Name: an efficient method to obtain dedifferentiated fat cells
Uploaded: 2016-07-15T13:00:00.000+00:00
Duration: 6 min 11 s
Description: Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

This research was supported in part by Program for Creating Start-ups from Advanced Research and Technology (START Program) from the Japan Society for the Promotion of Science (ST261006IP, TM) and by Program for the Strategic Research Foundation at Private Universities (2014-2019) (S1411018, TM) from the Ministry of Education, Sports, Science and Technology.

在整形外科，泌尿外科，小儿外科和日本大学板桥医院（东京，日本）的矫形外科的部门接受手术的患者中，获得了人类皮下脂肪样本。患者签署知情同意书，和医学的日本大学伦理委员会批准了这项研究。 1.组织准备<ol><li>从手术室到实验室使组织样品。 </li><li>洗涤1-2克脂肪组织用5ml的PBS（ - ）在室温（RT）下一次，在50ml管中，然后将在10cm的塑料盘的样品。 </li></ol> 2.胶原酶消化<ol><li> （ - ）从PBS取出样品，然后加入10毫升的无菌0.1％（重量/体积）的胶原酶溶液（胶原酶II型），以含有所述组织样本的菜。 </li><li>讳言与外科剪刀组织约15分钟，直到它到达一个泥组成ency。 </li><li>接着，消化在0.1％的组织糜（重量/体积）胶原酶在37℃下进行30分钟（每分钟60转）中的50ml管振荡器上轻轻搅拌溶液。 </li><li>过滤通过100微米的过滤器将消化的样品到50毫升管中。接着，通过10ml DCM中的（见材料的表 ）通过过滤器补充有2％FBS的。 </li></ol> 3.细胞分离<ol><li>离心过滤的细胞以100×g离心在RT 1分钟。 </li><li>在这一点上，制备溶于5ml DCM中补充有在50ml管中的2％FBS的。 </li><li>接着，绘制了使用1000微升吸管漂浮脂肪细胞，将细胞添加至含有50毫升管中。 </li><li>随后，轻轻摇晃50毫升管混合的细胞DCM网上平台。 </li><li>然后，离心管中，于100×g离心在RT 1分钟。 </li><li>用18G的针和20毫升注射器丢弃在管底部的介质。 </li><li>接下来，加入10 mL DCM的S有2％FBS upplemented向收集的细胞。 </li><li>通过轻轻摇晃50毫升管与细胞混合DCM。 </li><li>离心管中，于100×g离心在RT 1分钟。 </li></ol> 4.电镀细胞<ol><li>添加41毫升DCM或DMEM中补充有20％FBS的一个12.5厘米烧瓶中。 </li><li>接着，用200μl移液管添加40μl的脂肪细胞的烧瓶中。 </li><li>然后，填充用DCM或DMEM补充有20％FBS的烧瓶中。关键步骤:在这一点，确保细胞在整个介质传播。目视检查脱落的细胞，之后保持一个圆形培养皿的盖子瓶以保持其平整。 </li><li>最后，放置在培养箱内的烧瓶中，并把它在一个5％CO 2培养箱不受干扰在37℃下7天。 </li><li>孵化一个星期之后，反转瓶和检查DFAT细胞。接下来，评估如因为在这一点上fibrobl描述10 DFAT细胞生成能力AST样细胞（DFAT细胞）位于从脂肪细胞中不同的区域。 </li><li>加5毫升DCM中或DMEM之后除去前面的细胞培养基补充有20％FBS的烧瓶中。允许DFAT细胞改变介质后成长为一个星期。 </li><li>使用细胞计数器，计数各个细胞培养条件（DCM与DMEM）下产生DFAT细胞的数目。 </li></ol>

<ol>
	<li>Lanzoni, 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=Concise+review:+clinical+programs+of+stem+cell+therapies+for+liver+and+pancreas.">Concise review: clinical programs of stem cell therapies for liver and pancreas.</a> Stem Cells. 31 (10), 2047-2060 (2013).</li><li>de Girolamo, L., 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=Mesenchymal+stem/stromal+cells:+a+new+"cells+as+drugs"+paradigm.+Efficacy+and+critical+aspects+in+cell+therapy.">Mesenchymal stem/stromal cells: a new "cells as drugs" paradigm. Efficacy and critical aspects in cell therapy.</a> Curr. Pharm. Des. 19 (13), 2459-2473 (2013).</li><li>Lindroos, B., Suuronen, R., Miettinen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+of+adipose+stem+cells+in+regenerative+medicine.">The potential of adipose stem cells in regenerative medicine.</a> Stem Cell. Rev. 7 (2), 269-291 (2011).</li><li>Yan, J., Tie, G., Xu, T. Y., Cecchini, K., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+a+treatment+for+peripheral+arterial+disease:+current+status+and+potential+impact+of+type+II+diabetes+on+their+therapeutic+efficacy.">Mesenchymal stem cells as a treatment for peripheral arterial disease: current status and potential impact of type II diabetes on their therapeutic efficacy.</a> Stem Cell. Rev. 9 (3), 360-372 (2013).</li><li>Ringden, O., Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+as+treatment+for+chronic+GVHD.">Mesenchymal stromal cells as treatment for chronic GVHD.</a> Bone Marrow Transplant. 46 (2), 163-164 (2011).</li><li>Matsumoto, 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=Mature+adipocyte-derived+dedifferentiated+fat+cells+exhibit+multilineage+potential.">Mature adipocyte-derived dedifferentiated fat cells exhibit multilineage potential.</a> J. Cell. Physiol. 215 (1), 210-222 (2008).</li><li>Lessard, 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=Generation+of+human+adipose+stem+cells+through+dedifferentiation+of+mature+adipocytes+in+ceiling+cultures.">Generation of human adipose stem cells through dedifferentiation of mature adipocytes in ceiling cultures.</a> J. Vis. Exp. (97), (2015).</li><li>Lessard, 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=Characterization+of+dedifferentiating+human+mature+adipocytes+from+the+visceral+and+subcutaneous+fat+compartments:+fibroblast-activation+protein+alpha+and+dipeptidyl+peptidase+4+as+major+components+of+matrix+remodeling.">Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.</a> PLoS One. 10 (3), 0122065 (2015).</li><li>Peng, X., 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=Phenotypic+and+Functional+Properties+of+Porcine+Dedifferentiated+Fat+Cells+during+the+Long-Term+Culture+In+Vitro.">Phenotypic and Functional Properties of Porcine Dedifferentiated Fat Cells during the Long-Term Culture In Vitro.</a> Biomed. Res. Int. 2015, 673651 (2015).</li><li>Kono, S., Kazama, T., Kano, K., Harada, K., Uechi, M., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+properties+of+feline+dedifferentiated+fat+cells+and+adipose-derived+stem+cells.">Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells.</a> Vet. J. 199 (1), 88-96 (2014).</li><li>Bellin, M., Marchetto, M. C., Gage, F. H., Mummery, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cells:+the+new+patient.">Induced pluripotent stem cells: the new patient.</a> Nat. Rev. Mol. Cell Biol. 13 (11), 713-726 (2012).</li></ol>

The authors have nothing to disclose.

成熟脂肪细胞的进行体外分化，被称为天花板文化的过程，可以恢复到一个更原始的表型和增殖获得能力。这些细胞被称为去分化脂肪（DFAT）细胞。 DFAT细胞的多向分化潜能进行了评价。流式细胞术分析和基因表达分析表明，DFAT细胞相比的ASC 6是高度均匀的。事实上，DFAT细胞的细胞表面抗原轮廓是非常类似于在携带者6找到。因此，DFAT细胞可能功能类似于干细胞。 <p clas...

细胞治疗和组织工程是在再生医学1-5领域热点话题。虽然这些治疗方法具有很大的希望，一些技术问题目前阻碍其临床应用。在这方面，如在iPS细胞的产生，所有组织工程的治疗必须产生自由外部基因转导的细胞中，以维持患者的安全。因此，我们成功地制造人类细胞DFAT 6第一组。其他几个研究组已经自从采用我们的方法，以产生哺乳动物来源7-9 DFAT细胞，进一步凸显了模型的有效性。 以上几项研究的过程中，我们发现，细胞培养环境的质量可以通过调节细胞培养基的内容进行修改。这个发现导致增加DFAT电池生产的成功率，提高小区质量;在这两个关键因素有效地产生用于将来的临床试验的细胞。在这方面，一种改进的DFAT培养基（DCM中，一个介质类似于间充质干细胞培养基，其中包含重组人胰岛素，血清白蛋白，L-谷氨酸，几个脂肪酸和胆固醇）和DFAT细胞生成的方法和增殖，开​​发（约DCM的内容的更多信息可应要求提供）。与分化成多种细胞类型，包括脂肪形成，成骨和软骨形成细胞的能力中产生用此方法的高质量DFAT细胞。总之，这验证了细胞培养协议增强DFAT细胞的质量和可用于增强细胞治疗和组织工程的临床应用中十分有用。

<table><tbody><tr><td>CSTI303-MSC medium&amp;nbsp;</td><td>CSTI</td><td>87-671</td><td>This medium is defined as DCM in the text</td></tr><tr><td>PBS(-)</td><td>Wako</td><td>166-23555</td><td>It does not contain Mg&lt;sup&gt;2+&lt;/sup&gt; and Ca&lt;sup&gt;2+&lt;/sup&gt;</td></tr><tr><td>DMEM medium</td><td>Gibco</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>172012</td><td></td></tr><tr><td>Collagenase type II</td><td>Sigma</td><td>C-6885</td><td></td></tr><tr><td>Scissors</td><td>Takasago Medical Industry Co., Ltd</td><td>TKZ-F2194-1</td><td></td></tr><tr><td>Shaker</td><td>TAITEC</td><td>Bioshaker V.BR-36</td><td></td></tr><tr><td>Falcon Cell Strainer 100 &amp;mu;m Yellow</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>DL 352360</td><td></td></tr><tr><td>Falcon 12.5 cm&amp;sup2; Rectangular Canted Neck Cell Culture Flask with Blue Vented Screw Cap</td><td>CORNING LIFE SCIENCES&amp;nbsp;</td><td>353107</td><td></td></tr><tr><td>18 G needle</td><td>NIPRO</td><td>02-002</td><td></td></tr><tr><td>20 ml Syringe&amp;nbsp;</td><td>NIPRO</td><td>08-753</td><td></td></tr><tr><td>Z Series Coulter Counter</td><td>BECKMAN COULTER</td><td>383550</td><td></td></tr></tbody></table>

an efficient method to obtain dedifferentiated fat cells

组织工程和细胞治疗临床上具有很大的希望。在这方面，多能细胞，如间充质干细胞（MSC），可以用于治疗，在不久的将来，恢复功能受损的器官。尽管如此，一些技术问题，包括分离MSC和周围的放大的低效率的高度侵入性过程，目前阻碍潜在临床使用这些治疗方法的。这里，我们引入去分化的脂肪细胞（DFAT），MSC样细胞的产生一种高效的方法。有趣的是，DFAT细胞可以分化成多种细胞类型，包括脂肪形成，成骨和软骨形成细胞。虽然其他团体先前已经提出从成熟脂肪组织产生DFAT细胞的各种方法，我们的方法使我们能够更有效地生产DFAT细胞。在这方面，我们表明，DFAT培养基（DCM）中，FBS的补充有20％，是在产生DFAT细胞比的DMEM，补充有20％FBS的更有效。此外，通过我们的细胞培养方法生产的DFAT细胞可以再分化成几个组织类型。因此，一个非常有趣和有用的组织分化的研究模型。

<html>在这项研究中，DFAT细胞生成的方法和工具包进行了改进（ 图1）。我们的方法允许我们使用DCM和含有20％FBS的（ 图2A）的DMEM培养基中，以产生DFAT细胞。因此，我们比较DCM和DMEM中在产生DFAT细胞的效率。在这方面，相比于DMEM时，无论脂肪细胞的数量（ 图2A和B）的DCM中增强由三次DFAT细胞增殖。用20％相比，使用10％FBS的?...

Watch this Scientific Journal Video about An Efficient Method to Obtain Dedifferentiated Fat Cells at JoVE.com

An Efficient Method to Obtain Dedifferentiated Fat Cells

We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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Research

JoVE Journal

Developmental Biology

7.9K 次观看.  Nihon University. We have modified the conditions for DFAT cell generation and provide herein information regarding the use of an improved growth medium for the production of these cells.

视频: An Efficient Method to Obtain Dedifferentiated Fat Cells

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

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.

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 ...

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health. Still, there are significant limitations to available primary culture models, all of which have failed to faithfully recapitulate the adipose microenvironment or extend WAT viability beyond two weeks. The lack of a reliable primary culture model severely impedes research in WAT metabolism and drug development. To this end we have utilized NIH's standards of a microphysiologic system to develop a novel platform for WAT primary culture called 'SWAT' (sandwiched white adipose tissue). We overcome the natural buoyancy of adipocytes by sandwiching minced WAT clusters between sheets of adipose-derived stromal cells. In this construct, WAT samples are viable over eight weeks in culture. SWAT maintains the intact ECM, cell-to-cell contacts, and physical pressures of in vivo WAT conditions; additionally, SWAT maintains a robust transcriptional profile, sensitivity to exogenous chemical signaling, and whole tissue function. SWAT represents a simple, reproducible, and effective method of primary adipose culture. Potentially, it is a broadly applicable platform for research in WAT physiology, pathophysiology, metabolism, and pharmaceutical development.

White adipose tissue (WAT) has critical deficiencies in its current primary culture models, hindering pharmacological development and metabolic studies. Here, we present a protocol to produce an adipose microphysiological system by sandwiching WAT between sheets of stromal cells. This construct provides a stable and adaptable platform for primary WAT culture.

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

行为学

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

将人 iPSC 稳健分化为纯脂肪细胞群以研究脂肪细胞相关疾病

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

<meta charset="utf8"></head><body>用于纤维成脂祖细胞培养的小鼠心脏和骨骼肌消化

Fibro-Adipogenic Progenitor Isolation, Expansion, and Differentiation from the Spiny Mouse Model

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are tissue-resident cells that are able to differentiate into adipocytes, fibroblasts, and chondrocytes. Fibro-adipogenic progenitors are fundamental for orchestrating tissue regeneration as they are responsible for extracellular matrix remodeling after injury. This study focuses on investigating the specific role of fibro-adipogenic progenitors in spiny mouse cardiac repair and skeletal muscle regeneration. To this end, a protocol has been optimized for the purification of spiny mouse fibro-adipogenic progenitors by flow cytometry from enzymatically dissociated skeletal and cardiac muscle. The population obtained from this protocol is capable of expanding in vitro, and can be differentiated to myofibroblasts and adipocytes. This protocol offers a valuable tool for researchers to examine the distinctive properties of spiny mouse, and to compare them to the Mus musculus. This will provide insights that could advance the understanding of regenerative mechanisms in this intriguing model.

<meta charset="utf8"></head><body>作者聚焦：使用研究棘状小鼠模型的创新方案简化细胞分析

This protocol outlines the isolation of skeletal and cardiac muscle fibro-adipogenic progenitors (FAPs) from spiny mouse (Acomys) via enzymatic dissociation and fluorescence-activated cell sorting. The FAPs obtained from this protocol can be effectively expanded and differentiated to myofibroblasts and adipocytes.

来自 Spiny 小鼠模型的 Fibro-Adipogenic Progenitor 分离、扩增和分化

Due to its exceptional repair program, the spiny mouse is an emerging research model for regenerative medicine. Fibro-adipogenic progenitors are ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

人网膜和皮下脂肪组织 CD34+CD31+ 内皮细胞的分离、扩增和脂肪诱导

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

免疫与感染

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

用于研究基质细胞代谢串扰的人类3D细胞外基质-脂肪培养模型

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

生物工程

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 ...

一个有效的方法获取反分化脂肪细胞

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