我们要感谢詹妮弗彼得罗西诺和大卫DiSilvestro的编辑帮助。这项研究是由美国蛋包饭和奖多项10040042从诺和诺德制药公司，以及由食品创新中心，办公室国际事务中心高级功能食品的研究，并在创业俄勒冈州立大学以及支持由奖号20020728美国国家科学基金会授予EEC-0914790（LJL）。来自国家研究资源中心说明是由奖号R21OD017244（OZ）和UL1RR025755（OSUCCC）支持的项目，由卫生（OD）的主任，国家机构的办公室医学研究和资助和支持的美国国立卫生研究院路线图NCI P30CA16058。内容完全是作者的责任，并不一定代表美国国家研究资源中心和美国国立卫生研究院的官方意见。

该研究方案经美国俄亥俄州立大学伦理委员会。动物实验批准IACUC协议。所有步骤都在2级生物安全柜，带层流下进行。我们遵循所有标准的安全要求和程序。用于制备微胶囊的微胶囊化技术已经执行如所述17,18。 1.准备材料<ol><li>制备出10毫升2％藻酸钠溶液在高压灭菌生理盐水（0.9％NaCl的水）。准备在生理盐水0.05％PLL溶液。前一天制备这些溶液，并搅拌过夜。使用前用0.22微米的过滤器过滤的解决方案。 </li><li>制备50mM柠檬酸钠和100mM的CaCl 2中的0.9％NaCl溶液和高压灭菌。 </li><li>高压釜所有解决方案，针，电极和烧杯。彻底清洁的针具用导线将避免堵塞。 </li><li>确定细胞的适量的要用于胶囊（都需要良好的每平方厘米102的微胶囊，有大约每胶囊18 500个细胞）。使用将1ml海藻酸钠每2百万个细胞。 </li><li>准备一个"成纤维细胞生长培养基含10％小牛血清，和100U / ml青霉素/链霉素中高葡萄糖（4500毫克/升葡萄糖）的Dulbecco氏改良的Eagle氏培养基（DMEM）。 <ol><li>准备一个"分化培养基I&#39;含有10％胎牛血清，10μg/ ml的胰岛素，1μM的地塞米松，0.5mM的3-异丁基-1-甲基黄嘌呤，和100U / ml青霉素/链霉素的DMEM中。 </li><li>准备一个"分化培养基Ⅱ&#39;含有10％胎牛血清，10μg/ ml的胰岛素，并在DMEM 100U / ml青霉素/链霉素。 </li></ol></li><li>制备含有每10ml无线电免疫沉淀测定缓冲液一种蛋白酶抑制剂片剂裂解缓冲液（RIPA缓冲液）。 </li></ol> 2.海藻酸钠微珠准备（图1A） <ol><li>删除旧的媒体，从细胞培养瓶。冲洗细胞10毫升的PBS。带来的细胞在悬浮液用0.25％胰蛋白酶-EDTA（每一合流T175烧瓶2ml）中。 </li><li>计数使用血球细胞的细胞悬浮液。使用10个毫升一份从细胞悬浮液中，并根据制造商的说明计数细胞。离心剩余的细胞在离心培养基中于480×g离心，在室温下放置5分钟。 </li><li>暂停在海藻酸钠细胞沉淀步骤1.4中所述。传送海藻酸钠细胞溶液至5毫升注射器。 </li><li>除去溶液中的气泡，添加了23号针头和倒置注射器创建1毫升袋的空气。 </li><li>放置含144毫升下封装的针管口的100mM的CaCl 2溶液的小烧杯（180毫升）中。附加电极的封装与尖端approximately 2.5厘米的100mM的CaCl 2溶液的表面上方。 </li><li>放置紧紧包含在注射器泵的藻酸钠细胞溶液的注射器。连接橡胶管，以在注射器的开口。推动柱塞直到海藻酸钠细胞液通过导管进入一半。调整电压为5.4千伏。设置12.06毫米直径在注射器泵。调整速度至3毫升/小时。启动泵。打开封装并保持电压5.4千伏。 </li><li>关闭罩的窗口，并避免任何不必要的振动，直到形成藻酸盐微珠的末端。毕竟溶液通过针，与PLL涂层固化前为另外20分钟的100毫米氯化钙2溶液海藻酸钠球形微珠。 </li></ol> 3.微珠涂料与PLL（图1B） <ol><li>除去含有藻酸钠单元球状微珠的烧杯中，并转移这些为MicroBEADS到50毫升离心管中。取下微珠颗粒氯化钙溶液。 </li><li>通过加入30ml的0.9％NaCl的洗涤藻细胞球状微珠。摇动管轻轻用手。除去的0.9％NaCl，用25毫升吸管后的微珠已经通过重力沉淀。重复两次，总共3次洗涤。 </li><li>使用加入10ml 0.05％的PLL溶液每1毫升藻酸钠溶液。添加0.05％PLL和涡旋以每分钟1000转进行10分钟。 注意:通常情况下，10分钟是足够的PLL涂层。 </li><li>后形成PLL大衣，除去PLL解决方案，如在3.2中描述洗胶囊3倍。 </li></ol> 4.去除海藻酸钠核心（图1C） <ol><li>加入30毫升50mM柠檬酸钠溶液。等待5分钟，或直到所有的藻酸钠溶解。洗涤胶囊的三倍，在步骤3.1.1中描述。 </li><li>取出的0.9％NaCl，加入20毫升培养培养基的50ml管中和含有细胞所有胶囊转移到细胞培养瓶。处理标准细胞培养条件下18封装细胞。 </li></ol> 5. 体外应用到研究异种移植物和宿主细胞相互作用或代谢流入动力学/流出细胞之间（图2） <ol><li>培养宿主细胞的24孔板直到融合的联合培养。使用"成纤维细胞生长培养基"为培养脂肪前体细胞。 </li><li>转移到微胶囊24孔含有汇合板的宿主细胞。添加的微胶囊，以实现一个单层（102微胶囊/孔厘米2）。 </li><li>诱导前脂肪细胞分化分化培养基I.每48小时，改变媒体到分化培养基Ⅱ6天。裂解细胞，RIPA缓冲液。用50微克蛋白每使用条件印迹分析蛋白的表达。 </li></ol> 6. 在体内应用的Treatm肥胖的ENT（图3） <ol><li>混合4％异氟烷的氧气麻醉诱导和2％异氟醚与氧为维持麻醉。确认足够的麻醉深度脚趾捏。 <ol><li>取适量涂抹于眼止痒软膏，以保护角膜干燥。 </li></ol></li><li>使用包封的细胞被永久标记的人工荧光蛋白，如绿色荧光蛋白（GFP）。 </li><li>使用3毫升注射器和20号针头注入封装细胞。 <ol><li>注入0.5×10 6个细胞悬浮于0.2ml PBS中到每个IAB脂肪库将位于一个性腺和肾脏之间腹膜内区域， 如图3，使用PBS中的此量和细胞数为小鼠的平均重量40克。调整中具有不同重量或用于确定剂量依赖性影响小鼠细胞数量和体积。 </li></ol></li></ol>

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No conflicts of interest declared.

各种方法已被用于包封细胞，包括干燥，挤出，和乳液19。在该方法中，藻酸盐珠粒通过针挤出，然后涂上PLL和藻酸盐核心会溶解以完成封装。虽然这种方法已使用多年，形成具有所需尺寸和球状珠是仍然具有挑战性。胶囊的大小是高度依赖于褐藻酸钠溶液中，挤出机直径和针尖端和氯化钙溶液20之间的距离的粘度。针尖和氯化钙溶液的表面之间的距离越短，则较?...

慢性疾病1发病率越来越高已经加强了对治疗的细胞群2的移植。同源或异基因干细胞可用于这些应用2中最常用的细胞类型。然而，这些治疗不允许分化和干细胞的迁移植入后的控制，并没有成本效益。遗传修饰的细胞与有益功能的移植预期改善许多疾病的治疗。然而，遗传细胞修饰由宿主的免疫系统所识别，因此，这些处理需要免疫抑制3。细胞产生胰岛素的封装已经制定了张大夫4。该技术是基于细胞的藻酸盐微滴被浸入氯化钙溶液的封装。藻酸盐分子由甘露糖醛（M）和古洛糖醛酸的（G）和可被Ca被连接2+。凝胶化后，将珠粒悬浮聚-L-赖氨酸（PLL）的解决方案。在此步骤期间，PLL结合G和M中的藻酸盐分子其中规定的胶囊型的膜。胶囊的膜的孔隙率可以通过改变M和PLL浓度，孵育时间和温度来调节。 PLL的结合也依赖于类型和藻酸盐的浓度。交联的钙离子藻酸盐基质，是不稳定的，在生理环境中或在普通缓冲溶液具有高浓度的磷酸盐和柠檬酸根离子。这些缓冲器可以从藻中提取的Ca 2+和液化的核心。海藻酸钠核心的液化提供空间胶囊细胞运动和增长中。细胞封装在聚阴离子藻酸盐与聚阳离子多聚-L-赖氨酸（APL）是不可透过免疫球蛋白，但有营养素涌入毒素和流出。这些APL的特性使长期苏移植到基因不同的主机后rvival封装的细胞。 Elliott 等人报道注入59年后运转包封猪胰腺细胞中的人类患者的生存。 封装技术可分为微囊（3-800微米）和macroencapsulation（大于1000微米）。微囊比大包囊6更耐用。自从被发现由张医生和他的同事在1964年，微胶囊已被广泛用于生产合成代谢胰岛素的细胞，其他激素和生物活性分子7的封装。这些治疗面临的宿主组织，包括肝纤维化和免疫反应8几个挑战。最初，与生物聚合物的质量的副作用也得到了解决。然而，合成代谢细胞移植仍然发起的副作用，如纤维化，如激素overpr的结果一个专门的腺体之外oduction。 近几十年来，肥胖和2型糖尿病已达到流行病的程度9。成年的人超过30％的全球超重和肥胖10。增加腹内（IAB）脂肪形成增加的慢性炎症的发病率，促进2型糖尿病，心血管疾病，某些癌症和其他并发症11-13。若干条证据表明，与IAB脂肪相关发病可由特定脂肪细胞被避免。最近的研究已经表明，移植皮下脂肪细胞的成IAB区域可以改善代谢，降低肥胖和胰岛素抵抗的啮齿动物体内 14。有效减少肥胖和胰岛素抵抗的已用能够在热15,16的形式耗散能量的产热脂肪细胞相关联。脂肪细胞的产热变形可通过稳定转染来实现基因参与线粒体解偶联质子，如解偶联蛋白1（UCP1）或基因调节UCP1等生热基因15,16的表达。我们最近的研究表明，缺乏乙醛脱氢酶1 A1（ALDH1A1）导致的脂肪IAB的产热重塑，减少肥胖和胰岛素抵抗在这些小鼠17,18。值得注意的是，产热ALDH1A1缺陷的封装（ALDH1A1 - / - ）前脂肪细胞介导的肥胖野生型小鼠在IAB脂肪相同的治疗效果，这表明对于治疗的IAB脂肪18新的治疗机会。在实验环境中，封装细胞使研究人员能够研究具有成本效益的方式19项具体细胞群的影响。在这里，我们在肥胖的小鼠模型中进行讨论的产热分解代谢细胞系的封装和其实验室和治疗应用的方法。本协议描述牛逼用于微胶囊的生产（ 图1）重稀土元素的阶段:藻酸盐微球（ 图1A），形成聚阳离子多聚-L-赖氨酸（PLL）的微珠的表面（ 图1B）上的膜，并在除去的形成藻芯（ 图1C）。

<table><tbody><tr><td>Encapsulation device (VAR V1)</td><td>Nisco</td><td>LIN-0042</td><td>None</td></tr><tr><td>KD scientific syringe pump</td><td>KD scientific</td><td>780100Y</td><td>None</td></tr><tr><td>Olympus microscope&amp;nbsp;</td><td>Olympus Optical</td><td>IX70-S8F2</td><td>None</td></tr><tr><td>Sodium alginate</td><td>Sigma</td><td>MKBP8122V</td><td>None</td></tr><tr><td>Poly-L-lysine hydrobromide (PLL)</td><td>Sigma</td><td>020M5006V</td><td>None</td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>SLBJ2662V</td><td>None</td></tr><tr><td>Sodium citrate tribasic dihydrate</td><td>Sigma</td><td>030M0200</td><td>None</td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>SLBD2595V</td><td>None</td></tr><tr><td>Mini-PROTEAN TGX Gels</td><td>Bio-Rad</td><td>456-1093</td><td>None</td></tr><tr><td>ATGL primary antibody (from rabbit)</td><td>Cell Signaling</td><td>2138S</td><td>None</td></tr><tr><td>Secondary anti body (anti rabbit)</td><td>LI-COR</td><td>926-68071</td><td>None</td></tr><tr><td>Radio-Immunoprecipitation Assay (RIPA) buffer</td><td>Boston BioProducts</td><td>D25Y6Z</td><td>None</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>RNBD2893</td><td>None</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200-056</td><td>None</td></tr><tr><td>Cortizone 10 anti-itch ointment</td><td>Cortizone 10</td><td>C4029138</td><td>None</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11965-092</td><td>None</td></tr><tr><td>Newborn calf serum (CS)</td><td>Sigma</td><td>N4762</td><td>None</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma</td><td>F4135</td><td>None</td></tr><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Sigma</td><td>I0516</td><td>None</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>None</td></tr><tr><td>Insulin (bovine)</td><td>Sigma</td><td>I5879</td><td>None</td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>4693159001</td><td>None</td></tr></tbody></table>

encapsulation thermogenic preadipocytes for transplantation into adipose tissue depots

电池封装的开发是为了半透膜中俘获的活细胞。嫁接封装细胞所用的处理过的宿主组织中交换的低分子量代谢物，以达到长期存活。半透膜允许移入的封装细胞以避免排斥反应的免疫系统。封装过程是为了使生物活性化合物，诸如胰岛素，其他激素和细胞因子的受控释放。在这里，我们描述了用于分解代谢细胞，其消耗的脂质为产热和能量耗散（产热）的肥胖小鼠的腹腔内脂肪组织的封装的方法。产热分解代谢的细胞封装可能潜在地适用于肥胖的预防和治疗2型糖尿病。分解代谢细胞的另一个潜在的应用可​​以包括由醇或其他有毒的代谢物和环境污染物解毒。

图1显示了微珠生产的每一个步骤可以在显微镜下进行控制。 图2A示出了如何共同培养的脂肪细胞与包被细胞的一个单层。 图2B是使用脂肪细胞/微胶囊共培养物的定量研究的代表性示例是在第5裂解液描述采用Western blot脂肪细胞进行了分析。包封细胞没有在该实验分析。初级ATGL和β肌动蛋白抗体被用在1:1000稀释。 ATGL的比值β肌动蛋白示的三个独立实验的平均?...

Watch this Scientific Journal Video about Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots at JoVE.com

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

encapsulation-thermogenic-preadipocytes-for-transplantation-into

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.3K 次观看.  The Ohio State University. Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

视频: Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

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 ...

科研

行为学

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

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

米色/布里特细胞的间质血管细胞的分离和分化

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

生物学

Three-Dimensional Culture of Vascularized Thermogenic Adipose Tissue from Microvascular Fragments

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for the design of personalized microtissues for health screening and drug testing. Current strategies are often quite complex and fail to accurately fully depict the multicellular and functional properties of thermogenic adipose tissue. Microvascular fragments, small intact microvessels comprised of arteriole, venules, and capillaries isolated from adipose tissue, serve as a single autologous source of cells that enable vascularization and adipose tissue formation. This article describes methods for optimizing culture conditions to enable the generation of three-dimensional, vascularized, and functional thermogenic adipose tissues from microvascular fragments, including protocols for isolating microvascular fragments from adipose tissue and culture conditions. Additionally, best practices are discussed, as are techniques for characterizing the engineered tissues, and sample results from both rodent and human microvascular fragments are provided. This approach has the potential to be utilized for the understanding and development of treatments for obesity and metabolic disease.

Here, we present a detailed protocol outlining the use of microvascular fragments isolated from rodent or human fat tissue as a straightforward approach to engineer functional, vascularized beige adipose tissue.

微血管碎片血管化产热脂肪组织的三维培养

Engineering thermogenic adipose tissue (e.g., beige or brown adipose tissues) has been investigated as a potential therapy for metabolic diseases or for ...

生物工程

Fat-Covered Islet Transplantation Using Epididymal White Adipose Tissue

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this procedure. However, intraperitoneal islet transplantation has some limitations, including poor transplant efficacy, difficult graft detection ability, and a lack of graftectomy capability for post-transplant analysis. In this paper, "fat-covered islet transplantation", an intraperitoneal islet transplantation method that utilizes epididymal white adipose tissue, is used to assess the therapeutic effects of bioengineered islets. The simplicity of the method lies in the seeding of islets onto epididymal white adipose tissue and using the tissue to cover the islets. While this method can be categorized as an intraperitoneal islet transplantation technique, it shares characteristics with intra-adipose tissue islet transplantation. The fat-covered islet transplantation method demonstrates more robust therapeutic effects than intra-adipose tissue islet transplantation, however, including the improvement of blood glucose and plasma insulin levels and the potential for graft removal. We recommend the adoption of this method for assessing the mechanisms of islet engraftment into white adipose tissue and the therapeutic effects of bioengineered islets.

This fat-covered islet transplantation method is suitable for the detection of engrafted islets in the intraperitoneal cavity. Notably, it does not require the use of biobinding agents or suturing.

使用附睾白色脂肪组织进行脂肪覆盖的胰岛移植

Islet transplantation is a cellular replacement therapy for severe diabetes mellitus. The intraperitoneal cavity is typically the transplant site for this ...

医学

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

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

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

用于细胞表征和分泌特征的人类脂肪组织微分裂

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

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 ...

<meta charset="utf8"></head><body>人 SATMVEC：分离和脂肪细胞串扰研究

Studying Adipose Endothelial Cell/Adipocyte Cross-Talk in Human Subcutaneous Adipose Tissue

Microvascular endothelial cells (MVECs) have many critical roles, including control of vascular tone, regulation of thrombosis, and angiogenesis. Significant heterogeneity in endothelial cell (EC) genotype and phenotype depends on their vascular bed and host disease state. The ability to isolate MVECs from tissue-specific vascular beds and individual patient groups offers the opportunity to directly compare MVEC function in different disease states. Here, using subcutaneous adipose tissue (SAT) taken at the time of insertion of cardiac implantable electronic devices (CIED), we describe a method for the isolation of a pure population of functional human subcutaneous adipose tissue MVEC (hSATMVEC) and an experimental model of hSATMVEC-adipocyte cross-talk.
hSATMVEC were isolated following enzymatic digestion of SAT by incubation with anti-CD31 antibody-coated magnetic beads and passage through magnetic columns. hSATMVEC were grown and passaged on gelatin-coated plates. Experiments used cells at passages 2-4. Cells maintained classic features of EC morphology until at least passage 5. Flow cytometric assessment showed 99.5% purity of isolated hSATMVEC, defined as CD31+/CD144+/CD45-. Isolated hSATMVEC from controls had a population doubling time of approximately 57 h, and active proliferation was confirmed using a cell proliferation imaging kit. Isolated hSATMVEC function was assessed using their response to insulin stimulation and angiogenic tube-forming potential. We then established an hSATMVEC-subcutaneous adipocyte co-culture model to study cellular cross-talk and demonstrated a downstream effect of hSATMVEC on adipocyte function.
hSATMVEC can be isolated from SAT taken at the time of CIED insertion and are of sufficient purity to both experimentally phenotype and study hSATMVEC-adipocyte cross-talk.

<meta charset="utf8"></head><body>作者聚焦：通过皮下脂肪组织微血管研究洞察心脏代谢疾病

Here, we describe a protocol for isolating, culturing, and phenotyping microvascular endothelial cells from human subcutaneous adipose tissue (hSATMVECs). Additionally, we describe an experimental model of hSATMVEC-adipocyte cross-talk.

封装产热前脂肪细胞移植到脂肪组织仓库

摘要

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此视频中的章节

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

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

米色/布里特细胞的间质血管细胞的分离和分化

微血管碎片血管化产热脂肪组织的三维培养

使用附睾白色脂肪组织进行脂肪覆盖的胰岛移植

用于细胞表征和分泌特征的人类脂肪组织微分裂

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰

研究人类皮下脂肪组织中的脂肪内皮细胞/脂肪细胞串扰