这项工作得到了国家自然科学基金 （82172107）、广东省自然科学基金 （2021A1515011927， 2021A1515010918， 2020A1515110347）、深圳市医药研究基金 （SMRF.D2301015）、深圳市科技创新委员会（JCYJ20210324135014040、JCYJ20220530172807016、JCYJ20230807150908018、JCYJ20230807150915031）和龙岗区经济技术发展专项资金（LGKCYLWS2022007）。

样本来自中国广东省深圳市龙岗区妇幼保健院同意的健康捐献者。北京中医药大学深圳医院伦理委员会 （SZLDH2020LSYM-095） 和深圳市龙岗区妇幼保健院医学伦理委员会 （LGFYYXLLS-2020-005） 批准了人类样本用于研究。所有实验均根据批准的指南进行。材料 表中列出了试剂和所用设备的详细信息。
1. 人体脐带的采集
<ol><li>在无菌条件下从合作医院获取人脐带样本。 注意:选择 35 岁以下的供体，最好是初产妇，没有肝炎、性传播疾病或遗传疾病病史。乙型肝炎病毒、丙型肝炎病毒和巨细胞病毒的入院检查结果需要为阴性。选择剖宫产作为分娩方式。</li>
	<li>选择一块长度约为 10-15 厘米的笔直且完整的 UC。</li>
	<li>用止血钳闭合两端，并在剖宫产分娩足月新生儿后立即在产钳之间的内端用手术结结扎。（收集的 UC，如图 1 所示，步骤 1）。 注意:剖宫产手术确保采集物无菌。使用 0 或 2-0 缝合线的手术结以获得更好的抗拉强度。</li>
	<li>使用手术剪刀剪断镊子和结之间的绑扎的脐带（图 1，步骤 1）。</li>
	<li>用生理盐水冲洗以去除表面的任何污染血凝块，并立即转移到含有 20 mL 无菌磷酸盐缓冲盐水 （PBS） 的无菌 50 mL 塑料管中。 注意:转移过程中未使用抗生素。</li>
	<li>密封管子。将其放在聚苯乙烯盒中的冰上，并在 4 °C 下储存，然后再运输到实验室。 注意:在收集后 4 小时内解剖脐带。</li>
</ol>2. 从脐带中分离沃顿氏胶
<ol><li>按照以下步骤处理脐带:<ol><li>用手术器械、无菌包装的 90 mm 培养皿、1000 μL 移液器、无菌 PBS、75% 乙醇溶液和无异种成分的人 MSC 培养基准备无菌托盘。 注意:齿形镊子和蚊夹可以有效地处理滑行的绳索。</li>
		<li>在将所需物品放入生物安全柜内之前，对所需物品和工作区进行表面净化。 注意: 在开始工作之前，让工作区的空气吹扫几分钟。</li>
		<li>对试管表面进行消毒，并将样品取出到生物安全柜中的培养皿中。将带有结的样品浸入 75% 乙醇中 30 秒，然后在新的培养皿中用无菌 PBS 多次冲洗。 注意: 在浸入 75% 乙醇之前，必须将绳索结扎，浸入时间应限制在 30 秒至 1 分钟，以便对绳索进行完全消毒而不损坏。</li>
		<li>剪掉结，用手术剪刀和镊子将两个结之间的绳索横向剪成约 2 厘米长的短块。 注意: 带结的绳索末端应丢弃。UC 的长度不应太长或太短;这两种情况都可能增加操作的难度。</li>
		<li>使用 1000 μL 移液器用 PBS 灌注静脉（较大的血管），直到从脐带另一端流出的液体完全透明19。</li>
	</ol></li>
	<li>解剖脐带。<ol><li>将已切好的一块转移到新的培养皿中。 注意:两条动脉和一条静脉应在横截面8 上明显暴露（图 2B）。</li>
		<li>添加适量的 PBS 以在整个操作过程中保持电源线湿润。</li>
		<li>用镊子抓住脐带，并在蚊夹的帮助下沿其长轴拉出动脉。 注意: 如果动脉已撕裂，请尝试从脐带的另一端拉出，因为动脉壁具有很大的弹性。</li>
		<li>将镊子的一片刀片插入脐静脉，然后夹紧，确保镊子夹在较粗的一侧，将脐带水平固定，较粗的一面朝上。</li>
		<li>使用手术刀沿着镊子另一刀片的边缘在羊膜上皮层上从一端到另一端做一个线性切口（图3）。 注意:通过尽可能少地切割 WJ 来保持沃顿商学院果冻的完整性。</li>
		<li>从切割间隙的一个角开始。用齿夹提起羊膜上皮的一角，然后继续用角膜剪刀沿着上皮内表面水平切割。<ol><li>用夹子将沃顿氏胶和羊膜上皮分开，并逐渐扩大到 UC 一端的整个圆圈。纵向剥离上皮层（图 3）。 注意:沃顿胶是一种被致密的羊膜上皮包裹的粘液结缔组织8。整个操作应该顺利，但如果在剥皮过程中遇到困难，可以使用蚊夹进行钝性解剖。</li>
		</ol></li>
		<li>将镊子的两个刀片插入静脉内，夹住一部分 WJ，然后将其翻转以露出脐静脉的上皮（图 3）。</li>
		<li>很容易剥离静脉的上皮，因为内部张力在静脉上皮和血管周围 WJ 之间形成分离（图 3）。</li>
	</ol></li>
	<li>使用常规方法剖剖 UC 进行比较20.<ol><li>选择另一个已经切割的样品，其重量与之前的脐带几乎相同，然后将其转移到新的培养皿中。</li>
		<li>用剪刀沿脐静脉纵向剪断，并展开 UC 以露出血管和 WJ。</li>
		<li>去除脐静脉和两条动脉，然后用剪刀和手术刀将 WJ 从内皮层上取下。</li>
	</ol></li>
</ol>3. UC-MSC 的分离和培养
<ol><li>将收集的 WJ 放入预先标记的 90 mm 培养皿中，并用剪刀和镊子将其切成 1-3 mm 的小块。</li>
	<li>将底部带盖的培养皿倒置，并将其置于 37 °C 的 5% CO2 培养箱中 30 分钟，以加强碎片与塑料表面之间的附着。</li>
	<li>将培养皿翻转过来，轻轻加入适量的人 MSC 培养基以进一步培养。 注意: 使用最低速度以确保部件仍处于连接状态。</li>
	<li>每 3 天更换一次人 MSC 培养基。在前 7 天内每 2 天更换三分之二的培养基并观察纤维细胞生长的外观，更换完整的培养基，此后每天观察一次。 注意:前 7 天避免移动培养皿。在大约 4 天内观察到成纤维细胞样细胞的显着生长。</li>
	<li>传代培养直至汇合度达到 80%。 注意:此步骤需要 7-10 天。<ol><li>在水浴中将 PBS、0.25% 胰蛋白酶和无异种成分的人 MSC 培养基预热至 37 °C。</li>
		<li>用移液管吸出上清液后用 PBS 冲洗，然后用预热的 0.25% 胰蛋白酶分离细胞，直到可以通过敲击培养皿将细胞脱落。</li>
		<li>通过以 4:1 的比例混合预热的 MSC 培养基来灭活胰蛋白酶，收集细胞悬液，并在 4 °C 下以 300 x g 离心 5 分钟。 注:这些细胞被视为传代 0 （P0）。</li>
		<li>用移液管弃去上清液，将细胞重悬于人 MSC 培养基中，并以 1x104 个细胞/cm2 的接种密度接种到新的培养皿中进行增殖。</li>
	</ol></li>
	<li>使用血细胞计数器，在第一次、第二次和第三次传代扩增后计数两种方法的总细胞数。在显微镜下确定第一、第三和第五代的细胞形态。 注意:这些细胞的形态应与 P0 细胞相似。</li>
	<li>在第 5 次传代时使用细胞进行流式细胞术和其他实验。</li>
</ol>4. 流式细胞术表达细胞表面标志物
<ol><li>用 0.25% 胰蛋白酶分离第 5 代 （P5） 细胞，用细胞染色缓冲液洗涤细胞，并在 4 °C 下以 300 x g 离心 5 分钟。 用细胞染色缓冲液重悬沉淀，每个沉淀至 100 μL。</li>
	<li>按照制造商的说明，标记每个试管并加入适量的抗体，这些抗体与别藻蓝蛋白 （APC）、异硫氰酸荧光素 （FITC） 或藻红蛋白 （PE） 偶联:CD44-APC、CD73-APC、CD90-FIFC、CD105-FIFC、CD11B-PE、CD19-PE、CD43-PE、HLA-DR-PE21。在室温下避光孵育 15-20 分钟。</li>
	<li>将染色的细胞在 4 °C 下以 300 x g 离心 5 分钟，用移液管吸出上清液，并用细胞染色缓冲液洗涤一次。</li>
	<li>用 300 μL 细胞染色缓冲液重复细胞离心和重悬。</li>
	<li>通过流式细胞仪运行细胞，并根据制造商的说明分析抗体染色的细胞。</li>
</ol>5. 用细胞计数法测定细胞生长曲线
<ol><li>使用两种方法通过在人 MSC 培养基中稀释 P5 细胞来制备 P5 细胞的单细胞悬液。</li>
	<li>在 24 孔板中每孔接种 8,000 个细胞，每种方法总共接种 21 个孔。 注:接种前用移液管吹气和抽吸轻轻混合细胞。</li>
	<li>接种 24 小时后，从三个孔中收获细胞。使用血细胞计数器进行细胞计数以计算平均值。 注意:在细胞计数之前摇动板。可以延长振荡的持续时间以防止形成细胞聚集体。</li>
	<li>每 24 小时重复细胞计数，总持续时间为 7 天。在 X 轴上使用时间 （d） 和 Y 轴上的细胞计数 （x104/mL） 绘制生长曲线。</li>
</ol>

从人脐带中解剖和分离 WJ-MSC

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+Multifaceted+characterization+of+human+mesenchymal+stem+cells+for+use+in+regenerative+medicine.">Concise review: Multifaceted characterization of human mesenchymal stem cells for use in regenerative medicine.</a> Stem Cells Transl Med. 6 (12), 2173-2185 (2017).</li><li>Sypecka, M., Bzinkowska, A., Sulejczak, D., Dabrowski, F., Sarnowska, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+optimal+manufacturing+protocols+and+therapeutic+properties+of+mesenchymal+stem/stromal+cells+derived+from+Wharton's+jelly.">Evaluation of the optimal manufacturing protocols and therapeutic properties of mesenchymal stem/stromal cells derived from Wharton's jelly.</a> Int J Mol Sci. 24 (1), 652 (2022).</li><li>Binato, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+human+mesenchymal+stem+cells+during+in+vitro+culture:+Considerations+for+cell+therapy.">Stability of human mesenchymal stem cells during in vitro culture: Considerations for cell therapy.</a> Cell Prolif. 46 (1), 10-22 (2012).</li><li>Mushahary, D., Spittler, A., Kasper, C., Weber, V., Charwat, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+cultivation,+and+characterization+of+human+mesenchymal+stem+cells.">Isolation, cultivation, and characterization of human mesenchymal stem cells.</a> Cytometry A. 93 (1), 19-31 (2018).</li><li>Hendijani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture:+An+advantageous+method+for+isolation+of+mesenchymal+stem+cells+from+human+tissues.">Explant culture: An advantageous method for isolation of mesenchymal stem cells from human tissues.</a> Cell Prolif. 50 (2), 12334 (2017).</li></ol>

作者报告没有利益冲突。

MSC 代表了一个充满活力的研究领域，对再生医学具有深远影响22。它们的独特特性使它们成为科学探索的焦点，并有可能彻底改变各种疾病和伤害的治疗7。WJ-MSC 是 MSC 的一个独特子集，可以从位于血管间上皮和羊膜上皮之间的 UC 内的凝胶状结缔组织获得23。MSC 的临床应用在于获得足够的细胞数量24。然而，随着传代次数的增...

自 1991 年首次从沃顿商学院果冻 （WJ） 中分离出间充质干细胞 （MSC） 以来，这些多能干细胞因其再生特性和多系分化能力而受到研究人员的极大关注1。MSC 可以从各种来源分离，包括骨髓、外周血、牙髓、脂肪组织、胎儿（人类流产）和分娩相关组织2。脐带 （UC） 因其非侵入性、丰富的细胞产量和分化能力、高增殖率、分化潜能和免疫调节特性而成为一种很有前途的储存库3。胎儿 MSC 表现出很强的干性和免疫特性，使其成为过去二十年进行的临床试验和基础研究的主要焦点 2,4,5。与其他来源的 MSC（如骨髓或脂肪组织）相比，UC 来源的 MSC 具有卓越的治疗潜力 6,7。
UC 由羊膜上皮、三根血管（两条动脉和一条静脉）和称为 WJ3 的凝胶状物质组成。有趣的是，UC 构成了一个简单的脉管系统，仅由内皮和间皮组成，而不包括外膜;WJ 不含淋巴或神经8.UC 具有独特的结构，非常适合分段分离。UC-MSC 主要位于 WJ。MSCs 可以从 WJ 的不同隔室中分离出来，包括羊膜、亚羊膜（羊膜和亚羊膜也被指定为脐带膜区域）和 WJ 的血管周围区域8。WJ 的每个区域都有自己的结构、免疫组织化学特征和功能 3,6。
与来自其他地区的 MSC 相比，从 UC 的 WJ 分离的 MSC 被广泛认为具有更高的临床效用3。WJ-MSC 因其多线分化潜能、免疫调节特性、旁分泌作用、抗炎作用和免疫特权特性，已在临床前和临床环境中被广泛研究用于治疗各种疾病 2,3。WJ-MSC 已被证明在治疗一系列疾病方面具有前景，包括移植物抗宿主病 （GvHD）、移植物排斥反应、克罗恩病、自身免疫性疾病和心血管疾病 9,10,11,12,13,14。随着 WJ-MSCs 的临床需求不断增加，脐带供应短缺目前是其广泛应用的障碍。
WJ-MSC 的产量取决于用于细胞提取的方法15。虽然 WJ-MSC 可以通过外植体培养或酶消化分离，但后一种方法的传播时间较长，这可能会增加细胞损伤的风险并降低细胞活力16。然而，大量研究表明，外植体培养方法可提高细胞产量和活力，并且外植体组织释放的旁分泌因子也有助于促进细胞增殖17,18。
本研究采用独特的解剖方法获得整个 WJ，产生具有增强增殖能力、活力和数量的 MSCs，同时最大限度地减少对 WJ 的损害。这种创新方法为分离 WJ-MSC 提供了一种简化的策略，满足了 MSC 应用中的关键需求。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>APC anti-human CD44 Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>338806</td>
			<td></td>
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			<td>24-well cell culture plates</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
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			<td>APC anti-human CD73 (Ecto-5&#39;-nucleotidase) Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>344006</td>
			<td></td>
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			<td>APC Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend&#160;</td>
			<td>400122</td>
			<td></td>
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			<td>Autoclave</td>
			<td>HIRAYAMA</td>
			<td>HVE-50</td>
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			<td>Automatic Cell Counter</td>
			<td>Countstar</td>
			<td>FL-CD</td>
			<td></td>
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			<td>BAMBANKER Cryopreservation Solution</td>
			<td>Wako</td>
			<td>302-14681</td>
			<td></td>
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			<td>Cell Staining Buffer</td>
			<td>Biolegend&#160;</td>
			<td>420201</td>
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			<td>Centrifugal Machine</td>
			<td>Eppendorf</td>
			<td>5424R</td>
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			<td>Clean Bench</td>
			<td>Shanghai ZhiCheng</td>
			<td>C1112B</td>
			<td></td>
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			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>HERAcell 150i</td>
			<td></td>
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			<td>D-PBS</td>
			<td>Solarbio</td>
			<td>D1040</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro- thermostatic Blast Oven</td>
			<td>Shanghai JingHong</td>
			<td>DHG-9423A</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD105 Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>323204</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-human CD90 (Thy1) Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend&#160;</td>
			<td>400110</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry</td>
			<td>Beckman</td>
			<td>CytoFLEX</td>
			<td></td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Superior Marienfeld</td>
			<td>640410</td>
			<td></td>
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			<td>Intracellular Staining Permeabilization Wash Buffer (10&#215;)&#160;</td>
			<td>Biolegend&#160;</td>
			<td>421002</td>
			<td></td>
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			<td>Inverted Biological Microscope</td>
			<td>ZEISS</td>
			<td>Axio Vert. A1</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen Storage Tank</td>
			<td>Thermo Scientific</td>
			<td>CY50935-70</td>
			<td></td>
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			<td>Normal saline (NS)</td>
			<td>Meilunbio</td>
			<td>MA0083</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1032</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD11b Antibody</td>
			<td>Biolegend&#160;</td>
			<td>393112</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD19 Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>392506</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD34 Antibody</td>
			<td>Biolegend&#160;</td>
			<td>343606</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human CD45 Antibody</td>
			<td>Biolegend&#160;</td>
			<td>368510</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-human HLA-DR Antibody&#160;</td>
			<td>Biolegend&#160;</td>
			<td>307606</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG1, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend&#160;</td>
			<td>400114</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Mouse IgG2a, &#954; Isotype Ctrl (FC) Antibody</td>
			<td>Biolegend&#160;</td>
			<td>400214</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Electronic Balance</td>
			<td>Satorius</td>
			<td>PRACTUM313-1CN</td>
			<td></td>
		</tr>
		<tr>
			<td>Snowflake Ice Machine</td>
			<td>ZIEGRA</td>
			<td>ZBE 30-10</td>
			<td></td>
		</tr>
		<tr>
			<td>steriled 50 mL plastic tube</td>
			<td>Greniner</td>
			<td>227270</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic Water Bath</td>
			<td>Shanghai YiHeng</td>
			<td>HWS12</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 1:250</td>
			<td>Solarbio</td>
			<td>T8150</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraGRO-Advanced</td>
			<td>Helios&#160;</td>
			<td>HPCFDCGL50</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure and Pure Water Purification System</td>
			<td>Milli-Q</td>
			<td>Milli-Q Reference</td>
			<td></td>
		</tr>
		<tr>
			<td>Xeno-Free Human MSC Culture Medium</td>
			<td>FUKOKU&#160;</td>
			<td>T2011301</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of umbilical cord-derived mesenchymal stem cells with high yields and low damage

间充质干细胞 （MSC） 是一组具有显著再生和免疫调节特性的多能细胞。来自脐带 （UC） 的沃顿胶 （WJ） 作为 MSC 的杰出来源，在生物医学领域引起了越来越多的兴趣。然而，出现了供应有限和现有方法缺乏标准化等挑战。本文提出了一种通过从脐带解剖完整 WJ 来提高 MSC 产量的新方法。该方法采用平割去除上皮层，保持整个 WJ 的完整性，并导致收获的 MSC 的数量和活力增加。与传统的尖锐解剖方法相比，这种方法显着减少了 WJ 废物。为了确保 WJ-MSC 的纯度并最大限度地减少外部细胞影响，在翻转 UC 后利用内部张力剥离内皮。此外，在外植体培养过程中，培养皿短时间倒置，以改善粘附和细胞生长。比较分析证明了所提出的方法的优越性，与传统方法相比，WJ 和 WJ-MSCs 的产量更高，活力更好。两种方法中细胞表面标志物的相似形态和表达模式证实了它们在各种应用中的表征和纯度。该方法为 WJ-MSC 分离提供了一种高产量和高活力的方法，显示出 MSCs 临床应用的巨大潜力。

图 1 总结了收集和培养 UC-MSC 的程序及其后续分析。使用独特的方法将 UC 整齐地分为几个部分;主要流程的具体操作示意图如图 2 所示。常规监测和记录来自外植体培养物的细胞生长。在培养外植体后约 4 天观察到贴壁纺锤形细胞，并越来越多地爬行成涡状。这些细胞也被鉴定为 P0 并继续增殖以进行后续传代，同时在增殖和传代过程中保持均匀的大小?...

作者聚焦:干细胞来源的血管化类器官的进展和沃顿胶的优化分离以增强 MSC 生产

本研究提出了一种独特的钝性解剖程序，以保持沃顿胶 （WJ） 的完整性，从而减少受损的 WJ 并提高收获的间充质干细胞 （MSC） 的数量和活力。与传统的尖锐解剖方法相比，该方法表现出优异的产量和增殖能力。

高产量和低损伤分离脐带来源的间充质干细胞

Mesenchymal stem cells (MSCs) are a population of multipotent cells with remarkable regenerative and immunomodulatory properties. Wharton's jelly (WJ) ...

isolation-umbilical-cord-derived-mesenchymal-stem-cells-with-high

Research

JoVE Journal

Medicine

Isolation of Umbilical Cord-Derived Mesenchymal Stem Cells with High Yields and Low Damage

1.5K 次观看.  Beijing University of Chinese Medicine. 我们的研究使用人类干细胞来制造各种类器官。它们可以模仿人体器官。我们开发了独特的中胚层和内胚层共分化技术。它们使这些类器官具有丰富的血液网络。这些类器官将促进器官发育、再生医学、疾病模型、药物筛选和个性化医疗，例如类器官移植。我们的方案解决了从脐带中优化完整沃顿氏胶分离方面的研究空白，同时保留了其血管周...

视频: 作者聚焦:干细胞来源的血管化类器官的进展和沃顿胶的优化分离以增强 MSC 生产

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

胆固醇外流检测

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

科研

医学

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

瘦素受体<sup&gt;分贝</sup&gt; 2型糖尿病小鼠模型:胰岛分离和活细胞双光子成像完好胰岛

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

在锝-99m在马腱末端标记的骨髓间充质干细胞的体内成像和跟踪

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

丙戊酸在人脐带血源性 CD34 + 细胞造血干细胞体外扩增中的作用

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...