Name: immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)
Uploaded: 2016-08-11T14:00:00.000+00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

这项工作是由美国国立卫生研究院DK095137（THC到）的支持。我们感谢现任和前任实验室成员是谁所描述的方法的发展和成熟作出了贡献。

伦理学声明:密歇根委员会对动物的使用和注意事项大学（UCUCA）已批准按照指南实验动物的护理和使用（学院实验动物研究，国家研究理事会）的所有方法和协议。小鼠均保持在密歇根大学的动物饲养，并给予食物和水自由进入并保持在12小时黑暗/光照周期。 1.准备<ol><li>制备原代培养介质用DMEM，10％FBS，1×P / S / G，和1x抗生素/抗真菌剂。这将被用来保持组织中之前消化。将股票和分装在37ºC水浴。 </li><li>制备的III型胶原酶溶液5毫升在5毫克/毫升溶于HBSS（+钙+镁）为每种类型的脂肪垫的被隔离，最多5只小鼠。例如，从一个单一的基因型分离SQ和可见光时，使10毫升，如果野生型和突变型的SQ和可见光，使20毫升调节pH值至7.4，STE激怒过滤器。分装成50ml试管。从光一边远位置。 </li><li>使用70％的乙醇消毒手术区域。擦拭，并用蓝色的垫覆盖。喷上蓝色垫些乙醇。 </li><li>使用22克针牵制的吸水垫的保丽龙板，并用剪刀修剪。喷雾用乙醇垫然后设置蓝色垫板并与另一个蓝色垫，直到开始清扫覆盖。 </li><li>填充两个50ml试管用乙醇和发生在邻近于外科领域的立场。放置在乙醇剪刀和镊子。 </li><li>填另一50ml管中，用PBS加1×防反冲洗乙醇关闭手术工具。 </li><li>在油烟机，标签60毫米菜肴每个组织类型，并填写与文化传媒加热到37ºC。放置邻近于手术区板。 </li></ol> 2.皮下（SQ）脂肪垫的分离<ol><li>安乐死鼠标异氟醚和气胸过量。 </li><li>平缓鼠标喷用70％乙醇打下平卧。针脚的爪子泡沫板与22克针。 </li><li>使下腹部皮肤的一小截。拿着切的顶部，镊子，拿剪刀和皮肤腹膜分开。 </li><li>一旦皮肤是从腹部到胸部的腹膜分离，通过在沿所述主体的一侧的皮肤侧向切口反映皮肤从朝向头部腹膜程。 </li><li>反映剩余的皮肤保持腹股沟的脂肪远离身体和牵制到板22克针。 </li><li>切换到精细剪刀和镊子。抓住用钳子腹股沟脂肪垫在腹膜附近的起源和剪断掉皮肤和脂肪腹股沟向腹股沟避免污染皮肤的SQ进展之间。 </li><li>放置曝晒腹股沟脂肪垫在标有60mm培养皿。 </li></ol> 3.内脏（VIS）脂肪垫的分离<ol><li>以类似的方式执行步骤2.5，切开腹膜打开以暴露内脏脂肪垫和肠道。 </li><li>移动肠道远朝胸部。 </li><li>抓住VIS脂肪垫在远端，轻轻往上拉。解剖出VIS脂肪垫，同时注意排除附睾（或子宫，如果使用雌性小鼠）组织。 </li><li>发生在标盘，并重复步骤3.3为剩余的VIS垫。 </li></ol> 4.脂肪垫的胶原酶消化<ol><li>移动60毫米菜的组织培养罩，吸了媒体。 </li><li>剁碎脂肪垫用弯剪，然后添加到胶原酶溶液。请务必用乙醇和PBS冲洗剪刀和镊子组织类型之间。 </li><li>在300rpm和37ºC10-20分钟摇动直至组织被消化。如果SQ的一些作品仍然可见这是可以接受的。这将在以后的步骤来解决。 </li><li> 25毫升培养基添加到胶原酶溶液以停止collagenase治疗和吸管上下10次，10毫升血清吸管驱散消化的组织。 </li><li>使用100微米的细胞滤网株细胞。离心机在300×g下10分钟。 </li><li>小心倒出了媒体不要打扰颗粒和5毫升无菌水添加到颗粒，轻轻吸暂停细胞。等待2分钟，以溶解红细胞。 </li><li>通过一个新的100微米的细胞滤网加入25毫升含10％FBS和应变细胞介质。 </li><li>离心机在300×g下10分钟。倒出培养基，并在1ml培养基重悬沉淀。 </li><li>计数使用1血球细胞:1台盼蓝。 </li><li>板1×10 6个细胞在一个阱上的6孔板中。 </li></ol> 5.磁性细胞分离<ol><li>约4至6小时细胞粘附后，冲洗细胞两次用HBSS（-Ca，-Mg）。分离使用0.05％胰蛋白酶粘附细胞。稀释胰蛋白酶细胞悬浮于1ml分离缓冲液和自旋为5分钟，在300×g的。 </li><li>吸上清。悬浮细胞于500μl缓冲液中。删除10微升等分试样并使用血球计数细胞。 </li><li>自旋细胞在300×g下5分钟。 </li><li>吸完全上清。悬浮细胞在90微升缓冲液中。添加10微升抗SCA1-FITC初级抗体。拌匀并在黑暗中在4ºC孵育10分钟。 </li><li>洗涤细胞用1毫升缓冲液中，离心细胞，在300×g下5分钟。完全取出上清液。 </li><li>悬浮细胞在80μl的缓冲液中。加入20微升抗FITC微珠。拌匀并在黑暗中在4ºC孵育15分钟。 </li><li>洗涤细胞用1毫升缓冲液中，离心细胞，在300×g下5分钟。 </li><li>在500微升缓冲液中取出上清，重新悬浮颗粒。 </li><li>放置列在磁性支架并用500μl缓冲液冲洗柱。 </li><li>细胞悬液添加到列。避免气泡而吹打。 </li><li>收集未标记SCA1 - &lt;/ SUP&gt;细胞并用500μl的缓冲液洗涤柱3次。只有增加新的缓冲区时，水库是空的。避免气泡而吹打。 </li><li>从磁性支架上取下柱并放入15毫升锥形管中。 </li><li>加入1ml通过柱水库推柱塞提供缓冲柱和洗脱SCA1 +。 </li><li>降速细胞组分在300×克5分钟。重悬细胞于1ml培养基中。 </li><li>除去来自标记的和未标记的级分10微升等分试样并使用血球计数细胞。 </li><li>板1以及一个6孔板的每一个单元级分。 </li></ol> 6.流式细胞仪SCA1 高 ACSs中的免疫磁珠分离的验证<ol><li>冲洗细胞两次用HBSS（-Ca，-Mg），并解离用0.05％胰蛋白酶的细胞。 </li><li>离心细胞，在300×g离心5分钟。重悬细胞于1ml培养基和计数使用血球。 </li><li>获得"10 6 </SUP&gt;细胞于1ml培养基，并在300×g离心5分钟离心。 </li><li>除去上清液，并重复步骤6.3两次。 </li><li>悬浮细胞用1毫升2％山羊血清的+ 2％BSA中并在室温30分钟块。 </li><li>离心细胞，在300×g离心5分钟。 </li><li>除去封闭液。 </li><li>添加大鼠IgG2a的Alexa Fluor 647（0.25微克，1:400）或抗SCA1的Alexa Fluor 647（0.25微克，1:400），在100μlPBS中，用2％山羊血清和2％BSA + PBS在4℃下在黑暗中30分钟。 </li><li> 1毫升冷PBS加入细胞。离心机300 xg离心在4℃下5分钟。 </li><li>除去上清液，并重复步骤6.9两次。 </li><li>重悬细胞于1ml PBS中。通过100微米的细胞过滤细胞悬浮液中进行流式细胞术分析制剂。 </li></ol>

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在此，我们展示了隔离，并从不同的脂肪垫鼠携带者的免疫细胞分离和体外实验。所提出的方法是有效的的大量SCA1阳性的ASC的，这是过度的ASC 9,14的技术复杂和昂贵的FACS介导的隔离有利的快速隔离。不像FACS，免疫细胞分离不允许靶细胞群体的鉴定使用多个抗原。然而，如果表面抗原表征良好，利用免疫磁性分离的增加不依赖于使用的FACS设备15，其仍然不小院所许多生物学?...

干细胞抗原-1（SCA1，或Ly6A / E）最初被鉴定为通过造血细胞和间质干细胞5,6-表达的细胞表面标记。从小鼠脂肪库获得脂肪组织的基质血管级分（SVF）为含有成纤维细胞，巨噬细胞，血管内皮细胞，神经细胞，以及脂肪细胞祖细胞7的细胞的异质群体。脂肪细胞祖细胞，或脂肪干细胞（ASCs）是驻留在富含胶原蛋白的血管周围的细胞外基质（ECM）8非载脂细胞。大约SVF的50％由携带者，其特点为谱系阴性（林- ）和CD29 +:CD34 +:SCA1 + 9。大多数这些细胞是SCA1 +:CD24 -脂肪细胞祖细胞，其能够在体外脂肪细胞分化的;然而，只有细胞的级分（SVF的0.08％）构成SCA1 <sUP&gt; +:CD24 +细胞完全有能力和增殖分化为体内条件下9脂肪细胞。尽管使用SCA1 + SVF不脱离CD24识别CD24 +细胞的电位警告-的细胞，分离SCA1 +的ASC从用免疫细胞分离脂肪库是一种高效，实用的方法，以确定主脂肪细胞祖细胞的细胞自主表型。 在肥胖和糖尿病，组织纤维化和炎症的领域发挥在2型糖尿病3的开发和维护的关键作用。最近，德永等人 。表明，腹股沟（或皮下，SQ）和perigonadal（或内脏，VIS）隔离SCA1 高细胞C57BL6 / J脂肪库表现出体外 10个不同的基因签名和ECM重塑。 MMP14（MT1-MMP），膜 - t的典型构件YPE基质金属蛋白酶（MMP）家族通过其原活性1介导的白色脂肪组织（WAT）的开发。 可与分离和富集通过以下方案中的细胞进行的实验的例子包括三维培养，分化研究，胶原降解测定法，和RNA测序10,11。降解测定法应与酸提取胶原进行，以确保端肽11,12的保存。下面的协议将演示方法主要血管基质细胞从不同的脂肪库分离和使用免疫细胞分离脂肪细胞丰富的祖细胞。在细胞分选的有效性将用流式细胞仪，并通过使用SCA1-GFP小鼠表达SCA1 +细胞GFP，由SCA1启动子驱动13进行评估。

<table><tbody><tr><td>Type 3 Collagenase</td><td>Worthington Biochemical</td><td>LS004182</td><td>Tissue digestion</td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965-092</td><td>High-glucose culture medium</td></tr><tr><td>Pen/Strep/Glutamine (100x)</td><td>Gibco</td><td>10378-016</td><td>Media antibiotic</td></tr><tr><td>Anti-anti (100x)</td><td>Gibco</td><td>15240-062</td><td>Media antifungal</td></tr><tr><td>FBS</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>PBS (1x, pH 7.4)</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Trypsin (0.05%)</td><td>Gibco</td><td>25300-054</td><td></td></tr><tr><td>Cell strainer</td><td>BD Bioscience</td><td>352360</td><td>100-&amp;mu;m cell strainer</td></tr><tr><td>60 mm plates</td><td>BD Falcon</td><td>353004</td><td></td></tr><tr><td>Scissors</td><td>FST</td><td>14001-12</td><td>Large</td></tr><tr><td>Scissors</td><td>FST</td><td>14091-11</td><td>Fine, curved tip</td></tr><tr><td>Large Forceps</td><td>FST</td><td>11000-12</td><td></td></tr><tr><td>Fine Forceps</td><td>Any vendor</td><td></td><td></td></tr><tr><td>25G 5/8&amp;rdquo; needles</td><td>BD</td><td>305122</td><td></td></tr><tr><td>22G 1.5&amp;rdquo; needles</td><td>BD</td><td>305159</td><td></td></tr><tr><td>15 ml conical tubes</td><td>BD Falcon</td><td>352097</td><td></td></tr><tr><td>50 ml conical tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>MACS separation columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Anti-Sca1 microbead kit (FITC)</td><td>Miltenyi Biotec</td><td>130-092-529</td><td>FITC-anti-Sca1 1&amp;ordm;Ab and anti-FITC microbeads 2&amp;ordm;Ab</td></tr><tr><td>AutoMACS running buffer</td><td>Miltenyi Biotec</td><td>130-091-221</td><td></td></tr><tr><td>MiniMACS separator</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td></td></tr><tr><td>Blue chux pads</td><td>Fisher</td><td>276-12424</td><td></td></tr><tr><td>Absorbent pads</td><td>Fisher</td><td>19-165-621</td><td></td></tr><tr><td>Styrofoam board</td><td></td><td></td><td>Use from 50 ml tubes</td></tr><tr><td>70% ethanol</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Any vendor</td><td></td><td></td></tr><tr><td>Rat IgG2a Alexa Fluor 647</td><td>Invitrogen</td><td>R2a21</td><td></td></tr><tr><td>Rat IgG2a anti-mouse Sca1 Alexa Fluor 647</td><td>Invitrogen</td><td>MSCA21</td><td></td></tr><tr><td>Rat IgG2a R-PE</td><td>Invitrogen</td><td>R2a04</td><td></td></tr><tr><td>Rat IgG2a anti-mouse F4/80 R-PE</td><td>Invitrogen</td><td>MF48004</td><td></td></tr><tr><td>Round-bottom tube</td><td>BD Falcon</td><td>352058</td><td></td></tr><tr><td>HBSS (&amp;ndash;Ca, &amp;ndash;Mg)</td><td>Gibco</td><td>14175-095</td><td></td></tr><tr><td>HBSS (+Ca, +Mg)</td><td>Gibco</td><td>14025-092</td><td>For collagenase solution</td></tr><tr><td>Type I collagen (2.7 mg/ml in 37mm acetic acid</td><td>Prepare in house&lt;sup&gt;12&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>10x MEM</td><td>Gibco</td><td>11430-030</td><td></td></tr><tr><td>1 M HEPES</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>0.34 N NaOH</td><td>Prepare in house</td><td></td><td></td></tr><tr><td>Cover slips</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>Alexa Fluor 594 carboxylic acid, succinimidyl ester, mixed isomers</td><td>Invitrogen</td><td>A-20004</td><td></td></tr><tr><td>0.89 M NaHCO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>Gibco</td><td>25080-094</td><td></td></tr></tbody></table>

immunomagnetic separation of fat depot-specific sca1high adipose-derived stem cells (ascs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

从不同的脂肪垫SCA1 高的ASC的富集。 从SQ脂肪显示成纤维样分离出来的血管间质细胞，拉伸细胞形态，无论SCA1表达水平（ 图1A）。另一方面，VIS（eWAT衍生）SCA1 高和SCA1 低细胞证明其细胞形态明显的差异。像SQ（iWAT衍生）SCA1 高细胞，VIS（eWAT衍生）SCA1 高细胞显示拉伸，成?...

Watch this Scientific Journal Video about Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs at JoVE.com

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

immunomagnetic-separation-fat-depot-specific-sca1high-adipose-derived

Research

JoVE Journal

Developmental Biology

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

6.8K 次观看.  Biointerfaces Institute, University of Michigan. We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

视频: Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

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.

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 ...

科研

发育生物学

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

免疫与感染

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 ...

生物学

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

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

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

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

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

医学

Technique for Obtaining Mesenchymal Stem Cell from Adipose Tissue and Stromal Vascular Fraction Characterization in Long-Term Cryopreservation

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible source for regenerative clinical applications. Different protocols have been used to obtain adipose-derived stem cells. This article describes different steps of an improved time-saving protocol to obtain a more significant amount of ADSC, showing how to cryopreserve and thaw ADSC to obtain viable cells for culture expansion. One hundred milliliters of lipoaspirate were collected, using a 26 cm three-hole and 3 mm caliber syringe liposuction, from the abdominal area of nine patients who subsequently underwent elective abdominoplasty. The stem cells isolation was carried out with a series of washes with Dulbecco&#39;s Phosphate Buffered Saline (DPBS) solution supplemented with calcium and the use of collagenase. Stromal Vascular Fraction (SVF) cells were cryopreserved, and their viability was checked by immunophenotyping. The SVF cellular yield was 15.7 x 105 cells/mL, ranging between 6.1-26.2 cells/mL. Adherent SVF cells reached confluence after an average of 7.5 (&#177;4.5) days, with an average cellular yield of 12.3 (&#177; 5.7) x 105 cells/mL. The viability of thawed SVF after 8 months, 1 year, and 2 years ranged between 23.06%-72.34% with an average of 47.7% (&#177;24.64) with the lowest viability correlating with cases of two-year freezing. The use of DPBS solution supplemented with calcium and bag resting times for fat precipitation with a shorter time of collagenase digestion resulted in an increased stem cell final cellular yield. The detailed procedure for obtaining high yields of viable stem cells was more efficient regarding time and cellular yield than the techniques from previous studies. Even after a long period of cryopreservation, viable ADSC cells were found in the SVF.

The present protocol describes an improved methodology for ADSC isolation resulting in a tremendous cellular yield with time gain compared to the literature. This study also provides a straightforward method for obtaining a relatively large number of viable cells after long-term cryopreservation.

从脂肪组织和基质血管分数表征中获取间充质干细胞的技术在长期冷冻保存中的应用

Human mesenchymal stem cells derived from adipose tissue have become increasingly attractive as they show appropriate features and are an accessible ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident adipose progenitor cells (APCs) play a key role in adipose tissue homeostasis and can contribute to the tissue pathology. The growing use of single cell analysis technologies &#8211; including single-cell RNA-sequencing and single-cell proteomics &#8211; is transforming the stem/progenitor cell field by permitting unprecedented resolution of individual cell expression changes within the context of population- or tissue-wide changes. In this article, we provide detailed protocols to dissect mouse epididymal adipose tissue, isolate single adipose tissue-derived cells, and perform fluorescence activated cell sorting (FACS) to enrich for viable Sca1+/CD31-/CD45-/Ter119- APCs. These protocols will allow investigators to prepare high quality APCs suitable for downstream analyses such as single cell RNA sequencing.

We present a simple method to isolate highly viable adipose progenitor cells from mouse epididymal fat pads using fluorescence activated cell sorting.

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

脂肪组织来源的干细胞分离和表征

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

作者聚焦:分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

脂肪库专用SCA1的免疫磁性分离

摘要

探索更多视频

此视频中的章节

隔离和人力富集脂肪基质细胞的成骨增强

神经冠状原脂肪衍生干细胞的分离、培养和异位诱导，来自围阴脂肪组织

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

从人类Lipoaspirates脂肪来源干细胞的手动隔离

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

从脂肪组织和基质血管分数表征中获取间充质干细胞的技术在长期冷冻保存中的应用

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定