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 border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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&#39;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

Research

JoVE Journal

Medicine

9.2K 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

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

科研

医学

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

肺动脉瓣使用异位心脏移植小鼠模型移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Cerenkov Luminescence Imaging of Interscapular Brown Adipose Tissue

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely related to metabolism and energy expenditure, and its dysfunction is one important contributor for obesity and diabetes. Contrary to previous belief, recent PET/CT imaging studies indicated the BAT depots are still present in human adults. PET imaging clearly shows that BAT has considerably high uptake of 18F-FDG under certain conditions. In this video report, we demonstrate that Cerenkov luminescence imaging (CLI) with 18F-FDG can be used to optically image BAT in small animals. BAT activation is observed after intraperitoneal injection of norepinephrine (NE) and cold treatment, and depression of BAT is induced by long anesthesia. Using multiple-filter Cerenkov luminescence imaging, spectral unmixing and 3D imaging reconstruction are demonstrated. Our results suggest that CLI with 18F-FDG is a practical technique for imaging BAT in small animals, and this technique can be used as a cheap, fast, and alternative imaging tool for BAT research.

In this video report, we show the application of Cerenkov Luminescence Imaging (CLI) for interscapular brown adipose tissue in mice under activated and depressed conditions.

肩胛间褐色脂肪组织的切伦科夫发光成像

Brown adipose tissue (BAT), widely known as a &#8220;good fat&#8221; plays pivotal roles for thermogenesis in mammals. This special tissue is closely ...

Localization, Identification, and Excision of Murine Adipose Depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

Due to the drastic and negative connection between obesity and other comorbidities, research on the role adipose plays in disease and overall health is warranted. We present a protocol for the isolation and excision of adipose depots allowing for the study of adipose using in situ and in vitro methods.

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

Human Brown Adipose Tissue Depots Automatically Segmented by Positron Emission Tomography/Computed Tomography and Registered Magnetic Resonance Images

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in humans. Detecting BAT is typically confirmed by the uptake of the injected radioactive tracer 18F-Fluorodeoxyglucose (18F-FDG) into adipose tissue depots, as measured by positron emission tomography/computed tomography (PET-CT) scans after exposing the subject to cold stimulus. Fat-water separated magnetic resonance imaging (MRI) has the ability to distinguish BAT without the use of a radioactive tracer. To date, MRI of BAT in adult humans has not been co-registered with cold-activated PET-CT. Therefore, this protocol uses 18F-FDG PET-CT scans to automatically generate a BAT mask, which is then applied to co-registered MRI scans of the same subject. This approach enables measurement of quantitative MRI properties of BAT without manual segmentation. BAT masks are created from two PET-CT scans: after exposure for 2 hr to either thermoneutral (TN) (24 &#176;C) or cold-activated (CA) (17 &#176;C) conditions. The TN and CA PET-CT scans are registered, and the PET standardized uptake and CT Hounsfield values are used to create a mask containing only BAT. CA and TN MRI scans are also acquired on the same subject and registered to the PET-CT scans in order to establish quantitative MRI properties within the automatically defined BAT mask. An advantage of this approach is that the segmentation is completely automated and is based on widely accepted methods for identification of activated BAT (PET-CT). The quantitative MRI properties of BAT established using this protocol can serve as the basis for an MRI-only BAT examination that avoids the radiation associated with PET-CT.

The method presented here uses 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET-CT) and fat-water separated magnetic resonance imaging (MRI), each scanned following 2 hr exposure to thermoneutral (24 &#176;C) and cold conditions (17 &#176;C) in order to map brown adipose tissue (BAT) in adult human subjects.

人类的褐色脂肪组织的仓库的正电子发射断层扫描/计算机断层扫描自动分段及注册磁共振图像

Reliably differentiating brown adipose tissue (BAT) from other tissues using a non-invasive imaging method is an important step toward studying BAT in ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

A Technique for Subcutaneous Abdominal Adipose Tissue Biopsy via a Non-diathermy Method

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To obtain subcutaneous adipose tissue biopsies in the abdominal area, researchers and physicians use either a surgical or a needle-based technique. However, surgical subcutaneous fat biopsies can offer tissue samples that may provide a more comprehensive overview of the complexities of biological indices in white adipose tissue. Usually, a surgical adipose tissue biopsy includes a diathermy treatment for cauterizing blood vessels to prevent excessive bleeding. Nevertheless, side effects, such as flash fires and skin lesions in the tissue, have been reported after diathermy. Therefore, we aimed to standardize a surgical abdominal adipose tissue biopsy performed under local anesthesia using a non-diathermy method. We conducted 115 subcutaneous adipose tissue biopsies in healthy men using a non-diathermy abdominal surgical biopsy method. Our results showed three cases of excessive post-operation bleeding out of 115 operations (2.61%).In conclusion, our standardized subcutaneous abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men at the bedside, with minimal side effects.

We standardized an abdominal adipose tissue biopsy using a non-diathermy method performed under local anesthesia. Three cases of excessive post-operation bleeding out of 115 operations (2.61%) occurred.We conclude that an abdominal adipose tissue surgical biopsy using a non-diathermy method can be safely applied to healthy men.

经非透法皮下腹脂肪组织活检技术

Adipose tissue biopsies offer tissue samples that, upon analysis, may provide insightful overviews of mechanisms relating to metabolism and disease. To ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.

Here, we present a protocol to produce an adipose tissue-derived stromal vascular fraction and its application to improve knee functions by regenerating cartilage-like tissue in human patients with osteoarthritis.

制备脂肪组织基质血管部分用于潜在软骨再生的临床协议

Osteoarthritis (OA) is one of the most common debilitating disorders.&#160;Recently, numerous attempts have been made to improve the functions of the ...