该研究得到了 Conselho Nacional de Desenvolvimento Científico e Tecnologico （480807/2011-6） 和米纳斯吉拉斯州佩斯基萨保护基金 （APQ-01237-11） 的资助。这项研究部分由 PROPP UESC （073.6764.2019.0021079-85） 资助。MGAG 和 URS 分别感谢 Fundação de Amparo à Pesquisa do Estado da Bahia （FAPESB） 和 Coordenação de Aperfeiçoamento de Pessoal de Nível de Nível （CAPES） 颁发的奖学金。

所有动物实验均根据国际动物伦理准则进行，并由圣克鲁斯州立大学护理和使用机构委员会根据协议编号021/22批准。瑞士雄性小鼠（6-8周）购自动物育种，维护和实验实验室 - 圣克鲁斯州立大学（LaBIO-UESC）动物研究设施，维持在特定的无病原体条件下，自由接受水和食物，12小时光/暗循环。
注意:可以使用其他小鼠谱系;我们建议使用成年小鼠（6-8周），因为它们具有更发达的脂肪组织和具有高增殖活性的细胞。
1. 准备工作
注意:在继续之前，请准备以下所述的试剂。有关试剂和材料供应商信息，请参阅 材料表 。在所有实际程序中都必须佩戴个人防护设备，例如口罩、帽子、实验室外套和手套。
<ol><li>附睾脂肪组织摘除术<ol><li>预留手术材料:三套无菌镊子和剪刀、五根针头和一个装有 200 mL 70% 乙醇的烧杯。</li>
		<li>准备终末麻醉剂，混合氯胺酮（100mg / kg）和甲苯噻嗪（10mg / kg）。</li>
	</ol></li>
	<li>ADSCs文化<ol><li>基础培养基:制备补充有 10% 胎牛血清 （FBS）、50 μg/mL 庆大霉素、0.25 μg/mL 两性霉素 B、100 U/mL 青霉素和 100 mg/mL 链霉素的 Dulbecco 改良 Eagle 培养基 （DMEM） 高葡萄糖培养基。</li>
		<li>消化溶液:在0.25μm过滤器中灭菌的PBS中制备0.15%的II型胶原酶。 注意:没有必要使用基础培养基中描述的四种抗生素。这将取决于进行此操作的实验室的细胞培养条件。</li>
	</ol></li>
	<li>ADSCs表型表征<ol><li>在PBS中制备1%牛血清白蛋白（BSA）。</li>
		<li>稀释表 1中提到的抗小鼠抗体。</li>
		<li>在PBS中制备2%的甲醛。</li>
	</ol></li>
	<li>ADSCs成骨分化<ol><li>成骨分化培养基:制备补充有 5.67 M 抗坏血酸、10 nM 地塞米松、0.02 M β-甘油磷酸盐、10% FBS 和 1% 青霉素/链霉素的 DMEM。</li>
		<li>对于 Von Kossa 染色，制备以下溶液:70% 乙醇水溶液、5% 硝酸银水溶液、蒸馏水、5% 硫代硫酸钠水溶液和 1% 曙红 B 水溶液。</li>
	</ol></li>
	<li>成脂分化:<ol><li>成脂分化培养基:制备补充有0.5mM 3-异丁基-1-甲基黄嘌呤，200μM吲哚美辛，1μM地塞米松，10μM胰岛素，10%FBS和1%青霉素/链霉素的DMEM。</li>
		<li>对于油红染色，制备以下溶液:10%福尔马林在去离子水中，60%异丙醇在去离子水中，溶血色素（脂溶性染料）重氮染料（油红O溶液）在60%异丙醇中，和1%苏木精在去离子水中。</li>
	</ol></li>
	<li>软骨分化:<ol><li>软骨分化培养基:制备补充有10%FBS和1%青霉素/链霉素的软骨形成培养基。</li>
		<li>在PBS中制备10%的甲醛。</li>
		<li>对于蛋白聚糖和糖胺聚糖染色，制备以下溶液:杂聚糖染色剂（阿尔新蓝 1%）在乙酸、pH 2.5、石蜡、苏木精、乙醇系列水中稀释（100%、96%、80% 和 70%）。</li>
	</ol></li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>抗体/荧光基团</td>
			<td>稀释</td>
			<td>克隆</td>
			<td>终浓度 （μg/mL）</td>
			<td colspan="3">表达它的函数和细胞</td>
		</tr><tr><td>CD34-聚乙烯</td>
			<td>1:100</td>
			<td>内存34</td>
			<td>2</td>
			<td colspan="3">细胞粘附因子。造血干细胞。</td>
		</tr><tr><td>CD45-装甲运载火箭</td>
			<td>1:100</td>
			<td>30-F11型</td>
			<td>2</td>
			<td colspan="3">协助激活白细胞。在白细胞中表达。</td>
		</tr><tr><td>CD71-异硫氰酸荧光素</td>
			<td>1:100</td>
			<td>C2型</td>
			<td>5</td>
			<td colspan="3">控制细胞增殖过程中的铁摄取。增殖细胞、网织红细胞和前体细胞。</td>
		</tr><tr><td>CD29-异硫氰酸荧光素</td>
			<td>1:100</td>
			<td>公顷2/5</td>
			<td>5</td>
			<td colspan="3">粘附和活化、胚胎发生、白细胞、树突状细胞、血小板、肥大细胞、成纤维细胞和内皮细胞。</td>
		</tr><tr><td>CD90-PerCP</td>
			<td>1:100</td>
			<td>OX-7 （氧-7）</td>
			<td>2</td>
			<td colspan="3">信号，粘附。T淋巴细胞、NK、单核细胞、造血干细胞、神经元和成纤维细胞。</td>
		</tr></tbody></table>表 1:用于通过流式细胞仪对 ADSC 进行表型表征的抗体。 抗体列表，包括它们各自的荧光染料、稀释度、克隆和终浓度，以及它们在表达的细胞中的功能。
2. 方法
注意:脂肪组织分布在全身皮下或腹腔内位置。在小鼠中，用于实验的最常见脂肪组织包括皮下附睾、肠系膜和腹膜后22。这里显示了获得附睾脂肪组织的步骤（图1）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65722/65722fig01.jpg"> 图 1:从瑞士小鼠获得脂肪组织来源的干细胞的实验设计。 （A） 用针头将动物放在软木塞或聚苯乙烯泡沫塑料板上;（B） 用镊子提起皮肤，在腹部中央切开一个切口，然后将皮肤从腹膜上分离;（三）识别附睾上方的白色脂肪组织;（D）将组织转移到补充有10%FBS和1%抗生素的DMEM培养基中，用PBS彻底洗涤附睾脂肪组织，并将组织转移到消化液中;（E），将组织碎片和（F）在37°C下孵育1小时，每10分钟剧烈摇动一次;（G）计数细胞后，将板置于6孔板中，并在倒置显微镜下观察细胞形态。（F） 细胞形态将从圆形变为成纤维细胞样。比例尺 = 22.22 μm. <a href="https://www.jove.com/files/ftp_upload/65722/65722fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<ol><li>附睾脂肪组织摘除术 注意:请注意，所有程序都必须在无菌条件下在层流柜中进行，以确保纯净和无污染的细胞培养。<ol><li>通过腹膜内途径使用氯胺酮和甲苯噻嗪溶液（100和10mg / kg，如步骤1.1.2所述）对动物实施安乐死。 注意:可以使用其他安乐死方法23.通过确认没有心跳来确保动物已经死亡。</li>
		<li>将动物腹侧朝上放在覆盖有铝箔的软木或聚苯乙烯泡沫塑料板上，并使用无菌针头固定（图1A）。 注意:为避免污染，请在腹部区域喷洒 70% 的酒精。或者，用手术刀剃掉头发。</li>
		<li>用镊子在腹部区域中央提起皮肤，然后用无菌剪刀剪开。 注意: 为避免污染，需要两套无菌镊子和剪刀。第一种用于打开动物，切开皮肤和腹膜层;第二个用于拾取附睾脂肪组织。此外，在烧杯中保留 150 mL 70% 酒精，以便在两步之间清洁剪刀和镊子。</li>
		<li>将剪刀插入动物的皮肤下，然后打开它以将其与腹膜分离（图1B）。用镊子提起腹膜层，并用无菌剪刀剪开。</li>
		<li>使用另一套无菌镊子和剪刀，识别睾丸上方的附睾和附睾上方的白色脂肪组织。使用约2厘米大小的无菌镊子拉动整个附睾脂肪组织，并在非常靠近附睾的地方切开（图1C）。</li>
		<li>将脂肪放入含有基础培养基的50mL无菌锥形管中，并将其置于冰上（图1D）。在引擎盖中，继续执行下面所述的后续步骤。</li>
	</ol></li>
	<li>脂肪组织来源的干细胞 （ADSC） 分离 注意:在生物 II 类层流罩中处理样品，人员应穿实验室外套、手套和外科口罩。<ol><li>用PBS彻底清洗附睾脂肪组织。 注意:此步骤对于从样本中去除血液和其他颗粒至关重要。血液会抑制II型胶原酶并损害实验。</li>
		<li>使用无菌镊子，将附睾脂肪组织转移到含有 15-20 mL 消化溶液的 50 mL 管中，按步骤 1.2.2 制备。使用无菌剪刀将组织切碎，并将组织在37°C孵育1小时（图1E，F）。 注意:此步骤可以使用37°C的水浴或培养箱。每 10 分钟，必须剧烈摇晃悬浮液以促进消化。不要延长1小时的孵育时间。</li>
		<li>通过在基础培养基中1:1稀释样品来灭活胶原酶，并在4°C下以250× g 离心悬浮液10分钟以获得基质的维管部分。弃去上清液，将沉淀重悬于 1 mL 基础培养基中。</li>
		<li>在Neubauer室中使用1:10或1:100的稀释度，通过台盼蓝染料排除法检查细胞活力并计数细胞。 注:预期产量约为 0.5-100 万个细胞/g 处理过的脂肪组织，活力为 80%。</li>
		<li>将分离的细胞（0.3-50 万）接种在 6 孔板的一个孔中的 3 mL 基础培养基中。将细胞在37°C与5%CO2孵育过夜。</li>
		<li>第二天:从孔中取出培养基，用PBS洗涤细胞。加入 3 mL 基础培养基。每 2 天重复一次此过程，直到细胞达到 90% 汇合。 注意:始终在倒置显微镜下观察细胞的形态，从圆形变为成纤维细胞样（图1H）。</li>
	</ol></li>
	<li>脂肪组织来源的干细胞 （ADSC） 扩增 注意:一旦细胞生长~90%汇合，就按照下述方式进行传代培养。<ol><li>从孔中取出培养基，用PBS洗涤细胞。加入0.5mL /孔的胰蛋白酶/ EDTA（0.05%胰蛋白酶;200mg / L EDTA），并在37°C下孵育板3-5分钟以分离细胞。 注意:在胰蛋白酶/EDTA 中不要超过 5 分钟。细胞的完全脱离应在倒置显微镜下验证。可以通过小心地上下移液或轻敲板的侧面来加速该过程。</li>
		<li>通过在补充有 10% 胎牛血清 （FBS） 和 1% 抗生素的 DMEM 中以 1:1 的比例稀释样品来灭活酶。</li>
		<li>将细胞悬液分成 2 个孔，加入 3 mL 基础培养基（DMEM，补充有 10% FBS 和 1% 抗生素）。在37°C下用5%CO2孵育过夜。</li>
		<li>第二天更换培养基，然后每 2 天更换一次，直到汇合度达到 90%。</li>
		<li>重复该过程直到第三次传代，然后进行表型和功能表征。 注意:始终在倒置显微镜下观察细胞的形态，从圆形变为成纤维细胞状。</li>
	</ol></li>
	<li>脂肪组织来源的干细胞 （ADSC） 表征<ol><li>通过流式细胞术进行表型表征<ol><li>使用胰蛋白酶/EDTA 分离细胞，如步骤 2.3.1 至 2.3.2 中所述。</li>
			<li>将细胞收集在锥形管（50mL）中，并在4°C下以250× g 离心10分钟。 弃去上清液，将沉淀重悬于 1 mL PBS 中。</li>
			<li>如步骤 2.2.4 中所述计数细胞，并在 96 孔板中的 100 μL PBS 中接种 0.5-1 x10 6 个细胞。 注意:孔的数量将取决于荧光染料偶联物的颜色和细胞仪的滤光片/激光器。</li>
			<li>加入在PBS中的1%BSA稀释的抗体（表1）。 注意:没有必要使用 Fc 阻滞剂来避免非特异性结合，因为我们已经使用 BSA 来稀释抗体。保留一个未接收抗体的细胞样本，这些抗体将用作染色对照。根据实验室中可用的荧光染料和细胞仪，抗体可以在同一孔中结合。 表 1 中描述的所有抗体都可以在同一孔中添加，但 CD71 FITC 和 CD29 FITC 除外，由于荧光染料相同，它们必须在不同的孔中。</li>
			<li>将板在4°C下在黑暗中孵育30分钟。</li>
			<li>洗涤细胞，然后使用PBS完成体积至200μL，在4°C下以252× g 离心板10分钟，并弃去上清液。再次重复此步骤。</li>
			<li>通过在 PBS 中每孔加入 200 μL 2% 甲醛来固定细胞。将细胞储存在4°C的黑暗中，直到在流式细胞仪中进行分析。 注意:为获得最佳结果，请尽快在流式细胞仪上采集细胞。对于大多数荧光染料，标记物的荧光亮度在48小时后显着衰减。因此，不要超过 48 小时来读取板。建议采集 30,000 个事件。</li>
			<li>使用 图 2B 中所示的门控策略来选择 ASC 群体。</li>
		</ol></li>
		<li>功能表征:成骨分化<ol><li>使用胰蛋白酶/ EDTA分离细胞（如步骤2.3.1至2.3.2中所述），收集并离心细胞（如步骤2.4.1.2和2.4.1.2中所述），并计数它们（如步骤2.2.4中所述）。</li>
			<li>板，一式三份，每孔 2 x 105 个细胞，位于基底内侧的 6 孔板中（DMEM 补充有 10% FBS 和 1% 抗生素）;在37°C的5%CO2中孵育。 注意:需要两个 6 孔板，每个分化时间（14 天和 21 天）一个。</li>
			<li>第二天，将培养基更换为成骨培养基（步骤1.4.1）。 注意:保留 3 个孔仅用基础培养基培养，这将是分化的对照。</li>
			<li>在成骨培养基和对照细胞中培养细胞 14 天和 21 天，每 3 天更换一次培养基。继续进行Von Kossa染色（步骤1.4.2）以评估每个培养期结束时的矿化结瘤。</li>
			<li>从每个孔中吸出培养基，并用PBS洗涤细胞两次。在室温 （RT） 下用 2 mL 70% 乙醇固定细胞过夜。在流水中小心清洗细胞。</li>
			<li>加入 2 mL 5% 硝酸银溶液，并将板在紫外线下孵育 1 小时。用蒸馏水清洗细胞。加入 2 mL 5% 硫代硫酸钠，孵育 5 分钟。用蒸馏水清洗。</li>
			<li>用 2 mL 曙红复染 40 秒。用蒸馏水洗涤，直到去除染色溶液，并使用光学显微镜观察染色。</li>
		</ol></li>
		<li>功能表征:成脂分化<ol><li>使用胰蛋白酶/ EDTA分离细胞（如步骤2.3.1至2.3.2中所述），收集并离心细胞（如步骤2.4.1.2和2.4.1.2中所述），并计数它们（如步骤2.2.4中所述）。</li>
			<li>在基底内侧的 6 孔板中每孔板 2 x 105 个细胞（DMEM 补充有 10% FBS 和 1% 抗生素）;在37°C的5%CO2中孵育。 注意:需要两个 6 孔板，每个分化时间（14 天和 21 天）一个。</li>
			<li>第二天，将培养基更换为脂肪生成培养基（步骤2.5.1）。 注意:保留 3 个孔，仅使用基础培养基作为对照进行培养。</li>
			<li>在成脂培养基中培养细胞 14 天和 21 天，每 3 天更换一次培养基。在每个培养期结束时，进行油红O染色（步骤2.4.3.5-2.4.3.7），这是细胞内脂质积累的指示剂。</li>
			<li>从每个孔中吸出培养基。用PBS洗涤细胞两次。将细胞固定在 2 mL 10% 福尔马林中 1 小时。用PBS清洗一次，然后用水清洗一次。</li>
			<li>用 2 mL 油红 O 溶液在 60% 异丙醇中染色 5 分钟。用蒸馏水清洗一次。</li>
			<li>用 2 mL 1% 苏木精在蒸馏水中按 1:2 稀释 1 分钟进行复染。用蒸馏水洗涤直至去除染色溶液，并使用光学显微镜观察染色的样品。</li>
		</ol></li>
		<li>功能表征:软骨分化<ol><li>使用胰蛋白酶/ EDTA分离细胞（如步骤2.3.1至2.3.2中所述），收集并离心细胞（如步骤2.4.1.2和2.4.1.2中所述），并计数它们（如步骤2.2.4中所述）。</li>
			<li>将 5 x 105 ADSC 放入四个聚丙烯锥形管中，并以 450 x g 离心 5 分钟以形成沉淀。丢弃上清液。</li>
			<li>在软骨形成培养基（步骤1.6.1）或仅基础培养基（分化控制）中培养锥形管中的沉淀14和21天，在37°C的5%CO2中培养14和21天。每 3 天更换一次培养基，避免去除颗粒。 注:每个细胞沉淀是指每个培养时间，以及它们各自的对照品，仅用基础培养基生长。</li>
			<li>在每个培养时间点结束时，使用细尖镊子收集沉淀，并将它们置于 24 孔板中。继续进行蛋白聚糖和糖胺聚糖的组织学切片和染色（步骤2.4.4.5.-2.4.4.9）。 注意:每个颗粒都在一个单独的孔中处理。</li>
			<li>将沉淀固定在 2 mL 10% 缓冲甲醛中 30 分钟。弃去甲醛溶液，加入 2 mL 70% 乙醇。使用尖端从锥形管中取出颗粒，并将颗粒嵌入石蜡中。</li>
			<li>使用旋转石蜡切片机，制作 5 μm 组织学切片。将切片在60°C孵育15分钟，并将它们浸入二甲苯中两次（5分钟和15分钟）。</li>
			<li>通过将组织学切片浸入浓度降低（100%，96%，80%和70%）的酒精浴中各2分钟，然后用去离子水冲洗5分钟，使样品再水化。</li>
			<li>继续用1%的阿尔新蓝在乙酸（pH 2.5）中对样品进行染色30分钟。</li>
			<li>用苏木精复染1分钟，并用蒸馏水洗涤。用 DPX（组织学封片剂）或其他封片溶液封片，并使用光学显微镜观察样品。</li>
		</ol></li>
	</ol></li>
</ol>

来自小鼠附睾的脂肪组织来源干细胞的原代培养

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作者没有什么可透露的。

间充质干细胞可以从不同的组织中提取。尽管骨髓在小鼠和人类中都是间充质干细胞的常见来源25,26，但我们选择在这项研究中使用脂肪组织，因为它富含间充质干细胞、在体内的分布以及易于获取。作为一种替代方案，脂肪来源的干细胞可用于产生MSCs样永生化细胞27。
对于此处共享的协议的成功，提取的一些要?...

间充质干细胞 （MSC） 是分化成成骨细胞、软骨母细胞和脂肪细胞等细胞的成体多潜能细胞 1,2。这些细胞存在于多个器官中，因此，它们可以从骨髓、肌肉、脂肪、毛囊、牙根、胎盘、真皮、软骨膜、脐带、肺、肝脏和脾脏等成人组织中提取 3,4。
间充质干细胞对生理学和免疫系统的影响已有报道 5,6。这些细胞在人类和兽医学中都有望治疗多种疾病。间充质干细胞可以通过不同的机制控制炎症并促进血管生成和组织稳态，例如细胞间接触、可溶性因子和小细胞外囊泡 7,8,9,10。此外，间充质干细胞是免疫特权细胞，能够在免疫不相容的同种异体移植受体中存活，因为这些细胞显示出 I 类主要组织相容性复合体 （MHC） 分子的低表达，并且用于基于细胞的同种异体移植疗法11,12。低免疫原性与再生潜力相结合，使间充质干细胞成为细胞疗法的理想候选药物，例如移植物抗宿主病 （GvHD）13、系统性红斑狼疮 （SLE）14 和多发性硬化症15 等16,17。
尽管间充质干细胞存在于几种成体组织中，但脂肪组织比其他来源具有优势，例如易于收获，手术干预最少;可用电池数量多，扩增率高;使用易于执行的方案轻松进行体外扩增，无需特定设备和低成本材料 18,19,20。一旦提取，脂肪组织来源的干细胞 （ADSC） 必须按照国际细胞治疗学会 （ISCT） 21 的规定进行表征。因此，间充质干细胞必须表现出形态成纤维细胞样，对塑料培养物的粘附，表达高百分比 （≥95%） 的间充质标志物，如内分泌素 （CD105）、胞外-5'-核苷酸酶 （CD73） 和 Thy-1 （CD90），以及低百分比 （≤2%） 的造血标志物，如白细胞共同抗原 （CD45）、跨膜磷酸糖蛋白 （CD34）、糖脂锚定膜糖蛋白 （CD14）、整合素 α M （CD11b）、B 细胞抗原受体复合物相关蛋白 α 链 （CD79α） 或B 淋巴细胞表面抗原 B4 （CD19） 和 II 类人类白细胞抗原 （HLA-II）。此外，还需要进行功能表征，并且细胞应该能够分化为成骨细胞、软骨母细胞或脂肪母细胞21。
在这里，我们展示了如何使用机械解离和酶消化从附睾脂肪组织获得MSCs，用于 体外 研究以及ISCT预先确定的形态学表征。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>140 &#176;C High Heat Sterilization CO2 Incubator</td>
			<td>RADOBIO SCIENTIFIC CO. LTD, China</td>
			<td>C180</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1748</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>A9186</td>
			<td>BioReagent, suitable for detection of glycoproteins. 1% in acetic acid, pH 2.5</td>
		</tr>
		<tr>
			<td>Alcohol 70%</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>65350-M</td>
			<td>70% in water</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1662</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD29 FITC (Clone Ha2/5)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>555005</td>
			<td>Functions in the cell: Adhesion and activation, embryogenesis, Leukocytes, DC, platelets, mast cells, fibroblasts and endothelial cells</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD34 PE (Clone RAM34)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>551387</td>
			<td>Functions in the cell: Cell adhesion factor. Hematopoietic stem cells</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD45 APC (Clone 30-F11)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>559864</td>
			<td>Functions in the cell: Assists in the activation of leukocytes</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD71 FITC (Clone C2)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>553266</td>
			<td>Functions in the cell: Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes and precursors</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD90 PerCP (Clone OX-7)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>557266</td>
			<td>Functions in the cell: Signaling, adhesion. T lymphocyte, NK, monocyte, HSC, neuron, fibroblast</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic pipettes</td>
			<td>Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td>4700850N</td>
			<td>Finnpipette F1 Good Laboratory Pipetting (GLP) Kits</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (6-well)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z707759</td>
			<td>07 units sterile. TPP tissue culture plates</td>
		</tr>
		<tr>
			<td>Cell culture plates (96-well. Round or V bottom)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>CLS353077</td>
			<td>01 unit sterile. Wells, 96, Tissue Culture (TC)-treated surface, round bottom clear wells, sterile</td>
		</tr>
		<tr>
			<td>Chondrogenic medium</td>
			<td>Stem Pro Chondrogenesis Differentiation&#8211;Life Technologies</td>
			<td>A1007101</td>
			<td>TGF-&#946;2, TGF-&#946;3, dexamethasone, insulin, transferrin, ITS, sodium-l - ascorbate, sodium pyruvate, ascorbate-2-phosphate</td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Life Technologies, California, USA</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>cork or styrofoam board covered with aluminum</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>cotton</td>
			<td>Not applicable</td>
			<td></td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor</td>
			<td>Not applicable</td>
			<td></td>
			<td>03 units sterile</td>
		</tr>
		<tr>
			<td>DPX Mountant for histology</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>6522</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>D5523</td>
			<td>With 1000 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Eosin B</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>861006</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>47608</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle (0.3mm x 13mm)</td>
			<td>Not applicable</td>
			<td></td>
			<td>5 units</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I0200000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I3536</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>563935</td>
			<td>70% in H2O</td>
		</tr>
		<tr>
			<td>Ketamine-D4 hydrochloride solution</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>K-006</td>
			<td>1.0 mg/mL in methanol (as free base), certified reference material, Cerilliant&#174;</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>BR718620</td>
			<td>BRAND counting chamber BLAUBRAND Neubauer pattern. With clips, double ruled</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (0.1&#8211;10 &#956;L)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z645540</td>
			<td>Volume range 0.1&#8211;10 &#956;L, elongated, bulk pack. Sterile</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (1&#8211;10 mL)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z717401</td>
			<td>Volume range 1&#8211;10 mL, universal, bulk pack. Sterile</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (200 &#956;L)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z645516</td>
			<td>Maximum volume 200 &#956;L, graduated, ministack. Sterile</td>
		</tr>
		<tr>
			<td>Oil-Red O solution</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>O1391</td>
			<td>0.5% in isopropanol</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>107.151</td>
			<td>46&#8211;48, in block form</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>P4333</td>
			<td>Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 &#956;m filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline solution 1x (PBS).</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>P3813</td>
			<td>Powder, pH 7.4, for preparing 1 L solutions. Balanced and sterile</td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes (15 mL)</td>
			<td>Falcon, Fisher Scientific</td>
			<td>14-959-53A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes (50 mL)</td>
			<td>Falcon, Fisher Scientific</td>
			<td>14-432-22</td>
			<td>2 units sterile</td>
		</tr>
		<tr>
			<td>scalpel (optional)</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>85228</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>72049</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tweezer (15 cm)</td>
			<td>Not applicable</td>
			<td></td>
			<td>3 units sterile</td>
		</tr>
		<tr>
			<td>Swiss male mice (6&#8211;8 weeks)</td>
			<td>Bioterium, Santa Cruz State University</td>
			<td>021/22</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe (1 mL)</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Trypan Blue Dye</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>T8154</td>
			<td>0.4%, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (ethylenediaminetetraacetic acid)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR3263</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>G9422</td>
			<td>BioUltra, suitable for cell culture, suitable for plant cell culture, &#8805;99% (titration)</td>
		</tr>
	</tbody>
</table>

isolation, in vitro expansion, and characterization of mesenchymal stem cells from mouse epididymal adipose tissue

间充质干细胞 （MSCs） 作为一种新的治疗方法已被广泛研究，主要用于阻止炎症加剧，因为它们具有调节免疫反应的潜力。间充质干细胞是一种免疫特权细胞，能够在免疫互容的同种异体移植受者中存活，这些受体基于 I 类主要组织相容性复合物 （MHC） 分子的低表达以及使用基于细胞的疗法进行同种异体移植。这些细胞可以从多种组织中分离出来，最常用的是骨髓和脂肪组织。我们提供了一种简单的方案来分离、培养和表征小鼠附睾脂肪组织中的 MSC。附睾脂肪组织通过手术切除，物理破碎，并用 0.15% II 型胶原酶溶液消化。然后，在 体外培养和扩增原代脂肪组织来源的干细胞（ADSC）细胞，并通过流式细胞术进行表型表征。我们还提供了将ADSC分化为成骨细胞、产脂细胞和软骨生成细胞的步骤，然后对每个细胞谱系进行功能表征。此处提供的方案可用于 体内 和 离体 实验，作为替代方案，脂肪来源的干细胞可用于产生MSCs样永生化细胞。

根据此处介绍的方案从脂肪组织中提取的细胞显示出符合ISCT提出的MSCs最低标准的形态。该协议的概述如图 1 所示。从表型上看，ADSCs在细胞培养的第一天表现出对塑料和成纤维细胞样形态的粘附（图2A）。此外，它们均匀生长并形成殖民地。此外，ADSCs的造血标志物CD34（2.12%）和CD45（1.81%）表达低，CD71（42.6%）、CD29（74.2%）和CD90（55.1%）均为间充质细?...

Read this Scientific Article Protocol about Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells at JoVE.com

作者聚焦:利用脂肪来源的干细胞推进治疗方法

脂肪组织是间充质干细胞的极好来源。在这里，我们带来了从瑞士小鼠附睾脂肪组织中逐步提取、培养和表征脂肪组织衍生干细胞 （ADSC） 的过程。

小鼠附睾脂肪组织中间充质干细胞的分离、体外扩增和表征

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

我的研究重点是脂肪来源的间充质干细胞，作为一种与其他疗法相关或不相关的新方法，以改善几种疾病的炎症。我们证明，使用脂肪间充质干细胞与吡喹酮联合使用可显着控制曼氏血吸虫感染的肉芽肿反应，突出了这种联合治疗方法的优点。我们的方案解决了在无菌控制下细致收集附睾脂肪组织的研究空白，即涉及剧烈摇晃的物理处理，并确定了利用脂肪来源的干细胞的最佳时间。我们的方案具有显著的优势，特别是丰富的可用性和分离间充质干细胞的潜在强度。此外，间充质干细胞具有强大的治疗特性，包括抗炎和组织再生能力。

isolation-vitro-expansion-characterization-mesenchymal-stem-cells

Research

JoVE Journal

Biology

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

598 次观看.  State University of Santa Cruz - UESC. 我的研究重点是脂肪来源的间充质干细胞，作为一种与其他疗法相关或不相关的新方法，以改善几种疾病的炎症。我们证明，使用脂肪间充质干细胞与吡喹酮联合使用可显着控制曼氏血吸虫感染的肉芽肿反应，突出了这种联合治疗方法的优点。我们的方案解决了在无菌控制下细致收集附睾脂肪组织的研究空白，即涉及剧烈摇晃的物理处理，并确定了利用脂肪来源的干细胞的?...

视频: 作者聚焦:利用脂肪来源的干细胞推进治疗方法

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

成人内皮细胞集落形成祖细胞的分离和大型规模扩张

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

科研

生物学

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

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

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

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

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 &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

从人类Lipoaspirates脂肪来源干细胞的手动隔离

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

用于细胞表征和分泌特征的人类脂肪组织微分裂

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...