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Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier
使用纤维蛋白载体将小鼠毛囊源性干细胞移植到角膜   

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参见作者原研究论文

本实验方案简略版
Stem Cells
Jan 2011

Abstract

The goal of this protocol is to establish a procedure for cultivating stem cells on a fibrin carrier to allow for eventual transplantation to the eye. The ability to transfer stem cells to a patient is critical for treatment for a variety of disorders and wound repair. We took hair follicle stem cells from the vibrissae of transgenic mice expressing a dual reporter gene under the control of the Tet-on system and the keratin 12 promoter (Meyer-Blazejewska et al., 2011). A clonal growth assay was performed to enrich for stem cells. Once holoclones formed they were transferred onto a fibrin carrier and cultivated to obtain a confluent epithelial cell layer. Limbal stem cell deficient (LSCD) mice were used as the transplant recipient in order to test for successful grafting and eventual differentiation into a corneal epithelial phenotype.

Keywords: Holoclones (全克隆), Clonal growth (克隆生长), Hair follicle stem cells (毛囊干细胞), Transgenic mice (转基因小鼠), Fibrin carrier (纤维蛋白载体)

Background

Stem cells are widely used as a therapeutic tool, thus a means for delivery is essential. In fact, many researchers and companies are searching for the best way to deliver cells into the human body to optimize cell survival as well as integration into the host tissue. Injection methods have been widely used in animal models but often result in poor survival and integration. Techniques utilizing biomaterials and surgical devices are currently being employed. One technique that has been utilized to deliver stem cells is fibrin carriers. Fibrin gel is a degradable biopolymer that can adhere to native tissue allowing for cell attachment, migration and proliferation (Ehrbar et al., 2005). Fibrin gels have many advantages including biocompatibility, controlled degradation (Kjaergard et al., 1994; Sidelmann et al., 2000), uniform cell distribution and high cell seeding efficiency (Swartz et al., 2005). Fibrin gels have been utilized for treating skin burns (Pellegrini et al., 1999; Ronfard et al., 2000), junctional epidermolysis bullosa (Hirsch et al., 2017) and corneal damage (Pellegrini et al., 1997; Rama et al., 2010). The method described here uses a fibrin carrier to transplant hair follicle derived stem cells onto the ocular surface of a limbal stem cell-deficient mouse. Cell engraftment and differentiation was assessed for a 5-week period via fluorescent microscopy.

Materials and Reagents

  1. TISSEEL [Fibrin Sealant] (Baxter, catalog number: 1501261 )
  2. Insulin syringe (Fisher Scientific, catalog number: 14-829-1A)
    Manufacturer: BD, catalog number: 329420 .
  3. 6-well plates (Corning, Falcon®, catalog number: 353934 )
  4. Pipette tips (MidSci, Avant low binding tips)
  5. Transfer pipet
  6. HFSC discs
  7. Ethilon 10-0 nylon sutures (Ethicon, catalog number: 9006G )
  8. Microscope slides (Fisher Scientific, catalog number: 12-552-3 )
  9. Coverglass 22 x 50 (Fisher Scientific, catalog number: 12-548-5E )
  10. Thrombin
  11. C57BL/6 mice (THE JACKSON LABORATORY, catalog number: 000664 )
  12. K12rtTA/rtTA/tetO-cre/ROSAmTmG transgenic mice (see Notes)
  13. 0.9% saline (Fisher Scientific, catalog number: 23-535435 )
  14. BioGlo fluorescein sodium ophthalmic strips (Hub Pharmaceuticals, NDC 17238-900-11)
  15. Doxycycline chow (Custom Animal Diets, catalog number: AD3008 )
  16. Avastin (bevacizumab, Genentech, Inc.)
  17. Anti-inflammatory drops–Inflanefran forte (Allergan, NDC 11980-180)
  18. 16% paraformaldehyde (Electron Microscopy Sciences, catalog number: 15710 )
  19. Sodium borohydride (Sigma-Aldrich, catalog number: 71320 )
  20. DAPI (Thermo Fisher Scientific, InvitrogenTM, catalog number: D3571 )
  21. Sodium chloride (NaCl) (Fisher Scientific, catalog number: BP358-1 )
  22. Calcium chloride (CaCl2) (Acros Organics, catalog number: 349610025 )
  23. Dulbecco’s modified Eagle medium (DMEM) without calcium and magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 21068028 )
  24. Ham’s F12 Nutrient Mix (Thermo Fisher Scientific, GibcoTM, catalog number: 11765047 )
  25. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10082147 )
  26. Human recombinant epidermal growth factor (Merck, catalog number: GF144 )
  27. L-Glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030081 )
  28. Human corneal growth supplement (Thermo Fisher Scientific, GibcoTM, catalog number: S0095 )
  29. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  30. GibcoTM Amphotericin B (Thermo Fisher Scientific, catalog number: 15290026 )
  31. Sodium phosphate dibasic, anhydrous (Na2HPO4) (Sigma-Aldrich, catalog number: S7907 )
  32. Sodium phosphate monobasic, anhydrous (NaH2PO4) (Sigma-Aldrich, catalog number: S8282 )
  33. Analytical grade glycerol (Sigma-Aldrich, catalog number: G6279 )
  34. Mowiol 4-88 (Merck, catalog number: 475904 )
  35. Tris (Fisher Scientific, catalog number: BP152-1 )
  36. Ketamine/HCl 100 mg/ml (KetaJect; Henry Schein Animal Health, catalog number: 010177 )
  37. Xylazine AnaSed® 100 mg/ml (Santa Cruz Biotechnology, catalog number: sc-362949Rx )
  38. Fibrinogen solution (see Recipes)
  39. Thrombin solution (see Recipes)
  40. Stem cell media (see Recipes)
  41. 0.1 M Phosphate Buffer, pH 7.4 (see Recipes)
  42. Mowiol mounting medium (see Recipes)
  43. Ketamine/xylazine solution (see Recipes)

Equipment

  1. Algerbrush II Corneal rust ring remover (MicroSurgical Technology, catalog number: AM0100 )
  2. Inverted Fluorescence Microscope (Zeiss Observer Z1 with an apotome attachment) (ZEISS, model: AxioObserver Z1 )
  3. Suture Tying Forceps (Fine Science Tools, catalog number: 18025-10 )
  4. Microdissection scissors (Fine Science Tools, catalog number: 15000-00 )
  5. Epi-fluorescent stereomicroscope (ZEISS, model: Stemi SVII )
  6. BSL2 Laminar Flow Hood (Thermo Fisher Scientific, Thermo ScientificTM, model: 1300 Series A2 , catalog number: 1387)
  7. Hemocytometer (Hausser Scientific, catalog number: 3200 )
  8. CO2 Incubator (Thermo Fisher Scientific, Thermo ScienticTM, model: NAPCO Series 8000 WJ )
  9. Dissecting Scope (ZEISS, model: Stemi DV4 )
  10. Centrifuge (Hettich, model: Rotina 35 )

Software

  1. AxioVison 4.7

Procedure

  1. Preparation of fibrin carrier
    1. Thaw the TISSEEL Fibrin Sealant (2-ml pre-filled syringe) at 37 °C.
    2. Prepare diluted fibrin by adding 1 ml of Sealer Protein (fibrinogen) entire contents of the syringe to 1.16 ml of fibrinogen solution (Recipe 1).
    3. Prepare thrombin mix by adding 1 ml of thrombin (entire contents of the syringe) to 9 ml of thrombin solution (Recipe 2).
    4. Prepare diluted thrombin by adding 1.5 ml of thrombin mix (from Step A3) to 25 ml thrombin solution.
    5. To one well of a 6-well plate add 0.75 ml of diluted thrombin (from Step A3). Ensure that the diluted thrombin solution covers the entire well evenly.
    6. To the same well add 0.75 ml diluted fibrin. Add dropwise to the well quickly mixing with a pipette tip.
    7. Allow gel to set at room temperature. The final concentration of fibrinogen and thrombin is ~10 mg/ml and 3 IU/ml, respectively. The gel will set in approximately 5 min. Once the gel has set proceed to the cultivation of the cells.

  2. Cultivation on a fibrin carrier
    1. Seed hair follicle stem cells derived from a dual reporter mouse at 1 x 105 cells/ml onto a fibrin carrier and cultivate for 3 days in stem cell media (Recipe 3) at 37 °C, 5% CO2. A total volume of 2 ml is used for each well. Cells can be visualized using phase contrast microscopy (Figure 1)


      Figure 1. Scheme showing transplant of a fibrin carrier containing bulge derived hair follicle stem cells. Discs are prepared from hair follicle stem cells (HFSCs) from a dual reporter mouse cultivated for 3 days on a fibrin sheet by using the back of a pipet tip as a “cookie cutter”. Discs are then sutured to the eye of a limbal stem cell deficient mouse. Four sutures (red asterisks) are placed. Cells fluoresce red but not green as they do not yet express the corneal epithelial differentiation marker, Krt12.

  3. Total limbal stem cell deficiency (Figure 2)
    1. Anesthetize 6- to 8-week old C57BL/6 mice with ketamine (90 mg/kg) and xylazine (13 mg/kg) (Recipe 6) via intraperitoneal injection using a 28-G insulin syringe.
    2. Generate total limbal stem cell deficiency by removing the entire corneal and limbal epithelium using an Algerbrush II corneal rust ring remover with a 0.5 mm burr (Note 6). Hair follicle stem cell transplantation (see below) should immediately follow the removal of epithelial cells.
    3. Verify complete removal of corneal and limbal epithelium using fluorescein stain.
      1. Dip the tip of a fluorescein sodium ophthalmic strip in 0.9% saline for 20 times.
      2. Using a transfer pipet, add a drop of the fluorescein stain onto the eye. Let sit 30 sec.
      3. Extensively wash in 0.9% saline.
      4. Image using a fluorescent stereomicroscope.


      Figure 2. Scheme of limbal stem cell deficiency. An algerbrush is used to remove the entire corneal and limbal epithelium. The red circle depicts partial debridement. Fluorescein stain can be used to verify the complete removal of the epithelium. Note the absence of fluorescein stain in the partial removal (asterisk).

  4. Hair follicle stem cell transplantation
    1. Prepare ~7 mm fibrin gel containing HFSC discs by using the back of a P1000 pipet tip (see Figure 1). Place the discs into PBS (Note 5) using forceps (Figure 1).
    2. Suture an approximately 7 mm fibrin carrier containing HFSC onto the surface of a limbal stem cell-deficient mouse eye with the stem cells facing the recipient’s basement membrane. A suture was placed superiorly, inferiorly, nasally and temporally (Figure 1).
    3. Following surgical recovery, administer anti-inflammatory eye drops for the first five days followed by Avastin eye drops for the next 7 days. Eye drops should be administered once a day.

  5. Conversion to corneal epithelial cells
    1. Mice are fed doxycycline chow (1 g/kg) ad libitum until sample collection at 3 days, 1 week, 2 weeks, 3 weeks and 5 weeks.
    2. Euthanize mice and enucleate the eyes at the sample collection time points and fix in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (Recipe 4) at 4 °C overnight followed by PBS washes.
    3. Dissect the cornea using microdissection scissors.
    4. Treat the corneas with 0.2% sodium borohydride (Note 7) for 30 min at room temperature followed by washing in 0.1 M phosphate buffer, pH 7.4.
    5. Stain corneas with 4’6-diamidino-2-phenylindole (DAPI, 1 μg/ml), mount onto a microscope slide using cover glass and Mowiol medium (Recipe 5) and visualize using a Zeiss Observer Z1 microscope with an apotome attachment.

Data analysis

AxioVison 4.7. Z-stack images in the red (membrane tomato red), green (membrane EGFP) and blue (DAPI) channels were taken for each collection time point. Conversion of red to green was noted at each of the time points (Figure 3).


Figure 3. Engraftment and differentiation of the hair follicle bulge-derived stem cells into corneal epithelial cells. Single color images taken from a Z-stack depicting the cellular localization of membrane tomato red and membrane EGFP at 3 days and 5 weeks post-transplantation. Magnification x200. Reprinted with permission from Meyer-Blazejewska et al., 2011.

Notes

  1. Preparation of the fibrin gel and cultivation of the HFSC should be performed in a class II biological safety cabinet.
  2. Various dimensions of the fibrin carrier can be made as long as the ratio of diluted thrombin and diluted fibrin remain 1:1.
  3. All cell counts were performed using a hemocytometer.
  4. All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Cincinnati. Genetically modified mouse lines Krt12rtTA (Chikama et al., 2005), TetO-cre (Perl et al., 2002) and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J(ROSAmTmG) (Muzumdar et al., 2007) have been previously described. Compound transgenic mice were generated by breeding individual mouse lines to create K12rtTA/rtTA/tetO-cre/ROSAmTmG. This dual reporter mouse model uses the keratin 12 promoter (corneal epithelium-specific) to drive the expression of the Tet-on system. In conjunction with doxycycline and cre, the membrane tomato red hair follicle stem cells will turn green if they have differentiated into corneal epithelial cells.
  5. When creating and transferring the fibrin gel discs be sure to handle them with care as to avoid damaging the cells. Additionally, be sure the discs are placed in PBS with the HFSC facing upwards.
  6. When removing the epithelium with the Algerbrush care should be taken to avoid damaging the basement membrane. This can be done by holding the Algerbrush at a 45° angle and moving it in a circular, sweeping motion. Use only gentle pressure as the epithelium is relatively easy to remove.
  7. Care should be taken when handling sodium borohydride as it poses physical and health hazards.

Recipes

  1. Fibrinogen solution
    10% NaCl
    100 mM CaCl2
    0.9% NaCl
    ddH2O
  2. Thrombin solution
    10% NaCl
    100 mM CaCl2
    ddH2O
  3. Stem cell media
    3 parts DMEM/High glucose without Ca2+ or Mg2+
    1 part Ham’s F12
    10% FBS
    10 ng/ml EGF
    500 mg/L L-glutamine
    0.4 mM calcium chloride
    1x human corneal growth supplement
    10,000 U/ml penicillin
    10,000 μg/ml streptomycin
    25 μg/ml amphotericin B
  4. 0.1 M Phosphate Buffer, pH 7.4
    76 mM Na2HPO4 (anhydrous)
    27 mM NaH2PO4 (anhydrous)
  5. Mowiol mounting medium
    24 g analytical grade glycerol
    9.6 g Mowiol 4-88
    24 ml ddH2O
    48 ml 0.2 M Tris-HCl buffer, pH 8.5
  6. Ketamine/xylazine solution
    2 ml of ketamine (100 mg/ml)
    0.25 ml xylazine (100 mg/ml)
    7.75 ml PBS

Acknowledgments

This study was supported in part by grants from the NIH/NEI EY011845, Ohio Lions Eye Research Foundation to W.W.K. Authors do not have any conflicts of interest.

References

  1. Chikama, T., Hayashi, Y., Liu, C. Y., Terai, N., Terai, K., Kao, C. W., Wang, L., Hayashi, M., Nishida, T., Sanford, P., Doestchman, T. and Kao, W. W. (2005). Characterization of tetracycline-inducible bitransgenic Krt12rtTA/+/tet-O-LacZ mice. Invest Ophthalmol Vis Sci 46(6): 1966-1972.
  2. Ehrbar, M., Metters, A., Zammaretti, P., Hubbell, J. A. and Zisch, A. H. (2005). Endothelial cell proliferation and progenitor maturation by fibrin-bound VEGF variants with differential susceptibilities to local cellular activity. J Control Release 101(1-3): 93-109.
  3. Hirsch, T., Rothoeft, T., Teig, N., Bauer, J. W., Pellegrini, G., De Rosa, L., Scaglione, D., Reichelt, J., Klausegger, A., Kneisz, D., Romano, O., Secone Seconetti, A., Contin, R., Enzo, E., Jurman, I., Carulli, S., Jacobsen, F., Luecke, T., Lehnhardt, M., Fischer, M., Kueckelhaus, M., Quaglino, D., Morgante, M., Bicciato, S., Bondanza, S. and De Luca, M. (2017). Regeneration of the entire human epidermis using transgenic stem cells. Nature 551(7680): 327-332.
  4. Kjaergard, H. K. and Weis-Fogh, U. S. (1994). Important factors influencing the strength of autologous fibrin glue; the fibrin concentration and reaction time--comparison of strength with commercial fibrin glue. Eur Surg Res 26(5): 273-276.
  5. Meyer-Blazejewska, E. A., Call, M. K., Yamanaka, O., Liu, H., Schlotzer-Schrehardt, U., Kruse, F. E. and Kao, W. W. (2011). From hair to cornea: toward the therapeutic use of hair follicle-derived stem cells in the treatment of limbal stem cell deficiency. Stem Cells 29(1): 57-66.
  6. Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. and Luo, L. (2007). A global double-fluorescent Cre reporter mouse. Genesis 45(9): 593-605.
  7. Pellegrini, G., Ranno, R., Stracuzzi, G., Bondanza, S., Guerra, L., Zambruno, G., Micali, G. and De Luca, M. (1999). The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68(6): 868-879.
  8. Pellegrini, G., Traverso, C. E., Franzi, A. T., Zingirian, M., Cancedda, R. and De Luca, M. (1997). Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349(9057): 990-993.
  9. Perl, A. K., Wert, S. E., Nagy, A., Lobe, C. G. and Whitsett, J. A. (2002). Early restriction of peripheral and proximal cell lineages during formation of the lung. Proc Natl Acad Sci U S A 99(16): 10482-10487.
  10. Rama, P., Matuska, S., Paganoni, G., Spinelli, A., De Luca, M. and Pellegrini, G. (2010). Limbal stem-cell therapy and long-term corneal regeneration. N Engl J Med 363(2): 147-155.
  11. Ronfard, V., Rives, J. M., Neveux, Y., Carsin, H. and Barrandon, Y. (2000). Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix. Transplantation 70(11): 1588-1598.
  12. Sidelmann, J. J., Gram, J., Jespersen, J. and Kluft, C. (2000). Fibrin clot formation and lysis: basic mechanisms. Semin Thromb Hemost 26(6): 605-618.
  13. Swartz, D. D., Russell, J. A. and Andreadis, S. T. (2005). Engineering of fibrin-based functional and implantable small-diameter blood vessels. Am J Physiol Heart Circ Physiol 288(3): H1451-1460.

简介

该方案的目标是建立一种在纤维蛋白载体上培养干细胞以允许最终移植到眼睛的程序。 将干细胞转移给患者的能力对于治疗各种疾病和伤口修复至关重要。 我们在Tet-on系统和角蛋白12启动子(Meyer-Blazejewska等人,2011)的控制下从表达双报告基因的转基因小鼠的触须中取出毛囊干细胞。 进行克隆生长测定以富集干细胞。 一旦形成holoclones,将它们转移到纤维蛋白载体上并培养以获得融合上皮细胞层。 将角膜缘干细胞缺陷(LSCD)小鼠用作移植受体以测试移植成功和最终分化为角膜上皮表型。

【背景】干细胞被广泛用作治疗工具,因此递送手段是必不可少的。实际上,许多研究人员和公司正在寻找将细胞输送到人体内以优化细胞存活以及整合到宿主组织中的最佳方式。注射方法已广泛用于动物模型,但往往导致生存和整合差。目前正在使用利用生物材料和手术装置的技术。一种用于输送干细胞的技术是纤维蛋白载体。纤维蛋白凝胶是可降解的生物聚合物,其可粘附于允许细胞附着,迁移和增殖的天然组织(Ehrbar et al。,2005)。纤维蛋白凝胶具有许多优点,包括生物相容性,受控降解(Kjaergard等人,1994; Sidelmann等人,2000),均匀细胞分布和高细胞接种效率(Swartz ,2005)。已经将纤维蛋白凝胶用于治疗皮肤烧伤(Pellegrini等人,1999; Ronfard等人,2000),交界性大疱性表皮松解症(Hirsch等人, ,2017)和角膜损伤(Pellegrini等人,1997; Rama等人,2010)。这里描述的方法使用纤维蛋白载体将毛囊衍生干细胞移植到角膜缘干细胞缺陷小鼠的眼表面上。通过荧光显微镜评估细胞植入和分化5周的时间。

关键字:全克隆, 克隆生长, 毛囊干细胞, 转基因小鼠, 纤维蛋白载体

材料和试剂

  1. TISSEEL [Fibrin Sealant](Baxter,目录号:1501261)
  2. 胰岛素注射器(Fisher Scientific,目录号:14-829-1A)
    制造商:BD,目录号:329420。
  3. 6孔板(Corning,Falcon ,目录号:353934)
  4. 移液器吸头(MidSci,Avant低结合吸头)
  5. 移液管
  6. HFSC光盘
  7. Ethilon 10-0尼龙缝线(Ethicon,目录号:9006G)
  8. 显微镜幻灯片(Fisher Scientific,目录号:12-552-3)
  9. Coverglass 22 x 50(Fisher Scientific,目录号:12-548-5E)
  10. 凝血酶
  11. C57BL / 6小鼠(THE JACKSON LABORATORY,目录号:000664)
  12. K12 rtTA / rtTA / tetO-cre / ROSA mTmG转基因小鼠(见注)
  13. 0.9%盐水(Fisher Scientific,目录号:23-535435)
  14. BioGlo荧光素钠眼科条(Hub Pharmaceuticals,NDC 17238-900-11)
  15. 强力霉素食物(定制动物饮食,目录号:AD3008)
  16. 阿瓦斯丁(贝伐单抗,Genentech,Inc.)
  17. 抗炎滴剂 - Inflanefran forte(Allergan,NDC 11980-180)
  18. 16%多聚甲醛(Electron Microscopy Sciences,目录号:15710)
  19. 硼氢化钠(Sigma-Aldrich,目录号:71320)
  20. DAPI(Thermo Fisher Scientific,Invitrogen TM,目录号:D3571)
  21. 氯化钠(NaCl)(Fisher Scientific,目录号:BP358-1)
  22. 氯化钙(CaCl 2 2)(Acros Organics,目录号:349610025)
  23. Dulbecco改良Eagle培养基(DMEM)不含钙和镁(Thermo Fisher Scientific,Gibco TM,产品目录号:21068028)
  24. Ham's F12营养物混合物(Thermo Fisher Scientific,Gibco TM,目录号:11765047)
  25. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10082147)
  26. 人重组表皮生长因子(Merck,目录号:GF144)
  27. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM,目录号:25030081)
  28. 人角膜生长补充剂(Thermo Fisher Scientific,Gibco TM,目录号:S0095)
  29. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
  30. Gibco TM两性霉素B(Thermo Fisher Scientific,目录号:15290026)
  31. 磷酸二氢钠,无水(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907)
  32. 磷酸二氢钠,无水(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282)
  33. 分析级甘油(Sigma-Aldrich,目录号:G6279)
  34. Mowiol 4-88(Merck,目录号:475904)
  35. Tris(Fisher Scientific,目录号:BP152-1)
  36. 氯胺酮/盐酸100毫克/毫升(KetaJect;亨利沙因动物保健,目录号:010177)
  37. Xylazine AnaSed 100mg / ml(Santa Cruz Biotechnology,目录号:sc-362949Rx)
  38. 纤维蛋白原溶液(见食谱)
  39. 凝血酶溶液(见食谱)
  40. 干细胞培养基(见食谱)
  41. 0.1 M磷酸盐缓冲液,pH 7.4(见食谱)
  42. Mowiol安装介质(见食谱)
  43. 氯胺酮/赛拉嗪溶液(见食谱)

设备

  1. Algerbrush II角膜除锈环(MicroSurgical Technology,目录号:AM0100)
  2. 倒置荧光显微镜(蔡司观察员Z1与apotome附件)(蔡司,型号:AxioObserver Z1)
  3. 缝合绑镊子(精细科学工具,目录号:18025-10)
  4. 显微切割剪刀(Fine Science Tools,目录编号:15000-00)
  5. Epi-fluorescent立体显微镜(ZEISS,型号:Stemi SVII)
  6. BSL2层流罩(Thermo Fisher Scientific,Thermo Scientific TM,型号:1300系列A2,目录号:1387)
  7. 血细胞计数器(Hausser Scientific,目录号:3200)
  8. CO 2培养箱(Thermo Fisher Scientific,Thermo Scientic TM,型号:NAPCO Series 8000 WJ)。
  9. 解剖镜(蔡司,型号:Stemi DV4)
  10. 离心机(Hettich,型号:Rotina 35)

软件

  1. AxioVison 4.7

程序

  1. 纤维蛋白载体的制备

    1. 在37°C解冻TISSEEL纤维蛋白密封胶(2毫升预装注射器)。
    2. 通过将1ml Sealer蛋白(纤维蛋白原)全部内容物注射到1.16ml纤维蛋白原溶液(配方1)中来制备稀释的纤维蛋白。

    3. 加入1毫升凝血酶(注射器的全部内容物)至9毫升凝血酶溶液(方案2),制备凝血酶混合物。
    4. 通过将1.5ml凝血酶混合物(来自步骤A3)加入到25ml凝血酶溶液中来制备稀释的凝血酶。
    5. 向6孔板的一个孔中加入0.75ml稀释的凝血酶(来自步骤A3)。确保稀释的凝血酶溶液均匀覆盖整个孔。
    6. 在相同的孔中加入0.75ml稀释的纤维蛋白。逐滴添加到井中,快速与移液器吸头混合。
    7. 让凝胶在室温下凝固。纤维蛋白原和凝血酶的最终浓度分别为〜10mg / ml和3IU / ml。凝胶将在约5分钟内凝固。一旦凝胶已经开始进行细胞的培养。

  2. 在纤维蛋白载体上培养
    1. 将来自双报告小鼠的1×10 5细胞/ ml的毛囊干细胞种到纤维蛋白载体上并在干细胞培养基(方案3)中在37℃,5% CO <子> 2 。每个孔使用2ml的总体积。使用相差显微镜可以显现细胞(图1)


      图1.移植含有来自毛囊的毛囊干细胞的纤维蛋白载体的方案从纤毛蛋白片上培养3天的双报告小鼠的毛囊干细胞(HFSC)制备圆盘使用移液器尖端的背面作为“切割器”。然后将圆盘缝合到角膜缘干细胞缺陷小鼠的眼睛上。放置四根缝合线(红色星号)。细胞荧光红色但不是绿色,因为它们尚未表达角膜上皮分化标志物Krt12。

  3. 总的角膜缘干细胞缺乏症(图2)
    1. 使用28-G胰岛素注射器通过腹膜内注射麻醉具有氯胺酮(90mg / kg)和甲苯噻嗪(13mg / kg)的6-8周龄C57BL / 6小鼠(配方6)。
    2. 使用Alderbrush II角膜除锈器(0.5 mm毛刺)去除角膜和角膜缘上皮,产生全角膜缘干细胞缺陷(注6)。毛囊干细胞移植(见下文)应该立即去除上皮细胞。

    3. 使用荧光素染色验证角膜和角膜缘上皮的完全去除

      1. 将0.9%盐水中的荧光素钠眼科胶条尖端浸泡20次。
      2. 使用移液管,将一滴荧光素染剂滴在眼睛上。 让我们坐30秒。

      3. 在0.9%盐水中广泛清洗
      4. 使用荧光立体显微镜的图像。


    图2.角膜缘干细胞缺乏的方案 algerbrush用于去除角膜和角膜缘上皮。红色圆圈表示部分清创。荧光素染色可用于验证上皮完全去除。注意部分去除(星号)中没有荧光素染色。

  4. 毛囊干细胞移植
    1. 使用P1000移液枪头的背部准备含有HFSC圆盘的〜7 mm纤维蛋白凝胶(见图1)。
      使用镊子(图1)将光盘放入PBS(注5)。
    2. 将含有HFSC的约7mm纤维蛋白载体缝合到角膜缘干细胞缺陷小鼠眼睛的表面上,其中干细胞面对受体的基膜。
      缝线优越,下方,鼻腔和暂时放置(图1)。
    3. 手术恢复后,前5天给予抗炎眼药水,接下来7天使用阿瓦斯丁滴眼液。眼药水应该每天给药一次。

  5. 转化为角膜上皮细胞

    1. 小鼠在3天,1周,2周,3周和5周后进行多西环素饲料(1 g / kg)自由采食。
    2. 安乐死小鼠并在样品收集时间点将眼睛摘除,并在4℃下在0.1M磷酸盐缓冲液,pH7.4(方案4)中的4%多聚甲醛中固定过夜,然后进行PBS洗涤。

    3. 使用显微切割剪刀解剖角膜
    4. 用0.2%硼氢化钠(注7)在室温下处理角膜30分钟,然后用0.1M磷酸盐缓冲液(pH7.4)洗涤。
    5. 用4'6-二脒基-2-苯基吲哚(DAPI,1μg/ ml)染色角膜,用盖玻片和Mowiol培养基(配方5)固定在显微镜载玻片上,并用具有apotome附件的Zeiss Observer Z1显微镜观察。

数据分析

AxioVison 4.7。在每个收集时间点采集红色(膜番茄红),绿色(膜EGFP)和蓝色(DAPI)通道的Z-堆叠图像。
在每个时间点都记录了从红色到绿色的转换(图3)。


图3.毛囊隆起源干细胞在角膜上皮细胞中的植入和分化从Z-stack获得的单色图像描绘了膜番茄红和膜EGFP在3天的细胞定位和移植后5周。放大倍率x200。经Meyer-Blazejewska et al。许可重印。2011年。

笔记


  1. 制备纤维蛋白凝胶和培养HFSC应在II级生物安全柜中进行。
  2. 只要稀释的凝血酶和稀释的纤维蛋白的比例保持1:1,就可以制备纤维蛋白载体的各种尺寸。
  3. 所有细胞计数均使用血细胞计数器进行。
  4. 所有动物方案均由辛辛那提大学的动物管理和使用委员会批准。转基因小鼠系 Krt12rtTA (Chikama 等人,2005年), TetO-cre (Perl 等人 ,2002)和Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J(ROSAmTmG)(Muzumdar等,<2007>)。通过培养单独的小鼠系来产生化合物转基因小鼠以产生K12 rtTA / rtTA / tetO-cre / ROSA mTmG 。这种双报告小鼠模型使用角蛋白12启动子(角膜上皮特异性)来驱动Tet-on系统的表达。与多西环素和cre联合使用时,膜番茄红毛囊干细胞如果分化为角膜上皮细胞就会变绿。
  5. 当创建并转移纤维蛋白凝胶盘时,务必小心操作,以免损坏细胞。另外,确保光盘放置在PBS中,HFSC朝上。
  6. 当用Algerbrush去除上皮时,应注意避免损伤基底膜。这可以通过将Algerbrush按45°角移动并以圆形扫动来移动。
    。只用轻微的压力,因为上皮比较容易去除。
  7. 处理硼氢化钠时应小心,因为它会对身体和健康造成危害。

食谱

  1. 纤维蛋白原溶液
    10%NaCl
    100 mM CaCl 2 2/2 0.9%NaCl
    ddH <2>
  2. 凝血酶溶液
    10%NaCl
    100 mM CaCl 2 2/2 ddH <2>
  3. 干细胞培养基
    3份DMEM /不含Ca 2 +或Mg 2+ +的高葡萄糖 1份Ham's F12
    10%FBS
    10 ng / ml EGF
    500毫克/升L-谷氨酰胺
    0.4 mM氯化钙
    1x人体角膜生长补充剂
    10,000 U / ml青霉素
    10,000μg/ ml链霉素
    25μg/ ml两性霉素B
  4. 0.1 M磷酸盐缓冲液,pH 7.4
    76 mM Na 2 HPO 4(无水)
    27 mM NaH 2 PO 4(无水)
  5. Mowiol安装介质
    24克分析级甘油
    9.6克Mowiol 4-88
    24毫升ddH 2 O
    48ml 0.2M Tris-HCl缓冲液,pH8.5
  6. 氯胺酮/甲苯噻嗪溶液
    2毫升氯胺酮(100毫克/毫升)
    0.25毫升赛拉嗪(100毫克/毫升)
    7.75毫升PBS

致谢

这项研究部分得到了NIH / NEI EY011845,俄亥俄狮子眼科研究基金会和W.W.K的资助。作者没有任何利益冲突。

参考

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  4. Kjaergard,H.K。和Weis-Fogh,U.S(1994)。 影响自体纤维蛋白胶强度的重要因素;纤维蛋白浓度和反应时间 - 与商业纤维蛋白胶的强度比较。欧洲外科研究所(Eur Surg Res)26(5):273-276。
  5. Meyer-Blazejewska,E.A.,Call,M.K.,Yamanaka,O.,Liu,H.,Schlotzer-Schrehardt,U.,Kruse,F.E。和Kao,W.W。(2011)。 从头发到角膜:治疗使用毛囊干细胞治疗角膜缘干细胞缺陷。干细胞 29(1):57-66。
  6. Muzumdar,M. D.,Tasic,B.,Miyamichi,K.,Li,L。和Luo,L。(2007)。 全球双荧光的Cre记者鼠标 Genesis 45(9):593-605。
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引用:Call, M., Meyer, E. A., Kao, W. W., Kruse, F. E. and Schloetzer-Schredhardt, U. (2018). Murine Hair Follicle Derived Stem Cell Transplantation onto the Cornea Using a Fibrin Carrier. Bio-protocol 8(10): e2849. DOI: 10.21769/BioProtoc.2849.
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