这项研究是由卫生研究院资助R01-DE021683-01与健康研究资助R01-DE019434来MTL国家机构的国家机构的支持;霍华德·休斯医学研究所研究奖学金，以MTCDCW由ACS富兰克林·马丁教授研究奖学金，该Hagey实验室小儿再生医学和斯坦福大学儿童健康研究所教授学者奖的支持。

注：所有患者的样品与知情同意而获得的，和实验方案进行了审查并批准了斯坦福大学的机构审查委员会（议定书＃2188和＃9999）。 1.细胞分离培养： <ol><li>获得健康的女性患者在局部/全身麻醉进行腹部，胁肋，和/或大腿区域的选修lipoaspiration人类皮下脂肪组织。确保机构审查委员会（IRB）的批准已经从人体组织隔离携带者的协议获得的，并按照机构的安全防范措施，而这种材料的工作。 </li><li>以获得从lipoaspirated脂肪组织的SVF，先洗吸脂物三次1×无菌磷酸盐缓冲盐水等体积（PBS）中。小心吸弃底部水层。 </li><li>准备胶原酶消化缓冲液：0.075％I型胶原酶在汉克的平衡盐溶液（HBSS）中。制备FACS缓冲液：2％FBS，1％P188和1％青霉素 - 链霉素的PBS。过滤用市售0.22μm孔径快速流动聚醚砜过滤器这两种解决方案。 </li><li>消化洗涤脂肪组织，安全地在振荡水浴60分钟，在37℃（大约180摇动/分钟）添加的胶原酶消化缓冲等体积并放置消化容器中。 注：它是最好使用较大体积的消化容器比要求，因为这允许对最大消化振荡期间（ 即 ，加入250毫升的胶原酶消化缓冲，加入250毫升洗涤脂肪组织中的1L无菌烧瓶）。 </li><li>通过加入的FACS缓冲液等体积​​并允许坐在RT 5分钟中和酶的活性。接着，在离心机233×g离心20分钟，在4℃。 </li><li>小心吸弃上清，注意不要打扰高密度SVF颗粒。 </li><li>悬浮颗粒在5-10毫升RT红细胞裂解缓冲液，是根据粒料的大小。离开溶液坐在RT 5分钟，并离心在233×g离心5分钟，在室温。 </li><li>吸去上清液，并通过加入5-10毫升的传统的生长培养基的基础上，丸粒尺寸重悬沉淀（Dulbecco氏改良的Eagle培养基[DMEM] / 10％FBS / 1％青霉素 - 链霉素溶液〔笔链球菌]）。 </li><li>通过100微米的尼龙细胞过滤器过滤该悬浮液以去除细胞碎片。 </li><li>离心机在233×g离心5分钟，在4℃下，并弃去上清液而不扰乱沉淀。 </li><li>悬浮颗粒在5-10毫升的传统媒体增长的基础上，颗粒大小。 </li><li>放置2×10 6个细胞在15cm的标准培养皿，并建立原代培养物O / N在37℃/ 21％O 2，5％的CO 2。保持在亚汇合的水平，以防止自发分化，在37℃/ 21％O 2，5％CO 2中生长培养基。 注：在一个完全细胞密度汇合15厘米板是约4-6×10 6个细胞。 </li></ol> 2.染色<ol><li>染色前培养的ASCs在传统生长培养基，在37℃/ 21％O 2，5％的CO 2中 ，36小时/排序。 </li><li>从培养板使用的Accutase（按照制造商的协议）电梯携带者。 </li><li>一旦用FACS缓冲液（PBS，2％FBS和1％青霉素 - 链霉素）洗涤细胞。 </li><li>离心机在233×g离心5分钟，在4℃。 </li><li>弃去上清液而不扰乱沉淀。将细胞重悬于0.5-1毫升FACS缓冲液（取决于细胞数和抗体的期望的量）。 注：细胞悬浮液的体积，按照用FACS抗体生产商的建议，确定抗体浓度。然而，滴定抗体浓度，使用1起始浓度：100，通常建议，如果它是第一次使用的抗体。 </li><li>计数的总数目使用血球细胞。 </li><li>用于未染色样品与单色彩控制（根据所使用的荧光抗体的数量）在1.5ml的离心管储备100微升等分试样。 </li><li>添加适当的荧光标记的单克隆抗体（以预定的浓度，每制造商的说明），并孵育20-30分钟在冰上，避光。 <ol><li>标记成骨细胞亚群的稀释：抗CD90（APC抗 - 人CD90（Thy1肾炎））或抗CD105（FITC抗 - 人CD105（内皮糖蛋白））在FACS缓冲液以1:50的浓度。 </li><li>来标记其它的细胞群（ 例如 ，造血细胞和内皮细胞）的稀释：抗CD45（抗人CD45太平洋蓝），抗CD34（抗人CD34 APC），和/或抗CD31（抗人CD31 PE ）在FACS缓冲液以1:50的浓度。 </li><li>如果不使用直接标记的抗体，使用生物素化初级抗体用适当的链霉亲和共轭二进制抗体，如链霉亲和PE-Cy7的，在稀释度为1：100。 </li></ol></li><li>在4℃下填充管用FACS缓冲液，并离心在233×g离心5分钟，吸上清：温育后，洗细胞两次。 </li><li>通过一个70微米的尼龙细胞过滤悬浮细胞在400-500微升FACS缓冲和过滤样品以去除细胞团块。 </li><li>转移标记的细胞进入FACS管，带过滤器的顶部和保持样品在冰上进行分析的持续时间。 </li></ol> 3.荧光激活细胞分选注意：下面的步骤强制以前的知识在荧光激活细胞分选（FACS）或熟练的技术人员的协助。 <ol><li>使用适当的FACS软件，设计分析图检查前向散射（FSC），侧向散射（SSC），藻红蛋白（PE）-Texas红，蓝太平洋，异硫氰酸荧光素（FITC），别藻蓝蛋白（APC）以及其他任何使用荧光ð标记细胞。 </li><li>之前加载到细胞分选仪，简要地涡旋每个样品重悬细胞。 </li><li>与未染色样品的分析，以便确定门的总细胞群，以排除细胞碎片，并确定基线自发荧光展开。 </li><li>加入碘化丙啶（PI）至未染色样品，并将其进行分析，以可视化的总细胞群中活细胞的群体。构建解决这个人口大门。 </li><li>为了设置荧光补偿每个荧光染料的单色对照样品分析。 </li><li>染色样品设置门的位置进行排序分析。 </li><li>加入PI的染色样品，以染色死细胞。 </li><li>分类标记的细胞群。排序成为1.5毫升离心管中，或含有培养基锥形管（15ml）中。 </li><li>手动停止一旦已经获得了所需的细胞数量排序。 </li><li>我们荷兰国际集团的FACS，通过分析一小部分（ 例如 ，500个细胞）所获得的细胞群进行纯度检查。 注：此步骤确认排序细胞群的纯度。理想情况下，纯度应大于90％-95％。 </li><li>离心细胞，在233×g离心5分钟，在4℃下排序的完成和重悬后立即在1ml含有10％FBS的新鲜培养基中。 注意：使用介质的量可根据细胞沉淀的尺寸增大。 </li><li>板在明胶包被的板，以提高细胞生存力。取决于最终的细胞产量，板300000细胞每孔在6孔板中，每孔100,000个细胞在12孔板中。 </li></ol>

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没有一个作者有经济利益的任何产品，设备，还是在这个手稿中提到的药物。没有一个作家有任何竞争的财务权益报告。

目前，来自人体脂肪组织的SVF的ASC的均质亚群的分离仍然虽然期望目标的一个具有挑战性的。亲骨ASC亚隔离是特别理想的，因为这样的细胞可用于研究骨骼肌组织的形成和稳态。然而，脂肪组织的SVF窝藏显著异质对于干细胞的能力和分化潜能。11这种异质性的分子基础，不能从细胞的混合种群的理解，而是需要单细胞分析12用这种方法，表面标志物与先进的结构陶瓷的成骨分化能?...

The heterogeneous nature of stem cell populations is not yet fully understood and remains a major impediment to the development of clinically effective stem cell-based therapeutic applications. One of the most common ways to characterize a heterogeneous population of stem cells is to employ a cell sorting method, such as fluorescence-activated cell sorting (FACS), to separate cells based on their surface marker expression profiles. As sorting methods become more complex, it becomes possible to identify more distinct functional subpopulations of cells. Microfluidic-based technologies are becoming more and more frequently utilized in analysis of gene expression at the single cell level. Multiplexed quantitative polymerase chain reaction (qPCR) within a microfluidic chip allows for effective and reliable high-resolution, single cell transcriptional analysis.1-5
In a previous study using single cell transcriptional profiling of 48 genes, considerable transcriptional heterogeneity was observed among ASCs.6 However, the distribution of genes MSX2, BMP-5, BMP-7, ALP, OCN, RUNX2 exhibited a strong association with a cluster of cells possessing highly osteogenic transcriptional profiles. To isolate cells according to this osteogenic gene expression profile, surface antigen expression patterns were correlated with transcription patterns and surface marker expression of endoglin (CD105) was subsequently discovered to closely correlate with enhanced osteogenic differentiation potential of ASCs. Independent of CD105 expression, expression of surface receptor Thy-1 (CD90), a glycosyl-phosphatidylinositol-linked membrane protein previously shown by Chen et al. to be associated with osteoprogenitor cells, was also correlated with osteogenic gene expression.6,7 These findings provide the opportunity to prospectively isolate subpopulations within the larger heterogeneous pool of ASCs with increased osteogenic capacity for cell-based bone tissue engineering applications.

<table border="0" cellpadding="0" cellspacing="0" width="618">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Name of Reagent/Material</td>
			<td style="width:152px;">Company</td>
			<td style="width:112px;">Catalog Number</td>
			<td style="width:79px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Disposable 250 mL Conical Tubes</td>
			<td style="width:152px;">Corning (Thomas Scientific)</td>
			<td style="width:112px;">2602A43</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">15140-122</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">DMEM, high glucose, GlutaMAX Supplement</td>
			<td style="width:152px;">Gibco</td>
			<td style="width:112px;">10566-016</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS, pH 7.4</td>
			<td style="width:152px;">Gibco</td>
			<td>10010-023</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine - Antiseptic Povidone/Iodine Solution</td>
			<td style="width:152px;">Purdue&#160;</td>
			<td>PFC-67618015017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hank&#39;s Balanced Salt Solution, 1X</td>
			<td style="width:152px;">Cellgro</td>
			<td>21-023-CV</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum, Certified, US Origin</td>
			<td style="width:152px;">Gibco</td>
			<td>16000-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from&#160;Clostridium histolyticum</td>
			<td style="width:152px;">Sigma-Aldrich</td>
			<td>C0130-5G</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ACCUTASE Cell Detachment Solution</td>
			<td style="width:152px;">Stem Cell Technologies</td>
			<td>7920</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">APC Mouse Anti-Human CD90</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">559869</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">FITC Mouse anti-Human CD105 (Endoglin)</td>
			<td style="width:152px;">BD Pharmingen</td>
			<td style="width:112px;">561443</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="42">
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isolation and enrichment of human adipose-derived stromal cells for enhanced osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

使用CD90作为标记的细胞具有增强成骨结果在人的ASC（ 图1A，1B）的高度富集的群体的隔离。携带者沾满太平洋蓝偶联的抗人CD45，FITC缀合的抗人类CD105，和APC缀合的抗人CD90。排序后，纯度水平大于98％，如通过量化后的排序分析。 定义的基础上允许两种新型亚群的前瞻性孤立转录谱细胞群体。表征每个亚群有前途的（CD90 +和CD105 低 ）的成骨潜力，...

Watch this Scientific Journal Video about Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis at JoVE.com

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

隔离和人力富集脂肪基质细胞的成骨增强

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

isolation-enrichment-human-adipose-derived-stromal-cells-for-enhanced

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JoVE Journal

Developmental Biology

11.4K 次观看.  Stanford University School of Medicine. The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.