transfer to a new T25 flask with 3 ml complete medium.

    8. Place the flasks back in the cell culture incubator.


  6. Cryopreserve and revive

    The huPMCs cultured in our system can be cryopreserved and revived.

    1. Digest the huPMCs and pellet the cells by centrifuging as described above.

    2. Resuspend the cells in freezing medium. For each confluent T25 flask, the pellets can be resuspended in a 1 ml freezing medium in cryopreservation tubes.

    3. The cryopreservation tubes are transferred to a freezing container filled with isopropyl alcohol at room temperature. The container is then transferred to a -80°C freezer. The cryopreservation tubes can be stored in liquid nitrogen from the next day.

    4. For the revival, thaw the huPMCs in a 37°C water bath with minimum time (normally 1 min). Gently transfer the cells with a freezing medium to a 15 ml centrifuge tube. Add 4 ml pre-warmed complete culture medium into the tube drop by drop.

    5. Centrifuge at 200 × g for 5 min at 20°C and decant the supernatant. Resuspend the pellet in 4 ml complete culture medium and seed the cells into a T25 flask.

    6. Keep the flask in the incubator. The huPMCs usually reach 100% confluency within 3 days.


  7. Identification

    A group of Müller cell markers (CRALBP, VIMENTIN, SOX2, GS, and AQP4) can be detected from P3. Immunocytochemistry is suggested for identification.

    1. Digest P2 huPMCs as described above and seed the P3 huPMCs on EZ slides, 5,000 cells per well, with 500 ul complete medium. Culture the huPMCs in the 8-well EZ slide in the incubator at 37°C for 2 days.

    2. Replace the medium with 500 μl pre-warmed (37°C) PBS and leave the slide on a shaking bed (25 rpm) for 5 min.

    3. Replace the PBS with an additional 500 μl PBS and leave the slide on a shaking bed (25 rpm) for 5 minutes.

    4. (In a fume cupboard) Remove the PBS and fix the cells in 2% PFA for 30 minutes at room temperature.

    5. (In a fume cupboard) Remove the PFA. Wash the cells with PBS three times (as in Step G3), and the cells are ready for blocking.

    6. Replace PBS with 120 μl blocking solution per well. Block for 1 h at room temperature.

    7. Dilute primary antibodies with dilution solution (CRALBP 1:100, VIMENTIN 1:400, SOX2 1:200, GS 1:50, and AQP4 1:100). Centrifuge (13,000 × g, 5 min, 20°C) before application. Replace blocking solution with 120 μl of the desired antibody solution in each well. Incubate the slide with primary antibodies overnight on a shaking bed (25 rpm) at 4°C.

    8. Remove the primary antibodies. Rinse the cells with PBS three times (Step G3).

    9. Dilute secondary antibodies (donkey anti-mouse or rabbit, both 1:1,000) with dilution solution. Centrifuge (13,000 × g, 5 min, 20°C) before application. Replace PBS with 120 μl of the desired antibody solution in each well. Incubate the slide with secondary antibodies in the dark for 4 h at room temperature.

    10. Remove the secondary antibodies. Rinse the cells with PBS three times (Step G3).

    11. Stain with Hoechst 33342 (5.6 mg/L) for 5 min at room temperature. Avoid light.

    12. Remove the Hoechst. Rinse the cells with PBS three times (Step G3).

    13. Break the walls of the EZ slide as described in the manual. Mount the slide with Vectashield Antifade Mounting Medium. Immunofluorescence images can be taken using confocal microscopy immediately.

Notes

We have successfully cultured human primary Müller cells from retina donors aged 41 to 79 years old, following this protocol. The cultured cells were VIMENTIN/CRALBP/SOX2 positive (Figure 3). Other Müller cell markers positively tested include GS and AQP4 (data not shown). In most cases, huPMCs can be passaged successfully before P10 without a change of morphology and remain CRALBP positive.



Figure 3. Immunofluorescent staining of huPMCs (P3) with VIMENTIN (green), CRALBP (red) and Hoechst (blue) (A); CRALBP (red) and SOX2 (green) (B). Scale bar in A: 20 μm; B: 100 μm.

Recipes

  1. Complete medium

    DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% Penicillin-Streptomycin.

    Aliquot to 50 ml centrifuge tubes and store at 4°C. Avoid light and repeated opening. Use within a month.

  2. Sterile PBS

    Dissolve 5 PBS tablets in MQ water and fill up to 500 ml before autoclaving (0.14 M NaCl, 0.0027 M KCl, 0.010 M phosphate buffer pH 7.4).

    Aliquot in 50 ml centrifuge tubes and store at 4°C. Use within 3 months.

  3. Ethanol (80%, v/v)

    Add 1.6 L ethanol in a glass jar, then fill up to 2 L with MQ water.

  4. Freezing medium

    20% FBS and 10% DMSO in DMEM

  5. Blocking solution

    5%(v/v) Normal Donkey Serum, 0.5% Triton X-100 in PBS

  6. Dilution solution

    1% Normal Donkey Serum, 0.5% Triton X-100 in PBS

Acknowledgments

We acknowledge the Lions NSW Eye Bank and Australian Ocular Biobank tissue coordinators and scientists and eye donors and their families for the human donor eye tissue used in this project. This study was supported by the Ophthalmic Research Institute of Australia and a grant from the Lowy Medical Research Institute. This protocol was derived from Zhang et al. (2019) (DOI: 10.7554/eLife.43598).

Competing interests

The authors declare no competing interests.

Ethics

Human subjects: Human retinas were obtained from post-mortem donor eyes with ethical approval from the Human Research Ethics Committee of the University of Sydney (HREC#:16/282).

References

  1. Ahmad, I., Del Debbio, C. B., Das, A. V. and Parameswaran, S. (2011). Müller glia: a promising target for therapeutic regeneration. Invest Ophthalmol Vis Sci 52(8): 5758-5764.
  2. Giannelli, S. G., Demontis, G. C., Pertile, G., Rama, P. and Broccoli, V. (2011). Adult human Müller glia cells are a highly efficient source of rod photoreceptors. Stem Cells 29(2): 344-356.
  3. Limb, G. A., Salt, T. E., Munro, P. M., Moss, S. E. and Khaw, P. T. (2002). In vitro characterization of a spontaneously immortalized human Müller cell line (MIO-M1). Invest Ophthalmol Vis Sci 43(3): 864-869.
  4. Lupien, C. B. and Salesse, C. (2007). Characterization of two spontaneously generated human Müller cell lines from donors with type 1 and type 2 diabetes. Invest Ophthalmol Vis Sci 48(2): 874-880.
  5. Okawa, H., Sampath, A. P., Laughlin, S. B. and Fain, G. L. (2008). ATP consumption by mammalian rod photoreceptors in darkness and in light. Current Biol 18(24): 1917-1921.
  6. Reichenbach, A. and Bringmann, A. (2013). New functions of Müller cells. Glia 61(5): 651-678.
  7. Schnichels, S., Paquet-Durand, F., Löscher, M., Tsai, T., Hurst, J., Joachim, S. C. and Klettner, A. (2020). Retina in a dish: Cell cultures, retinal explants and animal models for common diseases of the retina. Prog Retin Eye Res. doi: 10.1016/j.preteyeres.2020.100880.
  8. Zhang, T., Zhu, L., Madigan, M. C., Liu, W., Shen, W., Cherepanoff, S., Zhou, F., Zeng, S., Du, J. and Gillies, M. C. (2019). Human macular Müller cells rely more on serine biosynthesis to combat oxidative stress than those from the periphery. eLife 8: e43598.

简介

[摘要] Müller 细胞是视网膜的主要神经胶质细胞,在维持氧化还原稳态和视网膜代谢方面起着至关重要的作用。永生化人 Müller 细胞系 (MIO-M1) 被广泛用作研究 Müller 细胞功能的体外模型,但它们可能与最初培养的人 Müller 细胞不完全相同。人类原代穆勒细胞 (huPMCs) 在培养中的使用受到复杂培养系统或特定年龄范围供体的要求的限制。我们已经使用我们建立的协议成功地培养了huPMC。细胞类型是纯的,培养的细胞强烈表达 Müller 细胞特异性标志物。培养的 huPMC 用于形态学、代谢、转录组学和功能研究。

图文摘要:

Ť imeline对人原代细胞穆勒(huPMC)培养



[背景] Müller 细胞是脊椎动物视网膜的主要大胶质细胞,负责视网膜氧化还原稳态并为视网膜神经元提供代谢支持(Reichenbach和Bringmann ,2013 年)。Müller 细胞也是治疗性再生的有希望的靶点(Ahmad等,2011)。2002 年表征了永生化的人类 Müller 细胞系 (MIO-M1),并已广泛用作研究人类 Müller 细胞的体外模型 (Limb等人,2002)。P人类细胞的培养物rimary可以提供更合适的体外模型比永生化细胞系为单-细胞研究(Schnichels,2020)。Ť他使用培养的人原代Müller细胞(huPMCs)的已被用于复杂培养系统的要求的限制(Giannelli等人,20 1 1 )捐赠者或特定年龄范围(Lupien和Salesse ,2007)。从使用我们的经修改协议培养捐献者的人视网膜组织是在消化之前黑暗冷藏过夜。然后,将组织切成小片,并牢固地压编到细胞培养瓶的底部。这些片用最少量的培养基培养一周,然后提供常规量的培养基。在几乎所有年龄组的供体中培养两到三周后出现细胞集落。我们的培养系统不需要生长因子或基质胶。用力按荷兰国际集团视网膜组织到烧瓶的底部大大提高了成功率,甚至在供体样品,其中所述验尸时间超过24 ħ 。我们已经成功地检测到下面的穆勒细胞标记小号:CRALBP ,波形蛋白,SOX2 ,GS和AQP4。

关键字:Müller细胞, 视网膜, 人类, 初代培养


材料和试剂

 
移液器吸头
Corning ® CellBIND ® 25 cm 2 (T25)带通风盖的矩形斜颈细胞培养瓶(Corning,目录号:3289)
5毫升平底管(螺旋盖)(Techno Plas,目录号:P5016SU)
无菌纱布拭子(LIVINGSTONE,目录号:GSS075X5PL)
1毫升一次性注射器(LIVINGSTONE,目录号:DSL001MLS)
18 G无菌皮下注射针(LIVINGSTONE,目录号:DN18GX1.5LV)
Falcon ® 60 mm TC 处理的 Easy-Grip 式细胞培养皿(Corning,目录号:353004)
Fisherbrand TM Pasteur Pipets (Fisher Scientific,目录号:22-209361)
挫败
血清移液管(Corning,Costar,目录号:4487)
15 ml离心管(Corning,目录号:430791)
50 ml 离心管(Corning,目录号:43 0291)
DMEM (Dulbecco氏米odified鹰米edium),高葡萄糖,GlutaMAX的TM小号补编第,丙酮酸(赛默飞,ģ IBCO ,目录号:10569-010)
胎b绵羊小号erum(FBS )(小号IGMA -A ldrich ,目录号:F9423)
青霉素小号treptomycin(S IGMA -A ldrich ,目录号:P4333)
的TrypLE TM小号选ë nzyme(1 × )(赛默飞,GIBCO,目录号:12563-029)
PBS 片剂(Medicago,目录号:09-2051-100)
CO 2独立介质(ThermoFisher,G ibco ,目录号:18045-088)
PBS 中的 2% PFA
EZ Slides (Millicell,目录号:PEZGS0816)
显微镜盖玻璃,矩形(Knittel,目录号:G417)
Triton X-100(默克,目录号:30632.4N)
驴血清(Millipore,S igma -A ldrich ,目录号:S30-100ML )
一抗:
CRALBP (A bcam,目录号:ab15051)
VIMENTIN(abcam,目录号:ab92547)
SOX2(Millipore,目录号:AB5603)
二抗:
驴抗兔 IgG(H+L),Alexa Fluor 488(Invitrogen,ThermoFisher,目录号:A-21206)
驴抗小鼠 IgG(H+L),Alexa Fluor 594(ThermoFisher,Invitrogen,目录号:A-21203)
Hoechst 33342核酸染色剂(ThermoFisher,目录号:H3570)
Vectashield Antifade Mounting Medium(Vector Laboratories,目录号:H-1000)
DMSO(二甲基亚砜)(S igma -A ldrich ,目录号:D2650)
冷冻保存管(Nunc CryoTubes)(Nunc,目录号:V7634)
I异丙醇(S igma -A ldrich ,目录号:W292907)
无菌标本罐(Livingstone,目录号:TP5744SLN)
完全培养基(见食谱)
无菌 PBS (见配方)
乙醇(80%,v/v )(见配方)
冷冻培养基(见ř ecipes)
B锁定解决方案(见配方)
稀释溶液(见配方)
 
设备
 
˚F orceps(眨眼医疗,katena,型号:HR142,镊子珠宝5号)
S剪刀(Eyeline,ACROfine,型号:AB-9921D )
凯利钳子(中号EDI Ť ools,目录号:SHF-2718 )
一个utoclave
37 ℃水浴
CO 2培养箱(37°C,5% CO 2 )
d issection罩
d issection显微镜
˚F UME橱柜
我ñ verted显微镜
共焦显微镜
小号HAK荷兰国际集团的床
P ipette
4°C冰箱
Ç entrifuge兼容与15 ml的锥形管小号
˚F reezing容器物(Thermo Scientific,目录号:5100-0001)
-80 °C 冰箱
液氮储存
 
程序
 
收集和预处理视网膜组织(第 0 天)
E是在取出角膜进行移植后从眼库中运输。使用CO 2独立中等至大气条件下输送所述组织。一个utoclave牛逼,他清扫工具是事先。在组织培养室中使用80% 乙醇 (v/v) 进行一般消毒。解剖捐赠者的眼睛并将视网膜从眼罩上分离。饿死不含FBS或生长因子的DMEM视网膜在冰箱和柯EP在黑暗过夜。杆状光感受器消耗更多能量以在黑暗中维持静息膜电位(Okawa et al. , 20 0 8 );因此,他们中的大多数人被认为是在黑暗中过夜后死亡的。
在不依赖CO 2 的介质中用无菌标本罐运输人类供体眼睛(角膜已经去除)。
使用剪刀和镊子,从眼罩中分离和去除虹膜、晶状体和玻璃体,轻轻地将神经视网膜与下面的视网膜色素上皮细胞分离(视频 1)。
 
 
视频 1.解剖神经视网膜
 
在含有 5 ml DMEM(用于 1-2 个 T25 烧瓶)的无菌平底管中收集 1 cm 2的人类神经视网膜。
用箔纸包裹管子避光并保持在4°C过夜。
 
消化和种子细胞(第 1 天)
消化会破坏组织结构。视网膜组织,针固定到该烧瓶中的小片,并CELLBIND ®表面促进细胞附着。
预暖的的TrypLE消化溶液和完全培养基(见[R在37℃水浴中在实验前ecipes)。
将组织转移到含有 5 ml 预热的 TrypLE 的新无菌平底管中以消化视网膜。在 CO 2培养箱中于 37°C孵育60 分钟。在消化过程中每 20 分钟反转一次管子。
使用无菌镊子对Ť消化的视网膜转让(BOT)到一个含有10ml完全培养基60毫米细胞培养皿中。
在解剖罩中的解剖显微镜下,用剪刀和镊子将组织切成小块(约 1 × 1 毫米)。
转移视网膜片放入使用无菌T25细胞培养烧瓶P asteur玻璃吸管用完全培养基的最小体积(图1A和图1B;视频2)。
 
 
视频 2. 转移、分配和修复视网膜碎片
 
弯曲无菌18号针以一个90 °使用止血钳角。分离解剖的视网膜碎片,并使用斜针将它们分布在烧瓶底部的周围,以防止结块(视频 2)。
使用18G的倾斜针用力按下视网膜片上的底部的T25烧瓶,以提高原代人穆勒细胞培养物的成功率; 将烧瓶放在显微镜下,找到视网膜碎片并用力按下注射器,使锋利的针头可以将微小的视网膜碎片固定在底座上。(图 1C和1D;视频 2)。将烧瓶垂直放置并在每个烧瓶中加入 2 ml 完全培养基。避免洗掉附着的视网膜碎片。
 
图1 Ť转让(BOT)视网膜升件到T25烧瓶(A和B)和用力按下视网膜件的底部(C和d)
 
将烧瓶垂直放置在细胞培养孵化器 (CO 2 , 37°C) 中 15 分钟,以使视网膜碎片更好地附着在 T-25 烧瓶的底部。
将 T25 烧瓶水平放置。培养基 (2 ml) 足以覆盖烧瓶底部,并将在培养过程中最大限度地减少组织脱离的机会。
尽量减少烧瓶的干扰。
 
增加中等音量(第7天)
在每个烧瓶中再加入 2 ml 完全培养基。
尽量减少烧瓶的干扰。
 
常规培养(从第 10 天开始)
在 huPMCs 集落从组织中出现之前大约需要2-3 周,再需要 2-3 周才能达到 90-100% 汇合(图 2)。
每周更换两次 4 ml 新鲜完全培养基。
 
 
图 2.培养菌落的形态。(A)c ^ olony 2后周文化的。(B)培养 6 周后的菌落,显示90-100% 汇合。比例尺:200 μm。
 
通道
huPMCs的消化通常需要更长的比对大多数细胞系(包括UDING MIO,ARPE19,或HEK293T)。1:1 开始。在 P1 后应用 1:2 到 1:3 传代。我们的实验室使用 P3 huPMCs 进行实验,尽管通常可以传代到 P10(取决于供体)。
取出培养基并用 3 ml 无菌 PBS 冲洗。
取出 PBS 并在每个 T25 烧瓶中加入 2 ml TrypLE 进行消化。
在细胞培养箱中用 TrypLE 孵育烧瓶 6-8 分钟(当超过一半的细胞在显微镜下从底部分离时)。
在烧瓶中加入 2 ml 完全培养基终止消化。
拆离的所有单元通过pipett荷兰国际集团和传输所有到15ml离心管中的液体。
通过离心机沉淀huPMCs荷兰国际集团(200 ×克,5分钟,和20℃)。
将沉淀重悬在 1 ml 完全培养基中,然后转移到装有 3 ml 完全培养基的新 T25 烧瓶中。
将烧瓶回在细胞培养孵化器。
 
冷冻保存和复活
在我们的系统中培养的 huPMCs 可以冷冻保存和复活。
消化huPMCs和通过离心机沉淀细胞荷兰国际集团如上所述。
在冷冻培养基中重悬细胞。对于每个汇合的 T25 烧瓶,可以将沉淀物重新悬浮在冷冻保存管中的 1 ml冷冻培养基中。
在室温下将冷冻保存管转移到装有异丙醇的冷冻容器中。然后将容器转移到 -80°C 的冰箱中。从第二天起,冷冻保存管可以储存在液氮中。
为了复兴,在 37 °C 水浴中以最短时间(通常为 1 分钟)解冻 huPMC 。用冷冻培养基轻轻地将细胞转移到 15 ml 离心管中。添加4米升预热的完全培养基到由下降管下降。
在 20°C 下以 200 × g离心5 分钟并倒出上清液。重悬在4米粒料升完全培养基和种子细胞到T25培养瓶中。
将烧瓶放在培养箱中。huPMCs 通常在 3 天内达到 100% 汇合。
 
鉴别
A组的穆勒细胞标记物(CRALBP,波形蛋白,SOX2,GS ,和AQP4)可以从P3检测。建议使用免疫细胞化学进行鉴定。
如上所述消化 P2 huPMCs 并将 P3 huPMCs 接种在EZ 载玻片上,每孔5 , 000 个细胞,500 ul 完全培养基。在 37°C 的孵化器中,将 huPMCs 培养在 8 孔 EZ 载玻片中 2 天。
替换500的介质μ升预温(37℃)的PBS ,并离开上的摇床(25 rpm)的滑动5分钟。
重新地方的PBS中的一个附加的500 μ l PBS并将载玻片放在摇床 (25 rpm) 上 5 分钟。
(我N A通风橱)拆下PBS并固定在2%PFA将细胞在室温下30分钟。
(我N A通风橱)拆下PFA。清洗细胞,用PBS 3倍(如在小号TEP ģ 3 ),和细胞准备用于阻断。
用120替换PBS μ升每封闭液孔。在室温下封闭 1 小时。
稀p与稀释溶液rimary抗体(CR ALBP 1:100,波形蛋白1:400,SOX2 1:200,GS 1:50 ,和AQP4 1:100) 。应用前离心(13,000 × g ,5 分钟,20 °C)。替换阻断与120溶液μ升的的每个孔中所需的抗体溶液。孵育在SHAK过夜与一抗滑动荷兰国际集团在4℃下的床(25 RPM)。
去除一抗。用 PBS 冲洗细胞3次(步骤 G3 )。
稀小号econdary抗体(驴抗-小鼠或兔,二者1:1 ,000)用稀释溶液。应用前离心(13,000 × g ,5 分钟,20 °C)。用120替换PBS μ升的的每个孔中所需的抗体溶液。在室温下用二级抗体在黑暗中孵育幻灯片 4 小时。
去除二抗。用 PBS 冲洗细胞3次(步骤 G3 )。
在室温下用Hoechst 33342 (5.6 mg/L)染色5 分钟。避光。
移除Hoechst 。用 PBS 冲洗细胞3次(步骤 G3 )。
按照手册中的说明打破 EZ 幻灯片的墙壁。使用Vectashield Antifade Mounting Medium 安装载玻片。可以立即使用共聚焦显微镜拍摄免疫荧光图像。
 
笔记
 
按照此协议,我们已成功从 41 至 79 岁的视网膜供体中培养出人类原代 Müller 细胞。培养的细胞呈波形蛋白/CRALBP/SOX2 阳性(图 3)。其他阳性的Müller 细胞标志物包括 GS 和 AQP4(数据未显示)。在大多数情况下,huPMCs 可以在 P10 之前成功传代而不会改变形态并保持 CRALBP 阳性。
 
 
图 3. huPMCs (P3)与 VIMENTIN(绿色)、CRALBP(红色)和 Hoechst(蓝色)(A)的免疫荧光染色;CRALBP(红色)和 SOX2(绿色)(B)。A 中的比例尺:20 μ m;B:100 μ米。
 
食谱
 
完全培养基
DMEM 补充有 10% 热灭活胎牛血清 (FBS) 和 1%青霉素-链霉素。
分装到 50 ml 离心管中,并在 4 °C 下储存。甲空隙光并重复打开。使用一个月内。
小号terile PBS
d issolve 5种PBS片剂在MQ水中和填充到autoclav之前500毫升荷兰国际集团(0.14 M氯化钠,0.0027米氯化钾,0.010磷酸盐缓冲液pH 7.4)。
分装在50 ml 离心管中,并在 4 °C 下储存。3个月内使用。
乙醇(80%,v/v)
甲DD在玻璃瓶1.6 L乙醇,然后填充到2升用MQ水。
˚F reezing媒体
DMEM 中的 20% FBS 和 10% DMSO
阻塞解决方案
5%(v/v) 正常驴血清,0.5% Triton X-100,在 PBS 中
稀释液
1% 正常驴血清,0.5% PBS 中的 Triton X-100
 
致谢
 
我们感谢新南威尔士狮子会眼库和澳大利亚眼科生物库的组织协调员、科学家和眼捐献者及其家人为本项目中使用的人类捐献眼组织提供的帮助。这项研究得到了澳大利亚眼科研究所和洛伊医学研究所的资助。该协议源自 Zhang等人。( 2019) (DOI: 10.7554/eLife.43598) 。
 
利益争夺
 
作者声明没有竞争利益。
 
伦理
 
人类受试者:在悉尼大学人类研究伦理委员会 (HREC#:16/282) 的伦理批准下,从死后捐赠者的眼睛中获得人类视网膜。
 
参考
 
Ahmad, I., Del Debbio, CB, Das, AV和Parameswaran, S. (2011)。Müller 胶质细胞:治疗再生的有希望的目标。Invest Ophthalmol Vis Sci 52(8) : 5758 - 5764。
Giannelli, SG, Demontis, GC, Pertile, G., Rama, P.和Broccoli, V. (2011)。成人 Müller 神经胶质细胞是杆状光感受器的高效来源。干Ç ELLS 29(2) :344 - 356。
Limb, GA, Salt, TE, Munro, PM, Moss, SE和Khaw, PT (2002)。自发永生化人 Müller 细胞系 (MIO-M1) 的体外表征。投资ø phthalmol V是小号CI 43(3) :864 - 869。
Lupien , CB和Salesse, C. (2007)。表征来自 1 型和 2 型糖尿病供体的两种自发产生的人类 Müller 细胞系。投资ø phthalmol V是小号CI 48(2) :874 - 880。
Okawa, H., Sampath, AP, Laughlin, SB和Fain, GL (2008)。哺乳动物杆状光感受器在黑暗和光照下的 ATP 消耗。Current B iol 18(24) : 1917 - 1921。
Reichenbach, A.和Bringmann, A. (2013)。缪勒细胞的新功能。神经胶质61(5) :651 - 678。
Schnichels, S.、Paquet-Durand, F.、Löscher, M.、Tsai, T.、Hurst, J.、Joachim, SC和Klettner, A.(2020 年)。培养皿中的视网膜:细胞培养物、视网膜外植体和视网膜常见疾病的动物模型。Prog R etin E ye R es 。doi:10.1016/j.preteyeres.2020.100880。
Zhang, T., Zhu, L., Madigan, MC, Liu, W., Shen, W., Cherepanoff, S., Zhou, F., Zeng, S., Du, J.和Gillies, MC (2019) . 与来自外周的细胞相比,人类黄斑部 Müller 细胞更多地依赖丝氨酸生物合成来对抗氧化应激。eLife 8 :e43598。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, Y., Zhang, T., Zeng, S., Yam, M., Gillies, M. C. and Zhu, L. (2021). Isolation, Culture, and Identification of Primary Müller Cells from Human Retina. Bio-protocol 11(19): e4179. DOI: 10.21769/BioProtoc.4179.
  2. Zhang, T., Zhu, L., Madigan, M. C., Liu, W., Shen, W., Cherepanoff, S., Zhou, F., Zeng, S., Du, J. and Gillies, M. C. (2019). Human macular Müller cells rely more on serine biosynthesis to combat oxidative stress than those from the periphery. eLife 8: e43598.
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