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Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue
将人外周角膜球体植入尸体人角膜组织   

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本实验方案简略版
Stem Cell Research & Therapy
Jun 2016

Abstract

Stem and progenitor cells isolated from human limbal tissue can be cultured in vitro as spheres. These spheres have potential for use as transplantable elements for the repopulation of corneal tissue (Mathan et al., 2016). Herein we describe the detailed protocol for the implantation of human corneal spheres into cadaveric human corneal tissue. This protocol describes the procedure for sphere formation and culture, preparation of tissue for sphere implantation, corneal limbus microsurgery and sphere implantation.

Keywords: Cell Culture (细胞培养), Cornea (角膜), Sphere-forming cells (球体形成细胞), Implantation (移植), Limbus (角膜缘)

Background

Previous research has focused on isolation of limbal cells which were exclusively epithelial (limbal stem cell) or stromal (keratocyte progenitor cell) in order to decipher their individual roles in corneal homeostasis and wound repair. This protocol aims to isolate limbal cells by their functional ability to form spheres in culture and which by their very nature will include a diversity of cells both epithelial and stromal which contribute to the formation of the limbal niche. Subsequent to isolation of these spheres we are investigating their potential use in corneal restoration after implantation. Here we describe an in-vitro surgical protocol for the implantation of these spheres into human corneal tissue and the downstream analysis.

Materials and Reagents

  1. Scalpel blade (ProSciTech, profile 11) (Swann Morton, catalog number: LSB11 )
  2. 20 x 100 mm cell culture dish (Corning, Falcon®, catalog number: 353003 )
  3. Cotton buds (Cotton Tips double ended, Protec Solutions, catalog number: 941001690389 )
  4. Transfer pipette (3 ml) (Interlab, catalog number: KJ622-1A )
  5. 5 ml tubes (Techno Plas, catalog number: P5016UL )
  6. 40 µm strainer (Corning, Falcon®, catalog number: 352340 )
  7. Glass coverslips (covergalss #1 30 mm diam.) (PST, catalog number: G430 )
  8. 6-well tissue culture plates (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 140675 )
  9. 35 x 10 mm cell culture dish (Corning, Falcon®, catalog number: 353001 )
  10. Fresh and frozen human cadaveric donor corneoscleral tissue obtained post-surgery from the New Zealand National Eye Bank
  11. 70% ethanol (EMD Millipore, catalog number: 1009832500 )
  12. Phosphate buffered saline (PBS) sterile (Sigma-Aldrich, catalog number: P4417-100TAB )
  13. Phosphate buffered saline and 10% glycerol (AnalaR NORMAPUR) (VWR, catalog number: 24388.320 )
  14. Dispase (Thermo Fisher Scientific, GibcoTM, catalog number: 17105041 )
  15. Collagenase (Blend Type L) (Sigma-Aldrich, catalog number: C8176 )
  16. Hyaluronidase (Sigma-Aldrich, catalog number: H3506 )
  17. Neurobasal-A Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10888022 )
  18. Human Epidermal Growth Factor (EGF) (PeproTech, catalog number: AF-100-15 )
  19. Human Fibroblastic Growth Factor (FGF) Basic (PeproTech, catalog number: 100-18B )
  20. B-27 Supplement, 50x (Thermo Fisher Scientific, GibcoTM, catalog number: 12587010 )
  21. N-2 Supplement, 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 17502048 )
  22. GlutaMAX, 100x (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )
  23. MEM with GlutaMAX (Minimum Essential Medium) (Thermo Fisher Scientific, GibcoTM, catalog number: 41090036 )
  24. Foetal bovine serum (New Zealand origin) (Thermo Fisher Scientific, GibcoTM, catalog number: 10091148 )
  25. Antibiotic/Antimycotic 100x (Anti/Anti) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  26. Supplemented Neurobasal-A medium (see Recipes)
  27. Standard culture medium (see Recipes)

Equipment

  1. Tissue culture hood (Heal Force, model: HFsafe 1800 )
  2. Straight scissors (World Precision Instruments, catalog number: 500216 )
  3. Fine forceps (World Precision Instruments, catalog number: 14142 )
  4. Orbital shaker (Ratek Instruments, model: MM1 )
  5. Centrifuge (Sigma Laborzentrifugen, model: 3-15 )
  6. Tissue culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: HereausTM HeraCell 150 )
  7. Laminar flow cabinet (Gelman, model: HLF 120 )
  8. Binocular Stereo microscope (Carl Zeiss, catalog number: 474110-9904 )
  9. Biological safety cabinet (Email Air Handling, catalog number: 1687-2340-618-3 )
  10. 2-20 µl Eppendorf® Pipette (Eppendorf, catalog number: 3120000038 )
  11. Feather MicroScalpel (pfm medical, catalog number: 200300715 )
  12. Crescent Bevel Up Ophthalmic Knife 2.3 mm (MANI, catalog number: MCU26 )
  13. NIKON Digital sight DS-UI camera (Nikon Instruments, model: DS-UI )
  14. Leica DMIL inverted contrasting microscope (Leica Microsystems, model: Leica DMIL )
  15. Leica DM-RA upright fluorescence microscope (Leica Microsystems, model: Leica DM-RA )

Software

  1. NIS-Elements Br Microscope Imaging Software version 3.0

Procedure

Note: This procedure has been described in previous work by our laboratory (Yoon et al., 2013), the following section however provides additional information on the process and describes the technique in a protocoled format.

  1. Sphere formation and culture (Video 1)

    Video 1. Corneoscleral tissue processing for Sphere Forming Assay. Corneoscleral tissue is rubbed with a cotton bud on the posterior side to remove endothelial cells, scraped on the anterior side to remove epithelial cells and washed in PBS. The corneal/limbal region is excised and cut into small pieces prior to enzymatic treatment for limbal cell isolation.

    1. Using sterile technique in a sterile tissue culture hood, prepare 1 container of 70% ethanol and 1 container of PBS for tools and 1 container of PBS for washing. Place straight scissors, scalpel blade and forceps in 70% ethanol for 10 min.
    2. Remove the above equipment from 70% ethanol and rinse briefly in sterile PBS to remove residual ethanol.
    3. Using forceps, place corneoscleral tissue into a 100 mm sterile Petri dish (the corneoscleral rim [Figure 1A] has an approximate 2-2.5 mm wide ring of corneal/limbal tissue, which when excised has an approximate wet weight of 55-57 mg).
    4. Using sterile cotton buds, rub the posterior surface of the corneal tissue to remove endothelial cells (Figure 1B).
    5. Using a crescent bevel up ophthalmic knife, gently scrape the anterior surface of the tissue to remove epithelial cells (Figure 1C).
    6. Using a sterile transfer pipette, wash tissue with PBS. Repeat epithelial scraping and washes until PBS washes run clear of cellular debris (Figure 1D).
    7. Excise the limbal region and exclude as much of the sclera as possible (Figure 1E). Rinse the excised limbus, mince the tissue using scissors (Figure 1F) and place into sterile 5 ml tubes.


      Figure 1. Corneoscleral tissue processing for Sphere Forming Assay. Corneoscleral tissue (A) is rubbed with a cotton bud on the posterior side to remove endothelial cells (B), scraped on the anterior side to remove epithelial cells (C) and washed in PBS (D). The corneal/limbal region is excised (E) and cut into small pieces (F) prior to enzymatic treatment for limbal cell isolation.

    8. Incubate in 1.2 U/ml Dispase for 40 min at 37 °C.
    9. Incubate in 2 mg/ml collagenase and 0.5 mg/ml hyaluronidase in MEM with Anti/Anti overnight at 37 °C on an orbital shaker.
    10. Strain the solution and cells using a 40 µm strainer to remove undigested material.
    11. Centrifuge filtrate for 7 min at 405 x g and wash cell pellet with PBS.
    12. Suspend cells in 2 ml supplemented Neurobasal-A medium.
    13. Prepare sterile glass coverslips in 6-well tissue culture plates.
    14. Seed the 2 ml suspension evenly into wells.
    15. Culture cells in a humidified incubator at 37 °C in 5% CO2.
    16. Carefully change 50% of medium twice weekly pipetting from the surface to avoid removing cells or newly forming spheres.
    17. Cells often become adherent to glass coverslips and aggregate into sphere like structures (Figure 2).


      Figure 2. Sphere formation. Phase contrast microscopy showing isolated cells initially adherent to glass coverslips after seeding (A), which over the course of days to weeks aggregate and form sphere like structures (B). Scale bar = 100 µm.

  2. Preparation of tissue for sphere implantation
    1. Under sterile conditions within a biological safety cabinet (Heal Force Model HF-safe 1800) donor corneoscleral rims, which have been in storage at -80 °C for over 3 months, are divided into 1/8th segments, stored in individual 5 ml containers and re-frozen at -80 °C.
    2. Each corneoscleral rim is subjected to a total of 3 freeze-thaw cycles in order to decellularise the tissue prior to use for implantation.
      Note: If complete decellularisation is required, LIVE/DEAD® 2 μM calcein AM and 4 μM ethidium homodimer-1 (Life Technologies) in standard culture medium can be used according to manufacturer directions to either confirm complete decellularisation or determine the need for additional freeze-thaw cycles.

  3. Corneal limbus microsurgery (Video 2)

    Video 2. Surgical excision of a wedge of limbal tissue. Fine forceps were used with the non-dominant hand to anchor the sclera and provide counter-traction as two diagonal ~3 mm incisions were made prior to gripping the apex of the incised tissue to cut away and remove the anterior tissue segment. Cornea-limbus-sclera demarcations have to be visually approximated.

    1. Using sterile technique in a laminar flow cabinet (Gelman model HLF 120 ), prepare 1 container of 70% ethanol and 1 container of sterile PBS for tools.
    2. Place fine forceps and MicroScalpel blade into the ethanol and leave for 10 min.
    3. Remove a thawed limbal rim segment and place into a Petri dish
    4. After washing tools in PBS, visualize the limbal rim using a binocular stereomicroscope.
    5. Using a MicroScalpel, make two diagonal ~3 mm incisions (Figure 3A), into the limbus with the apices intersecting such that a triangular wedge is created (Figures 3B and 3C). Using fine forceps grip the tissue at the apex and lift up the wedge (Figure 3D).


      Figure 3. Surgical excision of a wedge of limbal tissue. Fine forceps were used with the non-dominant hand to anchor the sclera and provide counter-traction as two diagonal ~3 mm incisions (A) were being made (B, C) prior to gripping the apex to cut away the incised tissue (D). Cornea-limbus-sclera demarcations have to be visually approximated.

    6. Cut away the anchoring tissue using the feather MicroScalpel while angling the blade anteriorly so that the depression created is shallower in the corneal direction (Figure 4).


      Figure 4. Removal of the wedge-shaped tissue. 3-D representation of the upward sloping floor of the wedge (A) that is created post excision (B) from the limbal region.

    7. Repeat to create additional incisions in tissue.
      Note: Visualization of the incisions are challenging initially to the untrained eye but will develop with practice. Good lighting will be helpful.
    8. The Petri dish is closed and the tissue is transferred to a class II biological safety cabinet.

  4. Sphere implantation
    1. Using sterile cotton buds, excess fluid is first blotted away from the corneoscleral tissue in which limbal wedge incisions have been made.
    2. Spheres are visualized in wells under a binocular stereomicroscope within a class II biological safety cabinet.
    3. Using a sterile 2-20 µl Eppendorf® Pipette set at a volume of 5 µl, gently aspirate spheres from culture.
    4. Visualize the incisions made in the corneoscleral tissue and eject the spheres and medium into the wedge incisions.
      Note: It is best to eject as minimum possible volume of culture medium as possible while ensuring that spheres have been ejected. Occasionally, difficulties arise with spheres floating off the tissue if the sphere implantation, specifically the ejection of sphere from the micropipette, is not performed gently.
    5. Transfer the corneoscleral rim with implanted sphere into a 35 x 10 mm cell culture dish and incubate at room temperature for 15 min epithelial side up.
    6. Invert the tissue so that the epithelium faces down.
      Note: Through personal experience, inverting the tissue has proven to be better than culturing tissue the right side up as the cornea usually floats and the spheres become not fully submerged in culture medium which does not seem facilitate cell migration and survival. With inverted culture however, spheres occasionally do not remain in situ and become detached from the tissue.
    7. Gently overlay the tissue with 2 ml of standard culture medium.
    8. Perform half medium changes every second day.
      The following image panel and legend has been reproduced from Mathan et al. (2016) (Figure 5).


      Figure 5. Implantation of peripheral corneal spheres into donor corneoscleral rims. Spheres (arrowheads) were implanted into wedge-shaped incisions made at the limbal region. Under stereomicroscopy, the corneoscleral rim with incisions (arrows) and implanted spheres can be clearly visualised (A). Combined phase contrast and fluorescence microscopy show an implanted sphere stained positively for live cells with LIVE/DEAD® stain (B). This signal is confined to the sphere and not detected in the surrounding tissue. A 40x DAPI stained 10 µm thick cross section of frozen-stored corneoscleral rim confirmed the absence of DAPI positive cell nuclei prior to implantation (C). Through phase contrast microscopy, the position of the spheres in the semi-transparent region of tissue is shown (D). Scale bars = 500 µm (A), 100 µm (B and D), 50 µm (C).

Data analysis

  1. To assess the viability of spheres and implanted cells in tissue, LIVE/DEAD® 2 μM calcein AM and 4 μM ethidium homodimer-1 (Life Technologies) in standard culture medium was used.
  2. Bright-field images, assessed using an SV6 Binocular Stereo microscope (Carl Zeiss), were captured using a NIKON Digital sight DS-UI camera (NIKON CORPORATION, Tokyo, Japan). Phase-contrast and fluorescence microscopy was performed using the following microscopes: Leica DMIL inverted contrasting microscope (Leica Microsystems, Wetzlar, Germany), 4x magnification 0.1 aperture, C PLAN with Leica Application Suite Version 4.4.0 Build 454; and Leica DM-RA upright fluorescence microscope (Leica Microsystems), 5x magnification 0.15 aperture, HC PL Fluotar and 40x magnification, 1.00 aperture, PL FLUOTAR Oil PH3 with NIS-Elements Br Microscope Imaging Software version 3.0 and images were captured using the NIKON Digital sight DS-UI camera (NIKON).
  3. The following image panel and legend has been reproduced from Mathan et al. (2016) (Figure 6).


    Figure 6. Peripheral corneal spheres implanted into corneoscleral tissue repopulate the ocular surface. LIVE/DEAD® staining (green) of implanted spheres, 5x magnification, at 0 h post implantation (A) and 4 days post implantation (B), show cell migration from the spheres appearing as green streaks out from the sphere. At 7 days post implantation (C), the entire corneal bed appears repopulated with live cells. Representative cells at the leading migratory edge of the corneal surface (D) and cells on the corneal surface taken from the region indicated by the single asterisk in panel C (E) show differing morphology. Representative cells over limbal region (F) and sclera (G) (taken from the region indicated by the double and triple asterisks respectively) show a different cell migration pattern and morphology to that observed in the corneal tissue. Scale bars = 1,000 µm (A, B, E). Scale bars = 100 µm (C, D, F, G). 

Recipes

  1. Supplemented Neurobasal-A medium
    Neurobasal-A medium supplemented with 2 ng/ml EGF, 1 ng/ml FGF, 1/50 B27, 1/100 N2, 2 µg/ml Heparin and 2 mM GlutaMAXTM
  2. Standard culture medium
    MEM (1x) GlutaMAX supplemented with 10% fetal calf serum and 1/100 Anti/Anti

Acknowledgments

The authors would like to thank the tissue donors and their families for their special gift to scientific research. They would also like to acknowledge Associate Professor Dipika Patel for assistance with the surgical method. The work presented in this study was funded by grants from the Auckland.
Medical Research Foundation [1111010], Save Sight Society [3622588], The University of Auckland School of Medicine PBRF grant as well as a Faculty of Medical and Health Sciences Summer Scholarship Award and John Hamel McGregor Award both to JM.

References

  1. Mathan, J. J., Ismail, S., McGhee, J. J., McGhee, C. N. and Sherwin, T. (2016). Sphere-forming cells from peripheral cornea demonstrate the ability to repopulate the ocular surface. Stem Cell Res Ther 7(1): 81.
  2. Yoon, J. J., Wang, E. F., Ismail, S., McGhee, J. J. and Sherwin, T. (2013). Sphere-forming cells from peripheral cornea demonstrate polarity and directed cell migration. Cell Biol Int 37(9), 949-960.

简介

可以将从人角膜缘组织分离的茎细胞和祖细胞作为球体体外培养。 这些球体有可能用作角膜组织再造的可移植元件(Mathan等人,2016)。 这里我们描述了将人角膜球植入尸体人角膜组织的详细方案。 该协议描述了球形成和培养的过程,球体植入组织的制备,角膜缘显微外科手术和球体植入。
【背景】以前的研究集中在隔离角膜缘细胞,这些细胞完全是上皮细胞(角膜缘干细胞)或基质(角质细胞祖细胞),以破译他们在角膜内稳态和伤口修复中的各自角色。 该方案旨在通过其在培养物中形成球体的功能能力来分离角膜缘细胞,并且其本质将包括上皮和间质的多种细胞,其有助于角膜缘的形成。 在分离这些球体之后,我们正在调查其在植入后角膜修复中的潜在用途。 在这里,我们描述了一种用于将这些球体植入人角膜组织和下游分析的体外手术方案。

关键字:细胞培养, 角膜, 球体形成细胞, 移植, 角膜缘

材料和试剂

  1. Scalpel刀片(ProSciTech,配置文件11)(Swann Morton,目录号:LSB11)
  2. 20×100mm细胞培养皿(Corning,Falcon ®,目录号:353003)
  3. 棉花芽(Cotton Tips double ends,Protec Solutions,目录号:941001690389)
  4. 转移移液管(3 ml)(Interlab,目录号:KJ622-1A)
  5. (Techno Plas,目录号:P5016UL)
  6. 40μm过滤器(Corning,Falcon ®,目录号:352340)
  7. 玻璃盖玻片(覆盖物#1 30毫米直径)(PST,目录号:G430)
  8. 6孔组织培养板(Thermo Fisher Scientific,Thermo Scientific TM,目录号:140675)
  9. 35×10毫米细胞培养皿(Corning,Falcon ®,目录号:353001)
  10. 从新西兰国家眼库获得手术后新鲜和冷冻的人类尸体供体角膜巩膜组织
  11. 70%乙醇(EMD Millipore,目录号:1009832500)
  12. 磷酸盐缓冲盐水(PBS)无菌(Sigma-Aldrich,目录号:P4417-100TAB)
  13. 磷酸盐缓冲盐水和10%甘油(AnalaR NORMAPUR)(VWR,目录号:24388.320)
  14. Dispase(Thermo Fisher Scientific,Gibco TM ,目录号:17105041)
  15. 胶原酶(Blend Type L)(Sigma-Aldrich,目录号:C8176)
  16. 透明质酸酶(Sigma-Aldrich,目录号:H3506)
  17. Neurobasal-A培养基(Thermo Fisher Scientific,Gibco TM,目录号:10888022)
  18. 人类表皮生长因子(EGF)(PeproTech,目录号:AF-100-15)
  19. 人成纤维细胞生长因子(FGF)基础(PeproTech,目录号:100-18B)
  20. B-27 Supplement,50x(Thermo Fisher Scientific,Gibco TM ,目录号:12587010)
  21. N-2 Supplement,100x(Thermo Fisher Scientific,Gibco TM ,目录号:17502048)
  22. GlutaMAX,100x(Thermo Fisher Scientific,Gibco TM,目录号:35050061)
  23. MEM与GlutaMAX(最低必需培养基)(Thermo Fisher Scientific,Gibco TM ,目录号:41090036)
  24. 胎牛血清(新西兰来源)(Thermo Fisher Scientific,Gibco TM,目录号:10091148)
  25. 抗生素/抗真菌剂100x(抗/抗)(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
  26. 补充的Neurobasal-A培养基(见食谱)
  27. 标准培养基(参见食谱)

设备

  1. 组织文化罩(Heal Force,型号:HFsafe 1800)
  2. 直剪刀(世界精密仪器,目录号:500216)
  3. 精镊子(世界精密仪器,目录号:14142)
  4. 轨道摇床(Ratek Instruments,型号:MM1)
  5. 离心机(Sigma Laborzentrifugen,型号:3-15)
  6. 组织培养培养箱(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Hereaus TM HeraCell 150)
  7. 层流柜(Gelman,型号:HLF 120)
  8. 双目立体显微镜(Carl Zeiss,目录号:474110-9904)
  9. 生物安全柜(Email Air Handling,目录号:1687-2340-618-3)
  10. 2-20μlEppendorf ®移液器(Eppendorf,目录号:3120000038)
  11. 羽毛MicroScalpel(pfm medical,目录号:200300715)
  12. 新月斜角眼镜2.3毫米(MANI,目录号:MCU26)
  13. NIKON数码摄像机DS-UI相机(尼康乐器,型号:DS-UI)
  14. 徕卡DMIL倒置对比显微镜(Leica Microsystems,型号:Leica DMIL)
  15. 徕卡DM-RA立式荧光显微镜(Leica Microsystems,型号:徕卡DM-RA)

软件

  1. NIS-Elements Br显微镜成像软件3.0版

程序

注意:此过程已在实验室(Yoon等人,2013)的以前工作中进行了描述,但以下部分提供了有关过程的其他信息,并以协议格式描述了该技术。

  1. 球体形成与文化(视频1)

    Video 1. Corneoscleral tissue processing for Sphere Forming Assay. Corneoscleral tissue is rubbed with a cotton bud on the posterior side to remove endothelial cells, scraped on the anterior side to remove epithelial cells and washed in PBS. The corneal/limbal region is excised and cut into small pieces prior to enzymatic treatment for limbal cell isolation.

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

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    1. 在无菌组织培养罩中使用无菌技术,制备1个容器的70%乙醇和1个容器的PBS用于工具和1个容器的PBS用于洗涤。将直剪刀,手术刀刀片和镊子放在70%乙醇中10分钟
    2. 从70%乙醇中取出上述设备,并在无菌PBS中短暂冲洗以除去残留的乙醇
    3. 使用镊子将角膜巩膜组织放入100毫米无菌培养皿(角膜巩膜边缘[图1A])具有大约2-2.5毫米宽的角膜/角膜缘组织环,当切除时具有55-57毫克的近似湿重) 。
    4. 使用无菌棉花芽,擦拭角膜组织的后表面以去除内皮细胞(图1B)
    5. 使用新月形斜角眼刀,轻轻擦拭组织的前表面以去除上皮细胞(图1C)。
    6. 使用无菌转移移液管,用PBS清洗组织。重复上皮刮擦并洗涤直到PBS洗涤清除细胞碎片(图1D)。
    7. 消除角膜缘区域并排除尽可能多的巩膜(图1E)。冲洗切除的角膜缘,用剪刀切碎组织(图1F),放入无菌的5ml管中。


      图1.球形成测定的角膜巩膜组织处理角膜巩膜组织(A)在后侧用棉花揉搓以去除内皮细胞(B),在前侧刮去以去除上皮细胞(C)并在PBS(D)中洗涤。切割角膜缘(E)并切割成小片(F),然后进行角膜缘细胞分离的酶处理。

    8. 在1.2U / ml分散液中于37℃孵育40分钟。
    9. 在37℃,在轨道振荡器上,用抗/抗体在MEM中以2mg / ml胶原酶和0.5mg / ml透明质酸酶孵育过夜。
    10. 使用40μm过滤器将溶液和细胞溶解,以除去未消化的物质
    11. 将滤液在405g离心滤液7分钟,并用PBS洗涤细胞沉淀
    12. 将细胞悬浮于2ml补充的Neurobasal-A培养基中
    13. 在6孔组织培养板中制备无菌玻璃盖玻片。
    14. 将2ml悬浮液均匀地种植到孔中
    15. 在37℃,5%CO 2的潮湿培养箱中培养细胞。
    16. 从表面小心地更换50%的培养基每周两次移液,以避免移除细胞或新形成的球体
    17. 细胞常常粘附在玻璃盖玻片上并聚集成球形结构(图2)

      图2.球形形成。相位显微镜显示在接种后最初粘附到玻璃盖玻片上的分离的细胞(A),其在几天至几周的过程中聚集并形成球状结构(B)。比例尺= 100μm。

  2. 用于球体植入的组织的制备
    1. 在生物安全柜(Heal Force Model HF-safe 1800)的无菌条件下,供体角膜巩膜边缘在-80°C储存3个月以上,分为1/8 储存在单独的5ml容器中并在-80℃下再冷冻。
    2. 每个角膜巩膜边缘经受总共3次冻融循环,以便在用于植入之前使组织脱细胞。
      注意:如果需要完全去细胞化,可以根据制造商的指示,使用标准培养基中的LIVE / DEAD 2 /μ2钙钙荧光素AM和4μM乙二胺二聚体-1(Life Technologies)确认完全脱细胞化或确定需要额外的冻融循环。

  3. 角膜缘显微外科(视频2)

    Video 2. Surgical excision of a wedge of limbal tissue. Fine forceps were used with the non-dominant hand to anchor the sclera and provide counter-traction as two diagonal ~3 mm incisions were made prior to gripping the apex of the incised tissue to cut away and remove the anterior tissue segment. Cornea-limbus-sclera demarcations have to be visually approximated.

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    1. 在层流柜(Gelman model HLF 120)中使用无菌技术,制备1个容器的70%乙醇和1个容器的无菌PBS用于工具。
    2. 将精镊子和MicroScalpel刀片放入乙醇中放置10分钟
    3. 取出解冻的角膜缘缘并放入培养皿中
    4. 在PBS中洗涤工具后,使用双目立体显微镜观察角膜缘。
    5. 使用MicroScalpel,使两个对角线〜3mm的切口(图3A)进入角膜缘,顶点相交,从而形成三角形楔形物(图3B和3C)。使用细镊子抓住顶点的组织并提起楔子(图3D)。


      图3.角膜缘组织的手术切除使用非优势手使用精细镊子锚固巩膜,并提供反向牵引,因为两个对角线〜3mm的切口(A)正在(B,C),然后握住顶点以切除切开的组织(D)。角膜缘 - 巩膜分界必须视觉近似。

    6. 使用羽毛MicroScalpel切割锚定组织,同时向前倾斜刀片,使得创建的凹陷在角膜方向较浅(图4)。


      图4.楔形组织的移除从角膜缘区域切除(B)后产生的楔形物(A)的向上倾斜的地板的3-D表示。

    7. 重复以在组织中创建额外的切口。
      注意:切割的可视化最初对未经训练的眼睛具有挑战性,但会随着实践而发展。良好的照明将会有所帮助。
    8. 培养皿关闭,将组织转移到II类生物安全柜。

  4. 球体植入
    1. 使用无菌棉花芽,首先将过量的液体从已经形成角膜缘楔形切口的角膜巩膜组织中吸出。
    2. 球体可以在II类生物安全柜内的双目立体显微镜下观察。
    3. 使用无菌2-20μlEppendorf ®移液器设置为5μl的体积,轻轻吸取培养物中的球体。
    4. 可视化角膜巩膜组织中的切口,将球体和介质推入楔形切口。
      注意:尽可能地尽可能地排出培养基体积,同时确保球体被排出。偶尔会出现球体浮出组织的困难,如果球体植入,特别是球形从微量移液器喷出,则不会轻轻地进行。
    5. 将角膜巩膜边缘用植入的球体转移到35×10 5细胞培养皿中,并在室温下孵育15分钟上皮侧。
    6. 反转组织使上皮面朝下。
      注意:通过个人经验,翻转组织已被证明比右侧培养组织更好,因为角膜通常漂浮,球体不完全浸没在培养基中,这似乎不利于细胞迁移和存活。然而,使用倒置培养物,球体有时不会留在原位,并从组织中脱离出来。
    7. 轻轻地用2ml标准培养基覆盖组织。
    8. 每隔一天进行一次中等的变化。
      以下图像面板和图例已从Mathan等人复制。 (2016)(图5)

      图5.将外周角膜球植入供体角膜巩膜边缘将球体(箭头)植入在角膜缘区域形成的楔形切口中。在立体显微镜下,可以清楚地看到具有切口(箭头)和植入的球体的角膜巩膜边缘(A)。组合相差和荧光显微镜显示用LIVE / DEAD染色(B)对活细胞染色的植入球体。该信号被限制在球体上,在周围组织中不被检测。冷冻储存的角膜巩膜边缘的40倍DAPI染色的10μm厚的横截面证实在植入之前不存在DAPI阳性细胞核(C)。通过相差显微镜,显示球体在组织半透明区域的位置(D)。刻度棒=500μm(A),100μm(B和D),50μm(C)。

数据分析

  1. 为了评估球体和植入的细胞在组织中的活力,使用标准培养基中的LIVE / DEAD 2μM钙黄绿素AM和4μM乙锭同二聚体-1(Life Technologies)。
  2. 使用NV6双目立体显微镜(Carl Zeiss)评估的亮场图像使用NIKON Digital sight DS-UI相机(NIKON CORPORATION,Tokyo,Japan)捕获。使用以下显微镜进行相差和荧光显微镜:Leica DMIL倒置对比显微镜(Leica Microsystems,Wetzlar,Germany),4倍放大0.1孔,C PLAN与Leica Application Suite V4.0.0 Build 454;和Leica DM-RA立式荧光显微镜(Leica Microsystems),5倍放大率0.15孔径,HC PL Fluotar和40倍放大倍数,1.00孔径,PL FLUOTAR油PH3与NIS元件Br显微镜成像软件3.0版和图像捕获使用NIKON数字DS-UI摄像机(NIKON)。
  3. 以下图像面板和图例已从Mathan等人复制。 (2016)(图6)

    图6.植入角膜巩膜组织的外周角膜球重新植入眼表。植入球后5x放大,植入后0小时的LIVE / DEAD ®染色(绿色) A)和植入后4天(B),显示从球体出现的绿色条纹的细胞迁移。在植入后7天(C),整个角膜床似乎与活细胞重新填充。在角膜表面(D)的前导迁移边缘处的代表性细胞和从图C(E)中由单个星号表示的区域取得的角膜表面上的细胞显示不同的形态。代表性细胞超过角膜缘区域(F)和巩膜(G)(分别由双星和三星号表示的区域)显示与角膜组织中观察到的不同的细胞迁移模式和形态。刻度棒=1,000μm(A,B,E)。刻度棒=100μm(C,D,F,G)。

食谱

  1. 补充的Neurobasal-A培养基 补充有2ng / ml EGF,1ng / ml FGF,1/50B27,1 / 100N2,2μg/ ml肝素和2mM GlutaMAX 的神经巴巴-A培养基
  2. 标准培养基
    MEM(1x)补充有10%胎牛血清和1/100抗/抗
    的GlutaMAX

致谢

作者要感谢组织捐赠者及其家属对科学研究的特别礼物。他们还要感谢Dipika Patel副教授对外科手术的帮助。本研究中提供的工作由奥克兰的赠款资助。
医学研究基金会[1111010],拯救视力社会[3622588],奥克兰医学院PBRF补助金以及医学和健康科学学院夏季奖学金奖和约翰·哈默尔麦克格雷戈奖。

参考

  1. Mathan,JJ,Ismail,S.,McGhee,JJ,McGhee,CN和Sherwin,T。(2016)。
  2. Yoon,JJ,Wang,EF,Ismail,S.,McGhee,JJ和Sherwin,T。(2013)。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mathan, J., Ismail, S., McGhee, J. and Sherwin, T. (2017). Implantation of Human Peripheral Corneal Spheres into Cadaveric Human Corneal Tissue. Bio-protocol 7(14): e2412. DOI: 10.21769/BioProtoc.2412.
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