作者要承认并感谢那些额外的研究人员促成所描述的协议和先进的结构陶瓷，其隔离的发展，包括:H.博士彼得·洛伦茨博士，博Muzuno博士，医学博士，杰里黄教授，医学博士博士亚当·卡茨博士，威廉·富特雷尔博士，医学博士荣Zhang博士，渠务署署长，博士，拉里萨·罗德里格斯博士，医学博士，阿方索·泽尼博士，博士，博士和约翰·弗雷泽博士。给出的结果进行资助，一部分，由美国国立卫生研究院，包括NIAMS和NIDCR研究院的研究经费。

这里显示的协议描述的ASC的使用酶消化和差速离心通过整容手术获得lipoaspirates手动隔离。该协议最初是发表在该杂志组织工程于2001年1，其中所产生的细胞被称为，因为从lipoaspirates他们的隔离处理脂肪抽吸物细胞或PLA细胞。然而，长期解放军电池现已替换为术语脂肪来源的干细胞或先进的结构陶瓷 ，让外地在命名方面的某 ​​种一致性。细胞通过本协议中分离已显示由许多具有在体外和体内中胚层电势（综述见4）。此外，ASC的外胚层和内胚层的电位也被探讨4。隔离技术是简单明了，需要大约一个小时才能完成。所得到的细胞s的标准组织培养条件下培养。随着培养时间，ASC的人口变得更均匀，由成纤维细胞，扩张容易，表现出良好的生存能力在长期的文化。 注:所需设备的以下部分被列在本文的结尾。所有的玻璃器皿，培养基和试剂应通过高压灭菌或消毒，通过0.22微米过滤器过滤。无菌组织培养技术应遵循在任何时候。为了确保这一点，放置在生物安全柜中的所有材料应当通过喷雾用70％的乙醇溶液中，用干净的纸巾擦拭进行消毒。 1。收获前，请准备以下: <ol><li>无菌1X磷酸缓冲盐水（PBS）的lipoaspirates的洗涤。准备大约1升的1×PBS为每100毫升脂肪抽吸物中。高压灭菌的PBS中，所有的OW在使用前冷却至室温。作为一个选项，抗生素/抗真菌剂可被添加到终浓度为:100 IU / ml青霉素，100微克/毫升链霉素和2.50微克/毫升两性霉素B，一旦PBS中冷却。 </li><li>无菌0.075-0.1％胶原酶IA型 （ 溶组织梭菌 ）的脂肪抽吸物的酶消化。浓度将取决于胶原酶的质量和来源- 即原油与纯化。其他类型的胶原酶，可以使用（ 例如 II型胶原酶或IV）在相似的浓度。使用千毫升过滤系统用0.22μm的过滤器（ 图1A）中的1×PBS和无菌过滤器做好准备。然而，也可以使用配有瓶顶过滤器无菌的玻璃瓶中。准备和使用在室温下。胶原酶溶液可被存储在短期在4℃下直到需要。不要冷冻胶原酶溶液，因为它可能会减少它的活动。 </li><li>无菌控制培养基（CM）的灭活胶原酶和ASC人口的培养。为500毫升的CM，结合以下内容:500毫升的DMEM（4.5 g / L的葡​​萄糖，含L-谷氨酰胺），加入50ml胎牛血清（加热灭活），5 ml青霉素/链霉素（10,000 IU青霉素，10,000μg/ ml的链霉素）。作为一个选项，将5ml两性霉素B（250微克/毫升两性霉素B），也可加入。如果非无菌试剂用于需要此媒体的无菌过滤。放置在CM中使用地暖介质前37℃水浴中30分钟。 </li><li>无菌玻璃器皿和塑料器皿隔离 。高压灭菌的1000ml的玻璃烧杯中，并允许在使用前冷却至室温。此外，具有下列塑料器皿准备:无菌血清移液管（5毫升，10毫升和25ml），50ml离心管中，和组织培养处理过的培养皿上。 </li></ol> 2。由L隔离先进的结构陶瓷的ipoaspirates 最lipoaspirates被收集在一个容器中抽吸（ 见图1B），并且可以由三个不同的层组成:油1）的上层由于成熟脂肪细胞的裂解，2）中间层的脂肪组织，以及3）一底部，含液体清液盐水和污染的细胞类型，如红血细胞（红细胞）。顶部油层可能不存在于显著金额。如果存在的话，它应该被吸入作为所得到的ASC培养的油污染可能会影响培养物的存活率。底部清液前应处理删除，以减少ASC文化与红细胞的污染。这将使中间，脂肪组织层，然后将清洗和酶消化解放其细胞， 间质血管分数（SVF）。 <ol><li> 洗完脂肪抽吸物:打开在组织培养h时脂肪抽吸物容器OOD和精心倒出脂肪抽吸物进入无菌1升的玻璃烧杯中。允许的脂肪抽吸物的层分开，直至脂肪组织层被很好地分离（ 图1B）。 <ol><li>吸脱油层（如果存在）使用无菌玻璃移液管和底部盐水清液用10ml的血清吸液管。这将删除更大的大多数污染的红血细胞和生理盐水。 </li><li>评估所得到的脂肪组织层的体积，并加入无菌1×PBS中以相等的体积。用搅拌以混合脂肪抽吸与PBS用于吸吸管吸出物。让这两个层来解决。如上面所指出的，1×PBS中可补充有抗生素/抗真菌剂。 </li><li>用10毫升血清吸管吸出清液。 </li><li>继续按照步骤2.1.2概述洗脂肪组织部分。和2.1.3。直至脂肪层有一个黄色/金色。第吸Ë清液最后一面，留下的脂肪组织部分在玻璃烧杯中。为了确保足够的去除清液中，让脂肪抽吸物层后，最后一次洗涤和最后愿望之前结清5分钟。 </li></ol></li><li> 在脂肪抽吸物的酶消化:准备无菌胶原酶1A的解决方案作为一个过滤单元上文所述。准备一个容积等于该脂肪级分和无菌过滤器。 <ol><li>继胶原酶溶液的过滤，弃去上层过滤单元，仔细倒入洗净的脂肪比例进入胶原酶溶液然后拧紧瓶盖。 </li><li>在37℃水浴中放置胶原酶/脂肪混合物，并消化在37℃30分钟 。轻轻摇动胶原酶/脂肪混合物，每次5-10分钟，放回水浴。脂肪组织层应该采取一种"更流畅"的外观（FIGURË1B）为消化募集资金。额外的消化时间（ 即最多2小时），可如果脂肪组织层似乎仍然有在它坚固件肥使用。 </li></ol></li><li> 隔离的ASC的:将胶原酶消化/脂肪混合物放回生物安全柜。一定要消毒用70％乙醇，过滤器单元放置在背面机壳之前。 <ol><li>吸取25毫升等分含有SVF到无菌50ml离心管中清液的 。 </li><li>加25毫升CM的各管中。允许CM，在室温下在机柜温育5分钟以灭活胶原酶。 </li><li>离心10分钟，在1200×g离心 ，收集SVF作为粒料。 </li><li>在生物安全柜中，抽吸从每个管中的上清液。确保顶部油层和任何浮动脂肪细胞吸出这上清液（ <str翁&gt;图1B）。 </li><li>结合SVF颗粒进入使用30毫升CM 1离心管中，并平分了两个新的50 ml离心管中。离心机如在步骤2.3.3。并从2 SVF粒料（在图中未示出）吸出上清液。 可选:如果红细胞裂解需要，悬浮每个SVF沉淀在10ml的RBC裂解缓冲液 （160 mM的NH 4 Cl）的， 并在室温下孵育10分钟。离心机如在步骤2.3.3。和吸出上清液，得到SVF小球（在图中未示出）。 </li><li>结合这两个SVF粒料成使用5-10毫升CM（图1B）1新的离心管中。 </li><li>过滤掉较大的组织颗粒，移液管重悬浮沉淀的SVF到一个100微米的筛网过滤器放置在一个新的50毫升离心管的顶部，并允许通过重力流进行过滤。 </li><li>的悬浮等分移液器（过滤），其中包含了先进的结构陶瓷到组织培养处理的培养皿SVF颗粒。补充每道菜与CM的适当的音量。 </li><li>培养的细胞在CM中3-4天，在37℃，5％CO 2的无介质变化。 </li></ol></li></ol>根据这一初步培养时间，培养基可能会被吸入和任何污染的红血细胞，用无菌1XPBS洗轻轻取出。更换CM，并根据需要不断培养的ASCs。用于组织培养皿和如何再悬浮的SVF沉淀物中这些菜划分的类型将取决于细胞所需的常规组织培养以下的数量。 3。 ASC的人口特征:流式细胞术及多向分化一旦分离，应执行所述ASC人口的表征。传统的方法为这个包括流式细胞metry来表征细胞的细胞表面的CD抗原更新和体外分化，以确认它们的多向分化的能力。虽然多种细胞可以在先进的结构陶瓷进行评估，该协议勾勒的ASCs分化成脂肪细胞，成骨和软骨细胞谱系的细胞作为确认多向中胚层的潜力5的一种手段。 在先进的结构陶瓷的体外多向分化<ol><li> 先进的结构陶瓷的成骨分化:在此之前归纳，编写成骨培养基（OM），如下所示:要将一个一瓶500毫升的DMEM（4.5克/毫升的葡萄糖）加到25.0毫升青霉素/链霉素的FBS（5.0％终浓度）和5.0ml （10,000 IU / ml和10,000 mg / ml的终浓度，分别）。对此，添加下面的成骨诱导剂给予指示的终浓度地塞米松（0.1微米决赛），L-抗坏血酸-2 - 磷酸（50.0微米决赛）和β-磷酸甘油（10.0毫最终）。 <ol><li>在诱导前，收获和板培养的ASCs成所需的组织培养皿中，使约50％汇合的实现。对于每一个实验皿镀金，镀额外的菜非诱导控制。隔夜文化在CM中。 注:50％汇合建议，以允许继续扩散可能发生在诱导过程中的</li><li>诱导的日子，除去中，添加以下内容:OM到所需的成骨实验井和CM的对照孔。使用介质的体积将取决于组织培养皿的大小。对于12孔培养皿中，1.0毫升每孔介质就足够了。对于6孔培养皿中，加入2.0ml媒体每孔就足够了。为100mm平皿，应使用10.0毫升的媒体每皿。 </li><li>培养最少14天的细胞用适当的培养基中，每3-4变化天。高成骨ASC人群可能最早的7天感应显示外矿物质沉积的迹象。 </li><li>成骨分化（ 即细胞外的矿物沉积）可以使用冯·科萨组织学染色为磷酸钙得到证实。这个污点会产生棕黑色沉淀物识别的胞外钙磷酸盐（参见图2）的存在。 染色与冯·科萨试剂: <ol><li>从所述控制单元删除该OM从实验细胞和CM </li><li>固定在1×PBS中制备的4.0％多聚甲醛固定细胞60分钟。 </li><li>用1×PBS洗涤细胞2次。 </li><li>孵育的细胞用硝酸银2.0％（在水中制得） 在黑暗中 30分钟。 </li><li>除去硝酸银，洗涤2次，用蒸馏水和空气干燥的细胞。 </li><li>暴露的细胞暴露于UV光60分钟来开发卡尔cium磷酸盐沉淀。 </li><li>用1XPBS洗涤细胞数次。 </li><li>如果需要，染液的细胞用苏木精。 </li></ol></li></ol></li></ol><ol start="2"><li> 先进的结构陶瓷的成脂分化:在此之前归纳，编写成脂培养基（AM），如下所示:要将一个一瓶500毫升的DMEM（4.5克/毫升的葡萄糖）加到50.0毫升胎牛血清（10.0％终浓度）和5.0 ml青霉素/链霉素（10,000 IU / ml和10,000μg/ ml的终浓度，分别）。对此，添加下面的成脂诱导剂给予指示的终浓度地塞米松（1.0微米决赛），3 - 异丁基-1 - 甲基黄嘌呤（IBMX - 0.5毫米决赛），吲哚美辛（0.2毫米决赛）和胰岛素（10.0微米决赛）。 <ol><li>在诱导前，收获和板培养的ASCs成所需的组织培养皿中，使约80％汇合的实现。对于每一个实验皿镀金，镀加法人菜的非诱导控制。 。文化过夜CM 注:先进的结构陶瓷的成脂分化似乎是与增加的融合更加高效。 </li><li>诱导的日子，除去中，添加以下内容:上午到所需的成骨实验井和CM的对照孔。使用介质的体积将取决于组织培养皿的大小。对于12孔板，每孔1.0.ml媒体就足够了。对于6孔培养皿中，加入2.0ml媒体每孔就足够了。为100mm平皿，应使用10.0毫升的媒体每皿。 </li><li>培养最少14天的细胞用适当的介质的变化，每3-4天。高脂肪生成ASC人群可能最早的7天诱导表明脂质空泡形成的迹象。 </li><li>先进的结构陶瓷进行成脂分化将开发可在光学显微镜下很容易地可视化多个脂质空泡。此外，成脂分化可采用油红O染色组织学，将积累这些空泡内（ 见图2）进行确认。 染色与油红O: <ol><li>从实验和对照组细胞中取出纸张，解决这些问题，使用10％的正式钙固定液60分钟。为了准备这个固定液，结合以下:1.0克CaCl 2，25.0毫升16％多聚甲醛（4.0％最终）和75.0毫升蒸馏水水。 </li><li>用1×PBS洗涤细胞2次。 </li><li>用70％乙醇简要地洗涤细胞。 </li><li>孵育的细胞在室温下放置5分钟，与油红O溶液。要准备油红O解决方案，化解2.0克油红O粉50.0 1ml 70％乙醇50.0毫升的丙酮。 </li><li>用70％的乙醇中，然后洗涤细胞用自来水。 </li><li>不久后，由于染色随时间褪色染色的可视化的细胞。 </li></ol></li></ol></li></ol><ol start="3"><li>先进的结构陶瓷的软骨分化:软骨细胞分化是通过被称为"微团培养"高密度培养技术来实现。在此之前的文化，准备软骨中（CHM），如下所示:要将一个一瓶500毫升的DMEM（4.5克/毫升葡萄糖）加入5.0毫升胎牛血清（1％终浓度）和5.0 ml青霉素/链霉素（10,000国际单位/毫升和10,000μg/ ml的终浓度，分别）。对此，添加下面的软骨诱导剂给予指示的最终浓度:L-抗坏血酸-2 - 磷酸（50.0微克/毫升决赛），胰岛素（6.25微克/毫升决赛），转铁蛋白（6.25微克/毫升决赛）和转化生长因子β1 （10.0毫微克/毫升）。 <ol><li>收获的ASC和离心5分钟，1,200×g离心沉淀细胞。计数细胞以确定细胞悬浮液的密度。 </li><li>要准备微团颗粒，准备两个细胞悬液:在1×10 7细胞的密度编制CHM 1实验停牌的S /毫升，并在1×10 7个细胞/ ml的密度制备CM 1控制悬浮液。 <ol><li>放置在一个多孔盘的各个孔的细胞悬浮液的10.0微升"液滴"。允许在37°C，坚持2-3小时</li><li>轻轻地覆盖在液滴镀与任何CHM或CM小心不要游离细胞。 </li><li>培养细胞进行长达14天的CHM或者CM的变化在中每3-4天。在细胞培养CHM会凝结在这个时候，形成一个高密度微颗粒几乎没有细胞周围的单层。培养的对照细胞不会发生这种结露，很多人会发现作为一个单层。 </li><li>以确认软骨形成，修复粒料在4.0％多聚甲醛15分钟（在1x PBS中制备的）在室温下进行。染色使用的爱茜蓝染色的组织学的硫酸化蛋白聚糖的存在。 染色用阿尔辛蓝: <ol><li>洗PAraformaldehyde固定粒料一次在1×PBS中。 </li><li>孵育5分钟，在0.1N的HCl（pH值1.0）的丸粒，以降低其pH值。 </li><li>除去HCl，并添加1％爱茜蓝试剂（在0.1N HCl中，pH值1.0的方法制备）。孵育30分钟，在室温下进行。 </li><li>除去爱茜蓝试剂和用0.1N的HCl（pH值1.0）洗涤小球以去除过量的染色。 </li><li>用自来水洗涤额外可用于降低背景染色。 </li><li>作为一种替代方案中，微团粒​​料可以被收获，在10％福尔马林中固定过夜，石蜡包埋和切片染色爱茜蓝。 </li></ol></li></ol></li></ol></li></ol> 4。先进的结构陶瓷的流式细胞仪鉴定细胞表面的CD抗原分布的表征可以通过传统的流式细胞仪来完成。 <ol><li> 先进的结构陶瓷的制备用于流式细胞术:在此之前分析，准备流式细胞仪缓冲液（FCB），包括下列各项:1.0％BSA，2.0％FBS和在1x PBS制备的0.025％皂素。 <ol><li>收获的ASC和离心细胞5分钟，在1200×g离心，以产生沉淀。 </li><li>重悬细胞于无菌1XPBS并使用血细胞计数器计数细胞。确定悬浮液的细胞密度。 </li><li>划分悬挂成5×10 5细胞总离心沉淀等分。 </li><li>除去PBS上清液和固定细胞60分钟，在-20℃下用过量的冰冷的70％乙醇中。如果需要的话，这些细胞可以被存储在这个70％的乙醇在-20℃下冷冻，直到需要。 </li><li>下面固定，小心吸出乙醇，轻轻地两次与过量的FCB的洗涤沉淀。洗: <ol><li>添加过量的FCB，轻轻悬浮颗粒。 </li><li>离心5分钟，在1200×g离心，收集细胞一SA颗粒。 </li><li>小心吸FCB的上清液。 </li><li>重复。 </li></ol></li><li>来自各等份样品重悬在100μlFCB的取出最后的FCB洗上清。使用制造商的建议稀释添加所需的荧光标记的第一抗体。在冰上孵育细胞与抗体30分钟。 注意:如果荧光标记的第一抗体是不可用时，也可以使用非共轭的第一抗体。 </li><li>对于使用（ 例如 FITC，PE），每个荧光染料，培养细胞的等分试样用100毫升FCB含有同种型匹配的IgG作为阴性对照。 </li><li>在100毫升的FCB不含抗体作为一个额外的控制下孵育1等分试样。 </li><li>与FCB洗小区的每个等分试样2倍，如步骤4.1.5详述。继最后一次洗涤，重悬等分在100毫升的FCB，并进行​​流量分析。 注意:如果使用非共轭的第一抗体:在此之后的洗涤步骤在冰上孵育等分试样进行20分钟，使用的FCB补充有合适的荧光标记的第二抗体使用制造商的建议稀释。如在步骤4.1.5概述洗2次。 </li></ol></li><li> 先进的结构陶瓷流分析:对于流量分析，最低为10,000个事件应该被计算在内。未染色和同型匹配的对照组应使用前向散射（FSC）和侧向散射（SSC）来设置门。对于这一点，步骤4.1.8的无抗体对照组（ 即未染色的）应以消除碎片和死的ASCs被使用。 4.1.7步的同型匹配的抗体对照。应当用于建立正面荧光的区域。 </li></ol>

<ol>
	<li>Zuk, P. A., 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=Multi-lineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multi-lineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Engineering. 7 (2), 211-226 (2001).</li><li>Rodbell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+isolated+fat+cells.">Metabolism of isolated fat cells.</a> J. Biol. Chem. 239, 375-380 (1964).</li><li>Poznanski, W. J., Waheed, I., Human Van, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fat+cell+precursors.+Morphologic+and+metabolic+differentiation+in+culture.">fat cell precursors. Morphologic and metabolic differentiation in culture.</a> Lab Invest. 29 (5), 570-576 (1973).</li><li>Zuk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+Stem+Cells+in+Tissue+Regeneration:+A+Review.">Adipose-derived Stem Cells in Tissue Regeneration: A Review.</a> ISRN Stem Cells. , (2012).</li><li>Zuk, P. A., 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=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Mol. Biol. Cell. 13, 4279-4295 (2002).</li><li>Mitchell, J. B., 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=Immunophenotype+of+human+adipose-derived+cells:+temporal+changes+in+stromal-associated+and+stem+cell-associated+markers.">Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.</a> Stem Cells. 24 (2), 376-385 (2006).</li><li>Oedayrajsingh-Varma, M. J., 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=Phenotypical+and+functional+characterization+of+freshly+isolated+adipose+tissue-derived+stem+cells.">Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells.</a> Stem Cells Dev. 16 (1), 91-104 (2007).</li><li>Yoshimura, K., 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=Characterization+of+freshly+isolated+and+cultured+cells+derived+from+the+fatty+and+fluid+portions+of+liposuction+aspirates.">Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.</a> J Cell Physiol. 208 (1), 64-76 (2006).</li><li>Zannettino, A. C., 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=Multipotential+human+adipose-derived+stromal+stem+cells+exhibit+a+perivascular+phenotype+in+vitro+and+in+vivo.">Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo.</a> J Cell Physiol. 214 (2), 413-421 (2008).</li><li>Chung, M. T., 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=CD90+(Thy-1)+Positive+Selection+Enhances+Osteogenic+Capacity+of+Human+Adipose-Derived+Stromal+Cells.">CD90 (Thy-1) Positive Selection Enhances Osteogenic Capacity of Human Adipose-Derived Stromal Cells.</a> Tissue Eng Part A. , (2012).</li><li>Li, H., 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=Adipogenic+potential+of+adipose+stem+cell+subpopulations.">Adipogenic potential of adipose stem cell subpopulations.</a> Plast Reconstr Surg. 128 (3), 663-672 (2011).</li><li>Rada, T., Reis, R. L., Gomes, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+stem+cells+subpopulations+isolated+from+human+adipose+tissue+exhibit+different+chondrogenic+and+osteogenic+differentiation+potential.">Distinct stem cells subpopulations isolated from human adipose tissue exhibit different chondrogenic and osteogenic differentiation potential.</a> Stem Cell Rev. 7 (1), 64-76 (2011).</li><li>Heydarkhan-Hagvall, S., 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=Human+Adipose+Stem+Cells:+A+Potential+Cell+Source+for+Cardiovascular+Tissue+Engineering.">Human Adipose Stem Cells: A Potential Cell Source for Cardiovascular Tissue Engineering.</a> Cells Tissues Organs. 187 (4), 263-274 (2008).</li><li>Jack, G. S., 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=Processed+lipoaspirate+cells+for+tissue+engineering+of+the+lower+urinary+tract:+implications+for+the+treatment+of+stress+urinary+incontinence+and+bladder+reconstruction.">Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction.</a> J Urol. 174 (5), 2041-2045 (2005).</li><li>Banas, A., 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=Rapid+hepatic+fate+specification+of+adipose-derived+stem+cells+and+their+therapeutic+potential+for+liver+failure.">Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure.</a> J Gastroenterol Hepatol. 24 (1), 70-77 (2009).</li><li>Schreml, S., 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=Harvesting+human+adipose+tissue-derived+adult+stem+cells:+resection+versus+liposuction.">Harvesting human adipose tissue-derived adult stem cells: resection versus liposuction.</a> Cytotherapy. 11 (7), 947-957 (2009).</li><li>Oedayrajsingh-Varma, M. J., 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=Adipose+tissue-derived+mesenchymal+stem+cell+yield+and+growth+characteristics+are+affected+by+the+tissue-harvesting+procedure.">Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.</a> Cytotherapy. 8 (2), 166-177 (2006).</li><li>Ahmad, J., Eaves, F. F., Rohrich, R. J., Kenkel, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+American+Society+for+Aesthetic+Plastic+Surgery+(ASAPS)+survey:+current+trends+in+liposuction.">The American Society for Aesthetic Plastic Surgery (ASAPS) survey: current trends in liposuction.</a> Aesthet Surg J. 31 (2), 214-224 (2011).</li><li>Tierney, E. P., Kouba, D. J., Hanke, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+of+tumescent+and+laser-assisted+liposuction:+review+of+the+literature.">Safety of tumescent and laser-assisted liposuction: review of the literature.</a> J Drugs Dermatol. 10 (12), 1363-1369 (2012).</li><li>Mojallal, A., Auxenfans, C., Lequeux, C., Braye, F., Damour, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+negative+pressure+when+harvesting+adipose+tissue+on+cell+yield+of+the+stromal-vascular+fraction.">Influence of negative pressure when harvesting adipose tissue on cell yield of the stromal-vascular fraction.</a> Biomed Mater Eng. 18 (4-5), 193-197 (2008).</li><li>Matsumoto, D., 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=Influences+of+preservation+at+various+temperatures+on+liposuction+aspirates.">Influences of preservation at various temperatures on liposuction aspirates.</a> Plast Reconstr Surg. 120 (6), 1510-1517 (2007).</li><li>Francis, M. P., Sachs, P. C., Elmore, L. W., Holt, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+adipose-derived+mesenchymal+stem+cells+from+lipoaspirate+blood+and+saline+fraction.">Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction.</a> Organogenesis. 6 (1), 11-14 (2010).</li><li>Boquest, A. C., Shahdadfar, A., Brinchmann, J. E., Collas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+stromal+stem+cells+from+human+adipose+tissue.">Isolation of stromal stem cells from human adipose tissue.</a> Methods Mol Biol. 325, 35-46 (2006).</li><li>Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+isolation,+expansion+and+differentiation.">Adipose-derived stem cells: isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Dubois, S. G., 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=Isolation+of+human+adipose-derived+stem+cells+from+biopsies+and+liposuction+specimens.">Isolation of human adipose-derived stem cells from biopsies and liposuction specimens.</a> Methods Mol Biol. 449, 69-79 (2008).</li><li>Mosna, F., Sensebe, L., Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bone+marrow+and+adipose+tissue+mesenchymal+stem+cells:+a+user's+guide.">Human bone marrow and adipose tissue mesenchymal stem cells: a user's guide.</a> Stem Cells Dev. 19 (10), 1449-1470 (2010).</li><li>Zachar, V., Rasmussen, J. G., Fink, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+growth+of+adipose+tissue-derived+stem+cells.">Isolation and growth of adipose tissue-derived stem cells.</a> Methods Mol Biol. 698, 37-49 (2011).</li><li>Williams, S. K., McKenney, S., Jarrell, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+lot+selection+and+purification+for+adipose+tissue+digestion.">Collagenase lot selection and purification for adipose tissue digestion.</a> Cell Transplant. 4 (3), 281-289 (1995).</li><li>Wang, H., Van Blitterswijk, C. A., Bertrand-De Haas, M., Schuurman, A. H., Lamme, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+enzymatic+isolation+of+fibroblasts+for+the+creation+of+autologous+skin+substitutes.">Improved enzymatic isolation of fibroblasts for the creation of autologous skin substitutes.</a> In Vitro Cell Dev Biol Anim. 40 (8-9), 268-277 (2004).</li><li>Pilgaard, L., Lund, P., Rasmussen, J. G., Fink, T., Zachar, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+highly+defined+proteases+for+the+isolation+of+adipose+tissue-derived+stem+cells.">Comparative analysis of highly defined proteases for the isolation of adipose tissue-derived stem cells.</a> Regen Med. 3 (5), 705-715 (2008).</li><li>Kurita, 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=Influences+of+centrifugation+on+cells+and+tissues+in+liposuction+aspirates:+optimized+centrifugation+for+lipotransfer+and+cell+isolation.">Influences of centrifugation on cells and tissues in liposuction aspirates: optimized centrifugation for lipotransfer and cell isolation.</a> Plast Reconstr Surg. 121 (3), 1033-1041 (2008).</li><li>Poloni, A., 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=Human+dedifferentiated+adipocytes+show+similar+properties+to+bone+marrow-derived+mesenchymal+stem+cells.">Human dedifferentiated adipocytes show similar properties to bone marrow-derived mesenchymal stem cells.</a> Stem Cells. 30 (5), 965-974 (2012).</li><li>D'Andrea, F., 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=Large-scale+production+of+human+adipose+tissue+from+stem+cells:+a+new+tool+for+regenerative+medicine+and+tissue+banking.">Large-scale production of human adipose tissue from stem cells: a new tool for regenerative medicine and tissue banking.</a> Tissue Eng Part C Methods. 14 (3), 233-242 (2008).</li><li>Tallone, T., 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=Adult+human+adipose+tissue+contains+several+types+of+multipotent+cells.">Adult human adipose tissue contains several types of multipotent cells.</a> J Cardiovasc Transl Res. 4 (2), 200-210 (2011).</li><li>De Francesco, F., 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=Human+CD34/CD90+ASCs+are+capable+of+growing+as+sphere+clusters,+producing+high+levels+of+VEGF+and+forming+capillaries.">Human CD34/CD90 ASCs are capable of growing as sphere clusters, producing high levels of VEGF and forming capillaries.</a> PLoS One. 4 (8), e6537 (2009).</li><li>Haasters, F., 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=Morphological+and+immunocytochemical+characteristics+indicate+the+yield+of+early+progenitors+and+represent+a+quality+control+for+human+mesenchymal+stem+cell+culturing.">Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing.</a> J Anat. 214 (5), 759-767 (2009).</li></ol>

这个手稿的作者是由加州大学董事会，并授权给Cytori治疗所拥​​有的专利的共同发明人。

脂肪组织为先进的结构陶瓷的隔离可以有多种形式:从通过切除或抽脂来或者通过注射器抽取或抽吸辅助抽脂（ 如吸脂术）获得更小的碎片得到的固体片组织。是否有更多的SVF细胞（从而先进的结构陶瓷）可从切除或抽吸的脂肪样本获得的是不清楚冲突的研究已经提出了16，17。这是可能的，只要操作者熟练的分离技术包括两种形式的脂肪组织是多适于SVF细胞和先进的结构陶瓷的隔?...

在2001年，来自脂肪组织的多能干细胞的推定的人口在杂志组织工程1进行了说明。这些细胞被赋予由于其推导通过整容手术获得的名称处理脂肪抽吸物或PLA细胞处理脂肪抽吸物组织。在这篇文章中所描述的隔离方法是基于从脂肪组织2基质血管组分（SVF）的隔离现有的酶战略。将SVF已被定义为红细胞，成纤维细胞，内皮细胞，平滑肌细胞，周细胞和前脂肪细胞，尚未粘附到组织培养基质2，3一最小加工人口。这SVF随时间的培养，提出消除许多这些污染细胞群，从而导致粘附，成纤维细胞群。这些成纤维细胞已在文献中在过去40年被认定为被预脂肪细胞。然而，我们的研究小组证明，这些细胞具有多能的中胚层，并改名为贴壁SVF人口PLA细胞。众多其他研究小组随后的研究加入到这一潜在的，既暗示内胚层和外胚层电位（综述见4）。自那时起，无数的附加条款，这些细胞已在文献中出现。为了提供某种类型的共识，术语脂肪来源的干细胞或先进的结构陶瓷在第二届年度IFATS会上获得通过。因此，该术语ASC将在本文中被使用。 在这篇文章中所描述的协议是一个相对简单的过程，需要标准的实验室设备，并使用简单的试剂如磷酸缓冲液，标准组织培养基试剂和胶原酶。它可以产生的ASC取决于起始脂肪组织体积和随后的C量大量ulture时间。然而，如此大量的脂肪组织的处理可呈现可使用该协议来减轻至一定程度的一些物理问题。此外，该协议不要求无菌组织培养设施和批准的生物安全罩，因此必须使用经认可的组织培养设施。这个要求也可以降低ASC人口的效用在临床应用中，除非他们被隔离在良好生产规范设计的分离和扩增材料的临床应用（GMP）认证的工厂。作为一种替代方法，自动化系统，可以在手术室隔离的ASCs在一个封闭的系统将避免这个关键问题，并允许直接使用的ASC的而不需要任何随后的体外扩增 。迄今为止，有六个自动化系统，是市售的用于细胞从人体组织的隔离。这些系统使得可以以分离显著数量从它的立即下收获大量脂肪组织的ASCs的。这些先进的结构陶瓷可随后被重新引入到患者体内，适用于各种不必离开手术室再生的目的，而病人。除了这个协议描述的ASC的手动隔离，一个协议，用于使用Celution系统的ASC自动隔离在一个同伴文章还给出。

<table border="1">
	<tbody>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Reagent</td>
		</tr>
		<tr>
			<td>
				DMEM (Dulbecco&#39;s Modification of Eagle&#39;s Medium)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				10-013-CV</td>
			<td>
				with 4.5 g/ml glucose, L-glutamine, sodium pyruvate</td>
		</tr>
		<tr>
			<td>
				Penicillin/Streptomycin</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-002-CI</td>
			<td>
				10,000 IU/ml penicillin/10,000 &mu;g/ml streptomycin</td>
		</tr>
		<tr>
			<td>
				Amphotericin B</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				30-003-CF</td>
			<td>
				250 &mu;g/ml amphotericin B</td>
		</tr>
		<tr>
			<td>
				10X PBS (Phospho-buffered Saline)</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				25-053-CI</td>
			<td>
				without calcium, without magnesium</td>
		</tr>
		<tr>
			<td>
				Trypsin/EDTA</td>
			<td>
				Mediatech Cellgro</td>
			<td>
				20-031-CV</td>
			<td>
				0.25 % trypsin/2.21mM EDTA</td>
		</tr>
		<tr>
			<td>
				Collagenase type IA (from Clostridium histolyticum)</td>
			<td>
				Sigma</td>
			<td>
				C2674</td>
			<td>
				crude preparation; &lt;125 collagen digestion units/mg solid</td>
		</tr>
		<tr>
			<td>
				FBS (Fetal Bovine Serum) heat inactivated</td>
			<td>
				Gemini Bioproducts</td>
			<td>
				100106</td>
			<td>
				USDA source, heat inactivated</td>
		</tr>
		<tr>
			<td>
				10 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-104</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				25 ml serological pipettes</td>
			<td>
				Genesee Scientific</td>
			<td>
				12-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				50 ml polypropylene centrifuge tubes</td>
			<td>
				Genesee Scientific</td>
			<td>
				21-106</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				100 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-202</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				150 mm tissue culture dishes</td>
			<td>
				Genesee Scientific</td>
			<td>
				25-203</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				500 ml Stericup Filter Units</td>
			<td>
				Millipore</td>
			<td>
				SCGPU05RE</td>
			<td>
				PES membrane, 0.22 &mu;m pore</td>
		</tr>
		<tr>
			<td>
				Cell strainers</td>
			<td>
				FisherBrand</td>
			<td>
				22-363-549</td>
			<td>
				100 &mu;m nylon mesh</td>
		</tr>
		<tr>
			<td>
				dexamethasone - water soluble</td>
			<td>
				Sigma</td>
			<td>
				D-2915</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				L-ascorbic-acid 2 phosphate</td>
			<td>
				Sigma</td>
			<td>
				A-8960</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				&beta;-glycerophosphate disodium salt</td>
			<td>
				Sigma</td>
			<td>
				G-9422</td>
			<td>
				also known as glycerophosphate</td>
		</tr>
		<tr>
			<td>
				insulin</td>
			<td>
				Sigma</td>
			<td>
				I-6634</td>
			<td>
				made from bovine pancreas</td>
		</tr>
		<tr>
			<td>
				indomethacin</td>
			<td>
				Sigma</td>
			<td>
				I-7378</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				apo-transferrin</td>
			<td>
				Sigma</td>
			<td>
				T-4382</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				TGF&beta;1</td>
			<td>
				R&amp;D Systems</td>
			<td>
				240-B-002</td>
			<td>
				recombinant human</td>
		</tr>
		<tr>
			<td>
				Oil Red O</td>
			<td>
				Sigma</td>
			<td>
				O-0625</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Alcian Blue</td>
			<td>
				Sigma</td>
			<td>
				A-5268</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Silver nitrate</td>
			<td>
				Sigma</td>
			<td>
				S-0319</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Hydrochloric acid</td>
			<td>
				Fisher Scientific</td>
			<td>
				A144</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				Paraformaldehyde</td>
			<td>
				Fisher Scientific</td>
			<td>
				30525-89-4</td>
			<td>
				supplied as a 16 % stock</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				[header]</td>
		</tr>
		<tr>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				&nbsp;</td>
			<td>
				Equipment Needed</td>
		</tr>
		<tr>
			<td>
				Class II A/B Biosafety hood</td>
			<td>
				Thermo Scientific</td>
			<td>
				&nbsp;</td>
			<td>
				ensure hood has vacuum lines for aspiration</td>
		</tr>
		<tr>
			<td>
				Benchtop centrifuge</td>
			<td>
				Hermle Labnet</td>
			<td>
				Z383</td>
			<td>
				Swing-out rotor for 50 ml tubes required, capable of 1200 x g</td>
		</tr>
		<tr>
			<td>
				Water bath</td>
			<td>
				Fisher Scientific Isotemp</td>
			<td>
				S52602Q</td>
			<td>
				5-10L capacity, capable of 37 C</td>
		</tr>
		<tr>
			<td>
				Automated Pipette Aids</td>
			<td>
				Drummond Pipette Aid XL</td>
			<td>
				4-000-105</td>
			<td>
				&nbsp;</td>
		</tr>
		<tr>
			<td>
				CO2 Incubator</td>
			<td>
				Thermo Scientific</td>
			<td>
				Forma 310</td>
			<td>
				direct heat or water jacketed</td>
		</tr>
	</tbody>
</table>

	&nbsp;

manual isolation of adipose-derived stem cells from human lipoaspirates

2001年，研究人员在加州大学洛杉矶分校，描述从抽脂的脂肪组织，他们最初被称为脂肪抽吸物处理的细胞或PLA细胞成体干细胞的新的人口的隔离。从那时起，这些干细胞已被更名为脂肪来源的干细胞或先进的结构陶瓷和已移居到成为干细胞研究和再生医学领域最热门的成体干细胞的人群之一。数以千计的文章，现在描述了使用先进的结构陶瓷的各种再生动物模型，包括骨再生，周围神经损伤修复和心血管工程。最近的文章开始描述的用途无数在诊所先进的结构陶瓷。在这篇文章中所示的协议概述了从大量的整容手术获得lipoaspirates手动和酶隔离先进的结构陶瓷的基本程序。这个协议可以很容易地放大或缩小，以accommod吃脂肪抽吸物的体积，并且可以适于通过隔离abdominoplasties和其他类似的方法获得来自脂肪组织的ASC。

上述协议纲要描述了一个SVF从大量脂肪抽吸物样品的隔离手册，酶法。在这个SVF许多细胞群，包括ASC。许多研究建议标准组织培养条件下培养这种SVF会选择贴壁成纤维细胞的人口可能会主要由ASC类型。与此相一致，我们已经表明，使用流式细胞术和免疫荧光，即培养的SVF粒料变得基本上不含主要污染细胞类型，即红细胞，平滑肌细胞和内皮细胞系1的细胞。所得到的ASC人口相对均匀的外观?...

Watch this Scientific Journal Video about Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates at JoVE.com

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

在2001年，研究人员在加州大学洛杉矶分校所述的成体干细胞群的分离，称为脂肪来源的干细胞或的ASC，从脂肪组织。本文概述了先进的结构陶瓷的使用手册，酶消化协议使用胶原酶lipoaspirates隔离。

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 ...

manual-isolation-adipose-derived-stem-cells-from-human

Research

JoVE Journal

Biology

41.1K 次观看.  Cytori Therapeutics Inc. 在2001年，研究人员在加州大学洛杉矶分校所述的成体干细胞群的分离，称为脂肪来源的干细胞或的ASC，从脂肪组织。本文概述了先进的结构陶瓷的使用手册，酶消化协议使用胶原酶lipoaspirates隔离。 

视频: Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

从胎盘中的造血干细胞的分离和分析

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

科研

生物学

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

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 ...

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 of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

天真成人干细胞的分离从初级人类成纤维细胞培养

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

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 ...