Name: three-dimensional, serum-free culture system for lacrimal gland stem cells
Uploaded: 2022-06-02T13:00:00
Description: Watch this Scientific Journal Video about Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells at JoVE.com

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。

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
<ol><li>低压合成材料隔离<ol>
<li>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...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Animal(Mouse)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bal B/C</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57 BL/6J</td>
			<td>Laboratory Animal Center of Sun Yat-sen University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/ShiLtJ</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ROSA26mT/mG</td>
			<td>Model Animal Research Center of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic dehydrator</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blood counting chamber</td>
			<td>BLAU</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>CountStar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 constant temperature incubator</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ECL Gel imaging system</td>
			<td>GE healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electric bath for water bath</td>
			<td>Yiheng Technology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis apparatus</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence quantitative PCR instrument</td>
			<td>Roche</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen tissue slicer</td>
			<td>Lecia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal centrifuge</td>
			<td>CENCE</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted fluorescence microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laser lamellar scanning micrograph</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen container</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low temperature high speed centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettor</td>
			<td>Gilson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Panasonic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop ultraviolet spectrophotometer</td>
			<td>Thermo</td>
			<td>measure RNA concentration</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin slicing machine</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Amplifier</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pH value tester</td>
			<td>Sartorius</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 &#176;C Refrigerator</td>
			<td>Haier</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic culture oscillator</td>
			<td>ZHICHENG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue paraffin embedding instrument</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;-80&#176;C Ultra-low temperature refrigerator</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;-20&#176;C Ultra-low temperature refrigerator</td>
			<td>Thermo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra pure water purification system</td>
			<td>ELGA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Experiment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCG</td>
			<td>Sigma</td>
			<td>9002-61-3</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Sigma</td>
			<td>14158-65-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital Sodium</td>
			<td>Sigma</td>
			<td>57-33-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17018029</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>BD</td>
			<td>354235</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma</td>
			<td>D6429</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Sigma</td>
			<td>D0697</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sangon Biotech</td>
			<td>A500895</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Gibco</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050087</td>
			<td></td>
		</tr>
		<tr>
			<td>Human FGF10</td>
			<td>PeproTech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (Matrix gel)</td>
			<td>BD</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine Wnt3A</td>
			<td>PeproTech</td>
			<td>315-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine EGF</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>NEAA</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>R-spondin 1</td>
			<td>PeproTech</td>
			<td>120-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin Inhibitor (TI)</td>
			<td>Sigma</td>
			<td>T6522</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>&#160;T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
		<tr>
			<td>HE staining &#38; Immunostaining</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-Mouse IgG</td>
			<td>Thermo</td>
			<td>A-21202</td>
			<td>Used dilution: IHC) 2 &#956;g/mL, (IF) 0.2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 donkey anti-Rabbit IgG</td>
			<td>Thermo</td>
			<td>A-21206</td>
			<td>Used dilution: (IHC) 2 &#956;g/mL, (IF) 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 donkey anti-Mouse IgG</td>
			<td>Thermo</td>
			<td>A-10037</td>
			<td>Used dilution: (IHC) 2 &#956;g/mL, (IF) 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 donkey anti-Rabbit IgG</td>
			<td>Thermo</td>
			<td>A-10042</td>
			<td>Used dilution: (IHC) 2 &#956;g/mL, (IF) 4 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-AQP5 rabbit antibody</td>
			<td>Abcam</td>
			<td>ab104751</td>
			<td>Used dilution: (IHC) 1 &#956;g/mL, (IF) 0.1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-E-cadherin Rat antibody</td>
			<td>Abcam</td>
			<td>ab11512</td>
			<td>Used dilution: (IF)&#160; 5 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-Keratin14 rabbit antibody</td>
			<td>Abcam</td>
			<td>ab181595</td>
			<td>Used dilution: (IHC) 1 &#956;g/mL, (IF) 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-Ki67 rabbit antibody</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>Used dilution: (IHC) 1 &#956;g/mL, (IF) 1 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-mCherry mouse antibody</td>
			<td>Abcam</td>
			<td>ab125096</td>
			<td>Used dilution: (IHC) 2 &#956;g/mL, (IF) 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Anti-mCherry rabbit antibody</td>
			<td>Abcam</td>
			<td>ab167453</td>
			<td>Used dilution: (IF)&#160; 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>C6H8O7</td>
			<td>Sangon Biotech</td>
			<td>A501702-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric Acid</td>
			<td>Sangon Biotech</td>
			<td>201-069-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB Kit (20x)</td>
			<td>CWBIO</td>
			<td>CW0125</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo</td>
			<td>62248</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>BASO</td>
			<td>68115</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Mounting Medium</td>
			<td>Dako</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sangon Biotech</td>
			<td>A501912-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG antibody (HRP)</td>
			<td>Abcam</td>
			<td>ab6789</td>
			<td>Used dilution: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG antibody(HRP)</td>
			<td>Abcam</td>
			<td>ab6721</td>
			<td>Used dilution: 2 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>BASO</td>
			<td>517-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Histogel (Embedding hydrogel)</td>
			<td>Thermo</td>
			<td>HG-400-012</td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</td>
			<td>Guangzhou Chemistry</td>
			<td>KD10</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Hydrogen Peroxide Solution</td>
			<td>Guangzhou Chemistry</td>
			<td>7722-84-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Guangzhou Chemistry</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Na3C6H5O7.2H2O</td>
			<td>Sangon Biotech</td>
			<td>A501293-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>SHANGHAI YIYANG</td>
			<td>YY-Neutral balsam</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-immunized Goat Serum</td>
			<td>BOSTER</td>
			<td>AR0009</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sangon Biotech</td>
			<td>A601891-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>DAMAO</td>
			<td>200-001-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose</td>
			<td>Guangzhou Chemistry</td>
			<td>57-50-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate tribasic dihydrate</td>
			<td>Sangon Biotech</td>
			<td>200-675-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Guangzhou Chemistry</td>
			<td>IB11-AR-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.T.C. Compound</td>
			<td>SAKURA</td>
			<td>SAKURA.4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>DINGGUO</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Guangzhou Chemistry</td>
			<td>128686-03-3</td>
			<td></td>
		</tr>
		<tr>
			<td>RT-PCR &#38; qRT-PCR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Guangzhou Chemistry</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Guangzhou Chemistry</td>
			<td>865-49-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Sangon Biotech</td>
			<td>214-984-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol</td>
			<td>Guangzhou Chemistry</td>
			<td>67-63-0</td>
			<td></td>
		</tr>
		<tr>
			<td>LightCycler 480 SYBR Green I Master Mix</td>
			<td>Roche</td>
			<td>488735200H</td>
			<td></td>
		</tr>
		<tr>
			<td>ReverTra Ace qPCR RT Master Mix</td>
			<td>TOYOBO</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>TAKARA</td>
			<td>R10T1</td>
			<td></td>
		</tr>
		<tr>
			<td>Goldview (nucleic acid stain)</td>
			<td>BioSharp</td>
			<td>BS357A</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol</td>
			<td>Magen</td>
			<td>R4801-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Construction &#38; Cell Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>OXID</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>69-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma</td>
			<td>56-75-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free Plasmid Extraction Kit</td>
			<td>Thermo</td>
			<td>A36227</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma</td>
			<td>25389-94-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipo3000 Plasmid Transfection Kit</td>
			<td>Thermo</td>
			<td>L3000015</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Reaction Kit</td>
			<td>Thermo</td>
			<td>11791019</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Extraction Kit</td>
			<td>TIANGEN</td>
			<td>DP103</td>
			<td></td>
		</tr>
		<tr>
			<td>Trans5&#945; Chemically Competent Cell</td>
			<td>TRANSGEN</td>
			<td>CD201-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Trytone</td>
			<td>OXID</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>OXID</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and Sequence</td>
			<td>Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: AQP5 
			Sequence: 
			F: CATGAACCCAGCCCGATCTT 
			R: CTTCTGCTCCCATCCCATCC</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: &#946;-actin 
			Sequence: 
			F: AGATCAAGATCATTGCTCCTCCT 
			R: AGATCAAGATCATTGCTCCTCCT</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Epcam 
			Sequence: 
			F: CATTTGCTCCAAACTGGCGT 
			R: TGTCCTTGTCGGTTCTTCGG</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Krt5 
			Sequence: 
			F: AGCAATGGCGTTCTGGAGG 
			R: GCTGAAGGTCAGGTAGAGCC</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Krt14 
			Sequence: 
			F: CGGACCAAGTTTGAGACGGA 
			R: GCCACCTCCTCGTGGTTC</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Krt19 
			Sequence: 
			F: TCTTTGAAAAACACTGAACCCTG 
			R: TGGCTCCTCAGGGCAGTAAT</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Ltf 
			Sequence: 
			F: CACATGCTGTCGTATCCCGA 
			R: CGATGCCCTGATGGACGA</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: Nestin 
			Sequence: 
			F: GGGGCTACAGGAGTGGAAAC 
			R: GACCTCTAGGGTTCCCGTCT</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer: P63 
			Sequence: 
			F: TCCTATCACGGGAAGGCAGA 
			R: GTACCATCGCCGTTCTTTGC</td>
			<td>Synbio Tech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pLX302 lentivirus no-load vector</td>
			<td>Addgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pENRTY-mCherry</td>
			<td>Xiaofeng Qin laboratory, Sun Yat-sen University</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional, serum-free culture system for lacrimal gland stem cells

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。

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?...

Watch this Scientific Journal Video about Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells at JoVE.com

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

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。

用于泪腺干细胞的三维无血清培养系统

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

该方案为富集泪腺干细胞以再生和重建泪腺提供了有利的策略。泪腺干细胞显示出长期的扩增能力和分化的潜力。该技术的主要优点是泪腺干细胞培养基无血清，这表明泪腺修复的巨大价值 首先获得安乐死的6至8周龄BALB / c雄性小鼠并切割耳后的皮肤以暴露泪腺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，并去除泪腺。将泪腺浸入装有4毫升75%乙醇的6厘米培养皿中10秒钟，并立即用10毫升PBS溶液冲洗两次。将泪腺切成约一毫米立方的小碎片。将它们转移到无菌的15毫升离心管中，并在37摄氏度下用500微升每毫升25单位的分散酶和500微升的0.1%胶原酶I处理组织一小时。用一毫升0.05%胰蛋白酶EDTA在37摄氏度下处理泪腺碎片10分钟，并通过反复移液将碎片解离成单个细胞。通过70微米过滤器过滤悬浮液，将过滤器收集到无菌的15毫升离心管中，并在150倍G下离心5分钟。离心后，取出上清液，加入10毫升10毫升PBS溶液洗涤细胞沉淀，并将悬浮液以150倍G离心5分钟。重复洗涤细胞沉淀。然后，除去上清液，加入1毫升1：1比例的杜尔贝茨科改性鹰培养基和汉姆的F-12，以重悬细胞沉淀。在24孔板的孔中心加入20微升基质凝胶泪腺干细胞培养基质。使用移液器吸头将其扩大到直径约6至8毫米的圆圈，并将混合物在37摄氏度下孵育20分钟以预涂孔。使用细胞计数器确定细胞数量，并将40微升泪腺干细胞培养基或LGSCM加入总共10， 000个细胞到40微升基质凝胶中。用移液管轻轻混合。使用移液管小心地将80微升的混合物滴在24孔板的每个孔的预涂膜区域上。将混合物在37摄氏度下孵育20分钟，并加入600微升LGSCM。每两天更换一次每个孔中的培养基。经过七天的培养，在倒置显微镜下寻找直径为100至300微米的LGSC球体。使用该方案分离和培养Rosa26和NOD小鼠的原发性泪腺干细胞。培养7天后，从球体培养井中取出培养基。通过在20微升每毫升分散酶10单位和100微升10毫升PBS中在37摄氏度下孵育30分钟来分解LGSC球。将悬浮液转移到15毫升离心管中，并以150倍G离心4分钟。取出上清液。用一毫升0.05%胰蛋白酶EDTA在37摄氏度下处理球体五分钟，加入一毫升0.05%胰蛋白酶抑制剂并反复移液以中和胰蛋白酶并将球体解离成单细胞。在150倍G下离心五分钟，并除去上清液以沉淀LGSC。加入一毫升LGSCM以重悬泪腺干细胞沉淀并如上所述接种细胞。如果执行程序一，使用泪腺干细胞培养系统将LGSC的培养时间从7天延长至14天，以进行随机分化。对于程序二，在泪腺干细胞培养开始时将基质凝胶与LGSCM的比例从1：1更改为1：2，用于导管细胞的分化。将泪腺干细胞接种到基质中以进行诱导14天。对于程序三，传代后，用LGSCM 10%FBS代替LGSCM以分化腺泡细胞。诱导分化 14 天。经过一周的培养，Kr14和Ki67在泪腺干细胞形成的所有球体中表达。球体的直径达到100微米。苏木精和曙红染色在第七天显示出细胞性。原代培养和传代培养7 d后获得的泪腺干细胞富集因子表明，该方法富集的细胞具有较强的增殖能力。在这个系统中，泪腺干细胞可以传代超过40次，并且仍然保持干细胞特征。通过胎牛血清和低比例基质凝胶诱导泪腺干细胞形成更多芽。苏木精和曙红染色表明，胎牛血清可以诱导球体产生更多的空化结构。在NOD小鼠中原位注射罗莎泪腺干细胞后，在泪腺附近形成新的泪腺小叶。小叶大部分由AQP5高表达的成熟腺泡细胞组成，小叶内形成，AQP5表达率低。罗莎泪腺干细胞注射侧的泪液分泌量高于对照侧，但低于野生型小鼠基质凝胶及其相关混合物在使用前必须始终保持在冰上，并且与基质凝胶接触的微量离心管和吸头应预冷。该系统为进一步研究泪腺干细胞、泪腺修复和泪腺疾病的药物筛选提供了理想的模型。

Research

JoVE Journal

Biology

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

基于芯片的三维细胞培养，在灌注微生物反应器

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

科研

生物学

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

芯片尺寸支架的三维细胞培养的微细

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation of Mouse Salivary Gland Stem Cells

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

老鼠胎儿整肠文化系统<em&gt;前体内</em&gt;信号通路和绒毛开发的三维实时成像的操作

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

Manipulating the Murine Lacrimal Gland

操纵小鼠泪腺

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...