用于泪腺干细胞的三维无血清培养系统
Summary
用于成体泪腺（LG）干细胞的三维无血清培养方法对于诱导LG类器官形成并分化为腺泡或导管状细胞已经确立。
Abstract
基于泪腺（LG）干细胞的治疗是治疗泪腺疾病的有希望的策略。然而，缺乏可靠的无血清培养方法来获得足够数量的LG干细胞（LGSC）是进一步研究和应用的一个障碍。用于成人小鼠LGSC的三维（3D），无血清培养方法已经建立并在此处显示。LGSC可以连续传代并诱导分化为腺泡或导管样细胞。
对于LGSC原代培养，用分散酶，胶原酶I和胰蛋白酶 – EDTA消化来自6-8周龄小鼠的LGs。将总共1×104 个单细胞接种到24孔板的每个孔中的80μL基质凝胶泪腺干细胞培养基（LGSCM）基质中，并用20μL基质凝胶-LGSCM基质预涂。将混合物在37°C下孵育20分钟后固化，并加入600μLLGSCM。
对于LGSC维持，通过分散酶和胰蛋白酶- EDTA将培养7天的LGSC分解成单细胞。根据LGSC原代培养中使用的方法植入和培养单个细胞。LGSC可以传代40次以上，并连续表达干细胞/祖细胞标志物Krt14，Krt5，P63和巢蛋白。在LGSCM中培养的LGSC具有自我更新能力，可以在 体外 和 体内分化成腺泡或导管样细胞。
Introduction
泪腺干细胞（LGSC）维持泪腺（LG）细胞更新，是腺泡和导管细胞的来源。因此，LGSC移植被认为是治疗严重炎症损伤和水缺乏性干眼病（ADD）的替代方法1，2，3。Tiwari等人使用胶原蛋白I和基质凝胶分离和培养原代LG细胞，并补充了几种生长因子;然而，LG细胞不能连续培养4。使用二维（2D）培养，由You等人5和阿克曼等人分离出小鼠LG衍生的干细胞。6、发现表达干细胞/祖细胞标记基因的Oct4、Sox2、纳米和巢蛋白，并可进行传代培养。然而，没有明确的迹象表明这些细胞可以分化成腺泡或导管细胞，也没有移植实验来验证体内的分化潜力。
最近，通过流式细胞术从小鼠LG中分离出c-kit+ dim/EpCAM+/Sca1-/CD34–/CD45– 细胞，发现其表达LG祖细胞标志物，如Pax6和Runx1， 并在体外分化成导管和阿西尼。在具有ADDD的小鼠中，用这些细胞进行原位注射可以修复受损的LG并恢复LG2的分泌功能。然而，通过这种方法分离的干细胞数量很少，并且没有合适的培养条件来扩增分离的LGSC。综上所述，需要建立适当的培养体系，以有效分离和培养具有稳定和持续扩增的LGS成体LGSC，以研究LGSC在ADD处理中的应用。
来自干细胞或多能干细胞的类器官是一组在组织学上与相关器官相似的细胞，可以维持自己的更新。在Sato等人于2009年7成功培养小鼠肠道类器官后，基于佐藤的培养系统，连续培养来自其他器官的类器官，如胆囊8，肝脏9，胰腺10，胃11，乳房12，肺13，前列腺14和唾液腺15.由于成体干细胞在类器官培养中分化前的比例很高，因此三维（3D）类器官培养方法被认为是LG成体干细胞分离和培养的最佳选择。
本研究通过优化3D无血清培养方法建立了成年小鼠LGSC培养系统。结果表明，从正常和ADD小鼠培养的LGSC显示出稳定的自我更新和增殖能力。移植到添加的小鼠LG后，LGSC定植受损的LG并改善了泪液的产生。此外，从ROSA26mT / mG 小鼠中分离红色荧光LGSC并进行培养。本研究为LGSC 体外 富集和LGSC自体移植在临床应用中的附加治疗提供了可靠的参考。
Protocol
该协议中的所有实验都遵循中山大学动物试验伦理委员会的动物护理指南。所有与细胞相关的操作都将在单元操作室的超洁净工作台上进行。所有使用二甲苯的操作都将在通风橱中进行。 1. 初级培养 低压合成材料隔离 获取6-8周龄的BALB / c雄性小鼠，并切开耳朵后面的皮肤以暴露LG及其周围的结缔组织。在镊子的帮助下通过钝性解剖剥离结缔组织并去…
Representative Results
建立 3D、无血清培养系统本研究开发了含有EGF、Wnt3A、FGF10和Y-27632的小鼠LGSC，并通过3D培养方法成功分离和培养LGSC（图1A）。使用该方法已经建立了来自C57BL / 6小鼠，NOD / ShiLtJ小鼠，BALB / c小鼠和ROSA26mT / mG小鼠的LGSC的成功3D，无血清培养系统16。对于雄性小鼠，通过解离从两个LG获得1.5-2×10 6个细胞。培养一周后，在培养开始?…
Discussion
有完善的泪液干细胞分离和 体外 培养方法，用于泪液干细胞培养和LG损伤修复。沙托斯等人17 和阿克曼等人。6 .分别通过2D培养方法成功培养和传代大鼠和小鼠的泪液干细胞，使得可以移植泪液干细胞用于ADDD的治疗。对2D培养的LGs的干细胞18 和间充质干细胞19，20 的研究表明，移植这些…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
本研究由国家自然科学基金项目（第31871413号）和广东省科学技术两个计划（2017B020230002和2016B030231001）资助。我们非常感谢在研究期间帮助我们的研究人员以及在动物中心工作的工作人员在动物护理方面的支持。
Materials
| Animal(Mouse) | |||
| Bal B/C | Model Animal Research Center of Nanjing University | ||
| C57 BL/6J | Laboratory Animal Center of Sun Yat-sen University | ||
| NOD/ShiLtJ | Model Animal Research Center of Nanjing University | ||
| ROSA26mT/mG | Model Animal Research Center of Nanjing University | ||
| Equipment | |||
| Analytical balance | Sartorius | ||
| Automatic dehydrator | Thermo | ||
| Blood counting chamber | BLAU | ||
| Cell Counter | CountStar | ||
| CO2 constant temperature incubator | Thermo | ||
| ECL Gel imaging system | GE healthcare | ||
| Electric bath for water bath | Yiheng Technology | ||
| Electrophoresis apparatus | BioRad | ||
| Fluorescence quantitative PCR instrument | Roche | ||
| Frozen tissue slicer | Lecia | ||
| Horizontal centrifuge | CENCE | ||
| Inverted fluorescence microscope | Nikon | ||
| Inverted microscope | Olympus | ||
| Laser lamellar scanning micrograph | Carl Zeiss | ||
| Liquid nitrogen container | Thermo | ||
| Low temperature high speed centrifuge | Eppendorf | ||
| Micropipettor | Gilson | ||
| Microwave oven | Panasonic | ||
| Nanodrop ultraviolet spectrophotometer | Thermo | measure RNA concentration | |
| Paraffin slicing machine | Thermo | ||
| PCR Amplifier | Eppendorf | ||
| pH value tester | Sartorius | ||
| 4 °C Refrigerator | Haier | ||
| Thermostatic culture oscillator | ZHICHENG | ||
| Tissue paraffin embedding instrument | Thermo | ||
| -80°C Ultra-low temperature refrigerator | Thermo | ||
| -20°C Ultra-low temperature refrigerator | Thermo | ||
| Ultra pure water purification system | ELGA | ||
| Reagent | |||
| Animal Experiment | |||
| HCG | Sigma | 9002-61-3 | |
| PMSG | Sigma | 14158-65-7 | |
| Pentobarbital Sodium | Sigma | 57-33-0 | |
| Cell Culture | |||
| B27 | Gibco | 17504044 | |
| Collagenase I | Gibco | 17018029 | |
| Dispase | BD | 354235 | |
| DMEM | Sigma | D6429 | |
| DMEM/F12 | Sigma | D0697 | |
| DMSO | Sigma | 67-68-5 | |
| EDTA | Sangon Biotech | A500895 | |
| Foetal Bovine Serum | Gibco | 04-001-1ACS | |
| GlutaMax | Gibco | 35050087 | |
| Human FGF10 | PeproTech | 100-26 | |
| Matrigel (Matrix gel) | BD | 356231 | |
| Murine Noggin | PeproTech | 250-38 | |
| Murine Wnt3A | PeproTech | 315-20 | |
| Murine EGF | PeproTech | 315-09 | |
| NEAA | Gibco | 11140050 | |
| N2 | Gibco | 17502048 | |
| R-spondin 1 | PeproTech | 120-38 | |
| Trypsin Inhibitor (TI) | Sigma | T6522 | Derived from Glycine max; can inhibit trypsin, chymotrypsin, and plasminase to a lesser extent. One mg will inhibit 1.0-3.0 mg of trypsin. |
| Trypsin | Sigma | T4799 | |
| Y-27632 | Selleck | S1049 | |
| HE staining & Immunostaining | |||
| Alexa Fluor 488 donkey anti-Mouse IgG | Thermo | A-21202 | Used dilution: IHC) 2 μg/mL, (IF) 0.2 μg/mL |
| Alexa Fluor 488 donkey anti-Rabbit IgG | Thermo | A-21206 | Used dilution: (IHC) 2 μg/mL, (IF) 2 μg/mL |
| Alexa Fluor 568 donkey anti-Mouse IgG | Thermo | A-10037 | Used dilution: (IHC) 2 μg/mL, (IF) 2 μg/mL |
| Alexa Fluor 568 donkey anti-Rabbit IgG | Thermo | A-10042 | Used dilution: (IHC) 2 μg/mL, (IF) 4 μg/mL |
| Anti-AQP5 rabbit antibody | Abcam | ab104751 | Used dilution: (IHC) 1 μg/mL, (IF) 0.1 μg/mL |
| Anti-E-cadherin Rat antibody | Abcam | ab11512 | Used dilution: (IF) 5 μg/mL |
| Anti-Keratin14 rabbit antibody | Abcam | ab181595 | Used dilution: (IHC) 1 μg/mL, (IF) 2 μg/mL |
| Anti-Ki67 rabbit antibody | Abcam | ab15580 | Used dilution: (IHC) 1 μg/mL, (IF) 1 μg/mL |
| Anti-mCherry mouse antibody | Abcam | ab125096 | Used dilution: (IHC) 2 μg/mL, (IF) 2 μg/mL |
| Anti-mCherry rabbit antibody | Abcam | ab167453 | Used dilution: (IF) 2 μg/mL |
| C6H8O7 | Sangon Biotech | A501702-0500 | |
| Citric Acid | Sangon Biotech | 201-069-1 | |
| DAB Kit (20x) | CWBIO | CW0125 | |
| DAPI | Thermo | 62248 | |
| Eosin | BASO | 68115 | |
| Fluorescent Mounting Medium | Dako | S3023 | |
| Formalin | Sangon Biotech | A501912-0500 | |
| Goat anti-Mouse IgG antibody (HRP) | Abcam | ab6789 | Used dilution: 2 μg/mL |
| Goat anti-Rabbit IgG antibody(HRP) | Abcam | ab6721 | Used dilution: 2 μg/mL |
| Hematoxylin | BASO | 517-28-2 | |
| Histogel (Embedding hydrogel) | Thermo | HG-400-012 | |
| 30% H2O2 | Guangzhou Chemistry | KD10 | |
| 30% Hydrogen Peroxide Solution | Guangzhou Chemistry | 7722-84-1 | |
| Methanol | Guangzhou Chemistry | 67-56-1 | |
| Na3C6H5O7.2H2O | Sangon Biotech | A501293-0500 | |
| Neutral balsam | SHANGHAI YIYANG | YY-Neutral balsam | |
| Non-immunized Goat Serum | BOSTER | AR0009 | |
| Paraffin | Sangon Biotech | A601891-0500 | |
| Paraformaldehyde | DAMAO | 200-001-8 | |
| Saccharose | Guangzhou Chemistry | 57-50-1 | |
| Sodium citrate tribasic dihydrate | Sangon Biotech | 200-675-3 | |
| Sucrose | Guangzhou Chemistry | IB11-AR-500G | |
| Tissue-Tek O.T.C. Compound | SAKURA | SAKURA.4583 | |
| Triton X-100 | DINGGUO | 9002-93-1 | |
| Xylene | Guangzhou Chemistry | 128686-03-3 | |
| RT-PCR & qRT-PCR | |||
| Agarose | Sigma | 9012-36-6 | |
| Alcohol | Guangzhou Chemistry | 64-17-5 | |
| Chloroform | Guangzhou Chemistry | 865-49-6 | |
| Ethidium Bromide | Sangon Biotech | 214-984-6 | |
| Isopropyl Alcohol | Guangzhou Chemistry | 67-63-0 | |
| LightCycler 480 SYBR Green I Master Mix | Roche | 488735200H | |
| ReverTra Ace qPCR RT Master Mix | TOYOBO | – | |
| Taq DNA Polymerase | TAKARA | R10T1 | |
| Goldview (nucleic acid stain) | BioSharp | BS357A | |
| TRIzol | Magen | R4801-02 | |
| Vector Construction & Cell Transfection | |||
| Agar | OXID | – | |
| Ampicillin | Sigma | 69-52-3 | |
| Chloramphenicol | Sigma | 56-75-7 | |
| Endotoxin-free Plasmid Extraction Kit | Thermo | A36227 | |
| Kanamycin | Sigma | 25389-94-0 | |
| Lipo3000 Plasmid Transfection Kit | Thermo | L3000015 | |
| LR Reaction Kit | Thermo | 11791019 | |
| Plasmid Extraction Kit | TIANGEN | DP103 | |
| Trans5α Chemically Competent Cell | TRANSGEN | CD201-01 | |
| Trytone | OXID | – | |
| Yeast Extract | OXID | – | |
| Primers and Sequence | Company | ||
| Primer: AQP5 Sequence: F: CATGAACCCAGCCCGATCTT R: CTTCTGCTCCCATCCCATCC |
Synbio Tech | ||
| Primer: β-actin Sequence: F: AGATCAAGATCATTGCTCCTCCT R: AGATCAAGATCATTGCTCCTCCT |
Synbio Tech | ||
| Primer: Epcam Sequence: F: CATTTGCTCCAAACTGGCGT R: TGTCCTTGTCGGTTCTTCGG |
Synbio Tech | ||
| Primer: Krt5 Sequence: F: AGCAATGGCGTTCTGGAGG R: GCTGAAGGTCAGGTAGAGCC |
Synbio Tech | ||
| Primer: Krt14 Sequence: F: CGGACCAAGTTTGAGACGGA R: GCCACCTCCTCGTGGTTC |
Synbio Tech | ||
| Primer: Krt19 Sequence: F: TCTTTGAAAAACACTGAACCCTG R: TGGCTCCTCAGGGCAGTAAT |
Synbio Tech | ||
| Primer: Ltf Sequence: F: CACATGCTGTCGTATCCCGA R: CGATGCCCTGATGGACGA |
Synbio Tech | ||
| Primer: Nestin Sequence: F: GGGGCTACAGGAGTGGAAAC R: GACCTCTAGGGTTCCCGTCT |
Synbio Tech | ||
| Primer: P63 Sequence: F: TCCTATCACGGGAAGGCAGA R: GTACCATCGCCGTTCTTTGC |
Synbio Tech | ||
| Vector | |||
| pLX302 lentivirus no-load vector | Addgene | ||
| pENRTY-mCherry | Xiaofeng Qin laboratory, Sun Yat-sen University |
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Citazione di questo articolo
Chen, H., Huang, P., Zhang, Y. Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells. J. Vis. Exp. (184), e63585, doi:10.3791/63585 (2022).