Name: a microphysiologic platform for human fat: sandwiched white adipose tissue
Uploaded: 2018-08-15T14:00:00
Description: Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

提交人希望承认由路易斯安那州立的健康科学中心提供的机构支助, 资助了该项目。

所有的任务都是遵守 #8759 和 #9189 的协议, 如 LSUHSC 的 IRB 办公室批准的。所有动物的工作都是遵守协议 #3285 在 LSUHSC IACUC 办事处批准的。
1. 夹细胞片的播种
注: 见图 1。
<ol><li>种子干细胞在大约80% 融合在组织培养板材 (6 cm 或6井板材)。为每一个特警所需, 种子1常规组织培养井和1井相应大小的聚 (n-丙烯酰胺 (pNIPAAm) 涂层组织培养塑?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Institutes+of+Health.+Microphysiological+Systems+Program+focuses+on+a+critical+challenge+in+the+drug+discovery+pipeline.">The National Institutes of Health. Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline.</a> Stem Cell Research and Therapy. 4, (2013).</li><li>Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+and+potential+roles+of+microphysiological+systems+in+biology+and+medicine.">The relevance and potential roles of microphysiological systems in biology and medicine.</a> Experimental Biology and Medicine. 239 (9), 1061-1072 (2014).</li><li>. NIH-CASIS Coordinated Microphysiological Systems Program for Translational Research in Space (#RFA-TR-18-001) Available from: <a target="_blank" href="https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html">https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html</a> (2017)</li><li>Lau, F. H., Vogel, K., Luckett, J. P., Hunt, M., Meyer, A., Rogers, C. L., Tessler, O., Dupin, C. L., St Hilaire, H., Islam, K. N., Frazier, T., Gimble, J. M., Scahill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwiched+White+Adipose+Tissue:+A+Microphysiological+System+of+Primary+Human+Adipose+Tissue.">Sandwiched White Adipose Tissue: A Microphysiological System of Primary Human Adipose Tissue.</a> Tissue Engineering Part C: Methods. 24 (3), (2018).</li><li>Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+microfluidic+channels+by+UV-mediated+graft+polymerization+of+non-fouling+and+'smart'+polymers.">Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and 'smart' polymers.</a> Radiation Physics and Chemistry. 76, 1409-1413 (2007).</li><li>Lin, J. B., Isenberg, B. C., Shen, Y., Schorsch, K., Sazonova, O. V., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+poly(N-isopropylacrylamide)+grafted+onto+microtextured+poly(dimethylsiloxane)+for+aligned+cell+sheet+engineering.">Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.</a> Colloids and Surfaces B: Biointerfaces. 99, 108-115 (2012).</li><li>Turner, W. S., Sandhu, N., McCloskey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering:+Construction+of+a+Multicellular+3D+Scaffold+for+the+Delivery+of+Layered+Cell+Sheets.">Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets.</a> Journal of Visualized Experiments. (92), e51044 (2014).</li><li>Bunnell, B., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+Isolation,+expansion+and+differentiation.">Adipose-derived stem cells: Isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Akiyama, Y., Kikuchi, A., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+poly(N-isopropylacrylamide)+grafted+layer+on+polystyrene+surfaces+for+cell+adhesion/detachment+control.">Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control.</a> Langmuir. 20 (13), 5506-5511 (2004).</li><li>Fried, S. K., Russell, C. D., Grauso, N. L., Brolin, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+lipase+regulation+by+insulin+and+glucocorticoid+in+subcutaneous+and+omental+adipose+tissues+of+obese+women+and+men.">Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.</a> Journal of Clinical Investigation. 92 (5), 2191-2198 (1993).</li><li>Keipert, S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brite/beige+fat+and+UCP1+-+is+it+thermogenesis?.">Brite/beige fat and UCP1 - is it thermogenesis?.</a> Biochimica et Biophysica Acta. 1837 (7), 1075-1082 (2014).</li></ol>

本议定书详细介绍了干细胞在三明治人体白色脂肪组织中的应用;人类 ADSC 细胞系可以通过建立完善的协议15进行隔离。然而, 该系统可以适应个性化的研究要求 (如使用 3T3L 1 细胞三明治小鼠笏)。这个过程涉及处理主要的人体组织。应采用标准安全预防措施;作为 BSL-2 病原体 (如HIV、HepC) 处理人体组织。只在平衡计分卡下直接处理组织。根据机构安全标准, 佩戴所有合适的 PP...

脂肪组织是肥胖的主要器官, 其在美国1的直接每年医疗费用为1470亿美元和2100亿美元。脂肪组织的积聚也会导致其他主要死因, 如心脏病、II 型糖尿病和某些类型的癌症2。体外培养模型对代谢研究和药物开发是必不可少的, 但目前脂肪组织的研究模式有很大的不足。脂肪细胞是脆弱的, 浮力的, 和晚期分化, 不会坚持细胞培养塑料, 因此不能使用传统的细胞培养方法培养。自二十世纪七十年代以来, 一些方法被用来克服这些障碍, 包括使用玻璃盖玻片, 天花板文化, 悬浮培养, 细胞外基质3,4,5,6,7. 然而, 这些方法的特点是细胞死亡和分化, 通常只用于为期两周的研究期。此外, 这些模型不尝试重述本机脂肪微环境, 因为它们不维持完整的 ECM, 脂肪细胞和基质支持细胞之间的相互作用, 也不是在体内相互施加的收缩力单元. 笏。
在缺乏金标准的原发脂肪培养方法的情况下, 脂肪的研究主要依赖于分化前脂肪细胞 (diffAds)。DiffAds 是 multilocular, 黏附和新陈代谢活跃。相比之下, 原发性白脂肪细胞单房性, nonadherent, 并表现出相对较低的新陈代谢。目前的脂肪培养模型未能概括健康成熟脂肪组织的生理, 可能是缺乏 FDA 批准的药物直接靶向脂肪细胞的主要因素。事实上, 缺乏生理的体外器官模型是一个主要的问题, 在大多数器官和疾病。
美国国立卫生研究院 (NIH) 在其立场文件中宣布创建其 Microphysiological 系统 (MPS) 计划, 报告说, 在所有人类药物临床试验中, 2013 的成功率仅为第二阶段和50% 阶段的18%。临床试验8。MPS 方案的设计是为了直接解决在体外单一的能力, 以模型人类生理学。NIH 将松龙定义为由人类主要或干细胞组成的多细胞3D 结构中的培养系统, 这些细胞概括了器官功能。与简化模型的同类, 永生化细胞培养, 松龙应准确模型细胞, 药物细胞, 药物药物, 和器官-药物相互作用9。与短期初级文化方法不同的是, NIH 标准规定了4周文化8的 MPS 的可持续性。MPS 计划的进一步细节可以在 NIH 的 RFAs (#RFA TR-18-001)10中找到。
我们开发了一种简单、新颖、适应性强、价格低廉的脂肪 MPS, 称为 "夹心白脂肪组织" (斯瓦特)11。我们克服了脂肪细胞的自然浮力的 "夹" 切碎的原始脂肪组织之间的脂质基质干细胞 (干细胞) (图 1)。由此产生的3D 构造概括细胞接触和当地脂肪微环境的周围成熟脂肪体与自然脂肪细胞支持的人群。斯瓦特已经通过展示8周的生存能力、对外源信号的反应、脂肪分泌物和植入成为动物模型来证实。

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">10x HBSS</td>
			<td style="width:92px;">Thermofisher</td>
			<td style="width:115px;">14185052</td>
			<td style="width:393px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma-aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermofisher</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 Media</td>
			<td>Thermofisher</td>
			<td>11043023</td>
			<td>Phenol red-free&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250&#181;m Mesh Filter</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2&#181;m Syringe Filter</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL Luer-Lok syringes&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Metal Washers</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3g&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Incubated Orbital Shaker</td>
			<td>VWR</td>
			<td>10020-988</td>
			<td style="width:393px;">Samples should be pitched at 45&#176; angle to facilitate collagenase digestion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Block</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to 37-40&#176;C and placed under Biosafety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to ~75&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialized Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">Upcell Dishes 6cm of 6-multiwell</td>
			<td>Nunc</td>
			<td>174902&#160; or 174901</td>
			<td style="width:393px;">These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &#60;6cm for 6cm dish, &#60;3.5cm for 6-well plate; approximate mass: 6.7g for 6cm dish, 5.1g for 6-well plate</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Plastic Plunger Apparatus</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td>
		</tr>
	</tbody>
</table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

白色脂肪组织 (笏) 在调节体重和日常健康方面起着至关重要的作用。 尽管如此, 现有的主要养殖模式仍存在重大限制, 所有这些模型都未能忠实地重述脂肪微环境或延长笏的生存能力超过两周。 缺乏可靠的初级文化模型严重阻碍了笏代谢和药物开发的研究。为此, 我们利用 NIH 的 microphysiologic 系统标准, 为 "斯瓦特" (夹心白脂肪组织) 的主要文化开发了一个新的平台。我们克服脂肪细胞的自然浮力的夹碎笏簇之间的薄层之间的脂质基质干细胞。 在这个构造中, 笏样本在八周的文化中是可行的。 斯瓦特保持完整的 ECM, 细胞对细胞的接触, 和物理压力的体内笏条件;此外, 斯瓦特还保持了强健的转录剖面, 对外源化学信号的敏感性, 以及整个组织功能。斯瓦特是一种简单、可重复、有效的原发性脂肪培养方法。 潜在的, 它是一个广泛适用的平台, 研究在笏生理学, 病理生理学, 新陈代谢和药物发展。

斯瓦特的生存能力最初是由序列 brightfield 成像的单个笏簇 (n = 12) 大约7.6 周。在这段时间内, 簇仍然固定在单层上。微小的形态学变化观察到个别脂肪细胞翘曲轻微或移位的位置。然而, 脂肪细胞并没有成为 multilocular 随着时间的推移, 表明缺乏分化, 也没有表现出任何明显的迹象死亡, 如细胞起泡, 裂解, 或分裂 (图 2)。两周大的特勤组的脂肪细胞特征?...

Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

白色脂肪组织 (笏) 在其目前的主要培养模式中存在严重缺陷, 阻碍了药理学的发展和代谢研究。在这里, 我们提出一个协议, 以产生一个脂肪 microphysiological 系统的夹笏之间的基质细胞。该构造为原笏培养提供了一个稳定、适应性强的平台。

人体脂肪 Microphysiologic 平台: 夹心白色脂肪组织

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A Novel Digital Platform for a Monitored Home-based Cardiac Rehabilitation Program

一种新型的家庭心脏康复监控数字平台

Despite the evidence that cardiac rehabilitation (CR) reduces the risk of recurrent cardiac events, only a minority of eligible patients are willing to ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

测量个体果蝇黑色素气器酒精镇分时间的高通量方法

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

Tactile Vibrating Toolkit and Driving Simulation Platform for Driving-Related Research

触觉振动工具包和驾驶模拟平台，用于驾驶相关研究

Collision warning system plays a key role in the prevention of driving distractions and drowsy driving. Previous studies have proven the advantages of ...

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

使用妊娠期心理社会压力的Murine模型作为母亲和婴儿精神障碍的翻译相关范式

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...