这项工作是由理查德·托马斯白血病基金，塔塔纪念夫人信托，Northwick公园医院白血病研究信托基金和美国国立卫生研究院研究所（NIHR），英国。

1。脚手架制造和生物功能化支架<ol><li>制作在培养皿磁盘的形式聚氨酯支架（100-250毫米孔径，孔隙率90-95％），使用热致相分离的准备聚合物溶液中二恶烷（添加5wt％），其次是13进程的冻结和后续溶剂升华（ 图1A）。 </li><li>切成0.5×0.5×0.5毫米，事先与ECM蛋白涂层（ 图1B）立方体脚手架磁盘。 </li><li>预湿的支架，由乙醇（70％）中浸泡1分钟，然后转移到磷酸盐缓冲液（PBS）为20分钟。然后离心10分钟，在3630 XG，并增加蛋白质的解决方案。 </li><li>在20分钟在1420 XG蛋白溶液离心支架。 </li><li>为了疏通表面的毛孔，让接种的细胞深入渗透到脚手架，离心支架在910 XG更多的时间为10分钟在PBS。 </li><li>消毒8分钟曝光组合使用230伏，50赫兹，0.14，紫外线灯，浸泡在乙醇2小时（70％）的支架。在此之后，支架在PBS洗两次，然后加入Iscove改性杜尔贝科中等（IMDM培养），辅以30％小牛血清（FBS）和1％青霉素和链霉素（的P / S）。 3天在37°C和5％的CO 2，使用前放置在湿润的孵化器的支架。 24良好的组织培养板（每口井脚手架）被放置在无菌的支架。 </li></ol> 2。单核细胞的分离和脚手架直播<ol><li>从脐带血或白血病骨髓样本中提取人类的跨国公司，利用Ficoll分离Paque密度梯度离心（1500转35分钟）（ 图1C）。 </li><li>重悬在IMDM培养跨国公司含30％胎牛血清和1％，P / S </li><li>种子100μL跨国公司悬架（2×10 6细胞/支架）上使用一个微型的移液器支架。 </li><li>种子棚架5％CO 2下孵育10分钟解决支架的细胞在37°C。 </li><li>每口井补足的IMDM培养1.5毫升含30％胎牛血清和1％的P / S和地方的孵化器孔板在37°C和5％CO 2的实验时间。 </li><li>种子棚架接受所有媒体的日变化。 </li></ol> 3， 原位细胞增殖和形态：MTS，扫描电镜和Cytospins <ol><li> MTS实验：从文化非种子和两个种子棚架和地方在一个干净的新24井板。加入1毫升的媒体和MTS的解决方案，并为3小时孵育200μL，在37°C和5％的CO 2。 <ol><li>在96孔板和使用酶标仪在490 nm处测量吸光度，以8×100μL上清样品;的地方。 </li></ol></li><li> 扫描电子显微镜（SEM）：在不同时间点的日ê文化，消除从媒体与跨国公司接种支架，并固定在4分40 2.5％PBS缓冲戊二醛溶液°C间，然后用PBS洗两次。 <ol><li>脱水乙醇梯度系列（25，50，70，80，90，95，和100％），10分钟和4小时的无菌环境中干各的支架。 </li><li>部分标本，然后溅射涂层在氩气气氛的黄金前2分钟，扫描电镜评价，使用一个20千伏的加速电压。 </li></ol></li><li> cytospins：收集吸气文化在不同的时间点从脚手架的细胞2.0×10 4细胞/幻灯片。 <ol><li>到3分钟1500转离心玻片的细胞。 </li><li>赖特 - 姬姆萨染色的修改，并使用光学显微镜观察染色的幻灯片。的F-查看软成像系统，可用于拍照。 </li><li>洗滑动之前，显微镜下观察。 </li></ol></li><li> 中号<html> ultiphoton分析：在不同的时间点，直到进一步的分析与乙醇蒸气（70％）过夜，然后干燥，储存于-20°C间的固定支架。在此之前与多光子显微镜的部分脚手架为30微米厚的幻灯片，并用PBS湿的可视化。 <ol><li> PBS在室温下30分钟与10％的胎儿牛血清阻断种子棚架。 </li><li>孵育4°C在黑暗中一个主要的单克隆抗体1:50稀释：小鼠抗人CD71的支架过夜。 </li><li>在PBS洗样品三次，染色二抗：抗鼠的Alexa Fluor 488室温在黑暗中1小时。 </li><li>用PBS洗样品三次，使用水排放X60物镜共焦显微镜观察。 </li><li>由脉冲激光器在488 nm荧光的Alexa Fluor 488是兴奋。 volocity 5.3.2软件可用于后续的图像分析。 </li></ol></li></ol>jove_title“&gt; 4。流式细胞仪分析细胞人口<ol><li>在流式细胞仪的细胞分析（下称“财委会”）之前，与相应的免疫抗体检测标记的细胞。根据选定的抗体检测的样品准备。对于跨国公司检测的抗体以下组合：CD45-FITC/CD71-PE/CD235a-PE-Cy5。 </li><li>吸在不同的时间点和离心机的从脚手架细胞。一个约1×10 6个细胞的细胞沉淀溶于100μLFC缓冲液（PBS + 0.1％的叠氮化钠）和添加10μL每个抗体的荧光染料。 30分钟在4°C孵育细胞，用PBS洗两次，终于重新暂停在FC缓冲区。 </li><li>流式细胞仪染色装入样品与同型对照设置的“消极”的抗体表达和校准流式细胞仪控制与检测渠道。 </li><li>阅读的样品染色瓦特<html>随着三种不同的抗体和终于代表使用WinList软件数据。 </li></ol> 5。代表结果没有添加外源性生长因子对造血细胞生长动力学的一个例子如图2所示。由于造血功能的异质性，两种不同的细胞：正常和异常造血干细胞进行了阐述。 图2A中，人类CBMNC的细胞增殖是显而易见的，经过28天中文化图2B显示在模仿的人原代白血病细胞的生长动力学。测量细胞代谢活性的关系，以吸收使用MTS检测细胞增殖评估。在这两种情况下，细胞增殖，并在模型建立。生长动力学的差异观察，建立了文化的正常造血细胞的速度比白血病细胞。 _content“收获细胞的形态，即使经过28天的文化，在缺乏外源性生长因子是典型的正常造血干细胞（ 图3A）和白血病细胞（ 图3B）。支架的中央部分进行了分析后由SEM支架被从文化和显示的传播种子细胞整个脚手架，在集群和“利基，像”结构（ 图3A“与乙”）上立足。 图C显示的孔隙大小和分布在1非种子选手支架作为对照，经过28天的多光子显微镜被用来突出三维支架原位细胞内的分布，这表明在中央部分的脚手架红岛屿的存在标记的表达（ 图4）在红细胞CD71的，这是积极的。这证明了的成熟和成熟细胞杜的重要性环红细胞。最后，流式细胞仪检测细胞前播种图显示：人类CBMNCs和图5B说明异常造血细胞的原代白血病细胞中的造血细胞， 图5A代表正常的造血干细胞表型的差异。的CD235a各级+和CD45 +相应的红细胞和白细胞分别高于正常样本中突出的白血病hemoblastic性质的白血病。 <img alt="图1" src="/files/ftp_upload/3654/3654fig1.jpg" /> 图1。插图在PU脚手架制造和生物功能化所涉及的过程。一）PU溶解在二恶烷（添加5wt％）和热致相分离过程和随后的溶剂升华脚手架生产，由Safinia 等 13。二）脚手架磁盘，然后切成诠释Ø立方体0.5×0.5×0.5毫米，然后涂上与细胞外基质（ECM）的蛋白质离心。 c）跨国公司提取人脐CB或使用了微管密度梯度离心法和种子在PU支架（2×10 6细胞/支架）从骨髓的愿望。 <img alt="图2" src="/files/ftp_upload/3654/3654fig2.jpg" /> 图2细胞增殖测定，使用MTS检测：A）使用人类脐带血跨国公司，B）使用人原代白血病细胞。列显示在聚氨酯支架生长的细胞随着时间的推移，当无外源性细胞因子除了接种。 <img alt="图3" src="/files/ftp_upload/3654/3654fig3.jpg" /> 图3：细胞形态和支架周围分布，使用cytospins，scanni纳克电子显微镜。 （AB）的代表赖特 - 姬姆萨染色cytospins一）脊髓从聚氨酯棚架收集外周血单个核细胞经过28天的文化，和B）骨吸气白血病细胞收集聚氨酯支架，经过14天的文化。细胞因子条件下进行了两个实验。 （A&#39;-B“））种子脐血跨国公司和B&#39;），白血病细胞培养后28天，和C）控制支架无细胞的PU支架A的SEM照片代表中央部分”。 <img alt="图4" src="/files/ftp_upload/3654/3654fig4.jpg" /> 图4。多光子显微镜与脐血跨国公司的一个PU脚手架播种后28ð在文化和红标记CD71的染色。 <img alt="图5" src="/files/ftp_upload/3654/3654fig5.jpg" /> 图5。流式细胞仪三维点图染色CD45，CD71和CD235a表面表达标记。还提出了相对荧光强度比较获得的同型对照。 A组显示上述标记阳性的脐血单核细胞，B组显示了人类主要的白血病细胞。

<ol>
	<li>Orkin, S., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132, 631-644 (2008).</li><li>Spradling, A., Drummond-Barbosa, D., Kai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+find+their+niche.">Stem cells find their niche.</a> Nature. 414, 98-104 (2001).</li><li>Panoskaltsis, N., Mantalaris, A., Wu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+a+mimicry+of+bone+marrow+tissue+ex+vivo.">Engineering a mimicry of bone marrow tissue ex vivo.</a> J Biosci. Bioeng. 100, 28-35 (2005).</li><li>Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457, 92-96 (2009).</li><li>Mantalaris, A., Bourne, P., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+osteoclasts+in+a+three-dimensional+bone+marrow+culture+system.">Production of human osteoclasts in a three-dimensional bone marrow culture system.</a> Biochem. Eng. J. 20, 189-196 (2004).</li><li>Placzek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+bioprocessing:+fundamentals+and+principles.">Stem cell bioprocessing: fundamentals and principles.</a> J. R. Soc. Interface. 6, 209-232 (2009).</li><li>Dexter, T., Testa, N., Prescott, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 14, 387-405 (1976).</li><li>Piacibello, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+growth+factor+requirement+of+primitive+cord+blood+hematopoietic+stem+cell+for+self-renewal+and+amplification+vs+proliferation+and+differentiation.">Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation.</a> Leukemia. 12, 718-727 (1998).</li><li>Yoshida, T., Takagi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+processing+engineering+for+ex+vivo+expansion+of+hematopoietic+cells:+a+review.">Cell processing engineering for ex vivo expansion of hematopoietic cells: a review.</a> Biochemical Engineering Journal. 20, 99-106 (2004).</li><li>Lim, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intelligent+bioprocessing+for+haemotopoietic+cell+cultures+using+monitoring+and+design+of+experiments.">Intelligent bioprocessing for haemotopoietic cell cultures using monitoring and design of experiments.</a> Biotechnol. Adv. 25, 353-368 (2007).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+three-dimensional+scaffold+for+ex+vivo+biomimicry+of+human+acute+myeloid+leukaemia.">The development of a three-dimensional scaffold for ex vivo biomimicry of human acute myeloid leukaemia.</a> Biomaterials. 31, 2243-2251 (2010).</li><li>Mortera-Blanco, T., Mantalaris, A., Bismarck, A., Aqel, N., Panoskaltsis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cytokine-free+expansion+of+cord+blood+mononuclear+cells+in+three-dimensional+scaffolds.">Long-term cytokine-free expansion of cord blood mononuclear cells in three-dimensional scaffolds.</a> Biomaterials. 32, 9263-9270 (2011).</li><li>Safinia, L., Datan, N., Hohse, M., Mantalaris, A., Bismarck, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+methodology+for+the+effective+surface+modification+of+porous+polymer+scaffolds.">Towards a methodology for the effective surface modification of porous polymer scaffolds.</a> Biomaterials. 26, 7537-7547 (2005).</li></ol>

我们什么都没有透露。

这里介绍的体外三维培养系统使我们能够建立一个造血的三维仿生概括了原始的BM结构和独立外源性细胞因子的细胞表型。三维模型提供的结构和微环境，使正常和异常造血细胞的增殖在体内遇到类似的条件。 聚合物支架材料的选择代表了仿生学设计的关键步骤。生物材料中重要的优势，已导致越来越大的兴趣在聚合物模仿体内环境提供所需的建筑，结构...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue No</td>
		</tr>
		<tr>
			<td>Dioxan</td>
			<td>Invitrogen</td>
			<td>D20,186-3</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>Invitrogen</td>
			<td>12440-053</td>
		</tr>
		<tr>
			<td>Ficoll-Paque</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
		</tr>
		<tr>
			<td>MTS</td>
			<td>Promega</td>
			<td>G3580</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fluka Biochemika</td>
			<td>49624</td>
		</tr>
		<tr>
			<td>Wright-Giemsa</td>
			<td>Sigma-Aldrich</td>
			<td>WG32</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10108-165</td>
		</tr>
		<tr>
			<td>CD71</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32272</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11001</td>
		</tr>
		<tr>
			<td>CD45-FITC</td>
			<td>BD Pharmigen</td>
			<td>74895</td>
		</tr>
		<tr>
			<td>CD71-PE</td>
			<td>BD Pharmigen</td>
			<td>555537</td>
		</tr>
		<tr>
			<td>CD235a-PE-Cy5</td>
			<td>BD Pharmigen</td>
			<td>555570</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S-8032</td>
		</tr>
	</tbody>
</table>

ex vivo mimicry of normal and abnormal human hematopoiesis

造血干细胞需要一个独特的微环境，以维持血细胞的形成1;骨髓（BM）是一个复杂的三维（3D）组织其中造血称为壁龛2-4空间组织的细胞微环境监管。该组织的骨髓龛是正常或恶性骨髓5的功能或功能障碍的关键。因此， 在使用前体内模仿体内微的更好的理解，将有助于我们澄清6白血病的分子，细胞和微环境因素。 目前，造血干细胞在体外培养中的二维（2D）的组织培养瓶/要求要么与异体或异种基质细胞共培养或添加外源性细胞因子8孔板7。这些条件是人工从体内不同</eM&gt;微环境中，他们缺乏的三维细胞龛和揭露细胞，细胞因子浓度异常高，这可能会导致分化和全能性9,10损失。 在此，我们提出了一种新的三维骨髓培养系统，模拟在体内的三维生长环境，在缺乏外源性生长因子，并支持多向造血。在聚氨酯（PU）制成的这种系统使用的脚手架高度多孔促进高密度，跨越更高的比表面积比传统2D 11单层培养细胞的生长。我们的工作表明，该模型支持脐血（CB）的单核细胞（MNC）的12和外源性细胞因子的情况下，在原代白血病细胞的生长。这种新颖的3D模仿一个人的实验模型，研究造血和探索新的治疗白血病的发展提供了一个可行的平台。

Watch this Scientific Journal Video about Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis at JoVE.com

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

用脐血及白血病骨髓细胞三维培养一种造血系统。该方法的基础上使用涂有细胞外基质蛋白合成的多孔聚氨酯脚手架。这种支架是适应能力，以适应广泛的细胞。

体外拟态正常与异常的人造血

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation1; the bone marrow (BM) is a complex three-dimensional ...

ex-vivo-mimicry-of-normal-and-abnormal-human-hematopoiesis

Research

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Bioengineering

Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

15.4K 次观看.  Imperial College London. 用脐血及白血病骨髓细胞三维培养一种造血系统。该方法的基础上使用涂有细胞外基质蛋白合成的多孔聚氨酯脚手架。这种支架是适应能力，以适应广泛的细胞。

视频: Ex vivo Mimicry of Normal and Abnormal Human Hematopoiesis

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, each offering its own advantages and shortcomings5,6. Most prominently, due to the microscale device dimensions, a high modulus is required to facilitate implantation into living tissue. Conversely, the stiffness of the device should match the surrounding tissue to minimize induced local strain7-9. Therefore, we recently developed a new class of bio-inspired materials to meet these requirements by responding to environmental stimuli with a change in mechanical properties10-14. Specifically, our poly(vinyl acetate)-based nanocomposite (PVAc-NC) displays a reduction in stiffness when exposed to water and elevated temperatures (e.g. body temperature). Unfortunately, few methods exist to quantify the stiffness of materials in vivo15, and mechanical testing outside of the physiological environment often requires large samples inappropriate for implantation. Further, stimuli-responsive materials may quickly recover their initial stiffness after explantation. Therefore, we have developed a method by which the mechanical properties of implanted microsamples can be measured ex vivo, with simulated physiological conditions maintained using moisture and temperature control13,16,17. 
To this end, a custom microtensile tester was designed to accommodate microscale samples13,17 with widely-varying Young's moduli (range of 10 MPa to 5 GPa). As our interests are in the application of PVAc-NC as a biologically-adaptable neural probe substrate, a tool capable of mechanical characterization of samples at the microscale was necessary. This tool was adapted to provide humidity and temperature control, which minimized sample drying and cooling17. As a result, the mechanical characteristics of the explanted sample closely reflect those of the sample just prior to explantation.
The overall goal of this method is to quantitatively assess the in vivo mechanical properties, specifically the Young's modulus, of stimuli-responsive, mechanically-adaptive polymer-based materials. This is accomplished by first establishing the environmental conditions that will minimize a change in sample mechanical properties after explantation without contributing to a reduction in stiffness independent of that resulting from implantation. Samples are then prepared for implantation, handling, and testing (Figure 1A). Each sample is implanted into the cerebral cortex of rats, which is represented here as an explanted rat brain, for a specified duration (Figure 1B). At this point, the sample is explanted and immediately loaded into the microtensile tester, and then subjected to tensile testing (Figure 1C). Subsequent data analysis provides insight into the mechanical behavior of these innovative materials in the environment of the cerebral cortex.

Environmentally-controlled Microtensile Testing of Mechanically-adaptive Polymer Nanocomposites for ex vivo Characterization

A method is discussed by which the in vivo mechanical behavior of stimuli-responsive materials is monitored as a function of time. Samples are tested ex vivo using a microtensile tester with environmental controls to simulate the physiological environment. This work further promotes understanding the in vivo behavior of our material.

环境控制机械自适应聚合物纳米复合材料的微拉伸测试<em&gt;体外</em&gt;表征

Implantable microdevices are gaining significant attention for several biomedical applications1-4. Such devices have been made from a range of materials, ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

成像变性的胶原股<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;通过光引发杂交笼胶原蛋白仿生肽

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Cerebral Blood Flow-Based Resting State Functional Connectivity of the Human Brain using Optical Diffuse Correlation Spectroscopy

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a key hemodynamic parameter related to cerebral oxygen supply. Resting state low frequency fluctuations based on oxygenation contrast have been shown to provide correlations between functionally connected regions. The presented protocol uses optical diffuse correlation spectroscopy (DCS) to assess blood flow-based resting state functional connectivity (RSFC) in the human brain. Results of CBF-based RSFC in human frontal cortex indicate that intra-regional RSFC is significantly higher in the left and right cortices compared to inter-regional RSFC in both cortices. This protocol should be of interest to researchers who employ multi-modal imaging techniques to study human brain function, especially in the pediatric population.

This protocol demonstrates how to measure resting state functional connectivity in the human prefrontal cortex using a custom-made diffuse correlation spectroscopy instrument. The report also discuss practical aspects of the experiment as well as detailed steps for analyzing the data.

使用光学漫反射相关光谱，基于脑血流的休息状态功能连接

To obtain a comprehensive understanding of the human brain, utilization of cerebral blood flow (CBF) as a source of contrast is desired because it is a ...

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and geometric&#160;deformation. Our understanding of the internal relationships between these events has been limited because we can neither &#39;see&#39; inside the muscle nor precisely track the spatio-temporal nature of excitation-contraction dynamics. To resolve these problems, we have constructed a device that combines a suite of imaging modalities. Specifically, it integrates a brightfield microscope to measure local changes of sarcomere length and tissue strain, a fluorescence microscope to visualize the Ca2+ transient, and an optical coherence tomograph to capture the tissue&#39;s geometric&#160;changes throughout the time-course of a cardiac cycle. We present here the imaging infrastructure and associated data collection framework. Data are collected from isolated rod-like tissue structures known as trabeculae carneae. In our instrument, a pair of position-controlled platinum hooks hold each end of an ex vivo muscle sample while it is continuously superfused with nutrient-rich saline solution. The hooks are under independent control, permitting real-time control of muscle length and force. Lengthwise translation enables the piecewise scanning of the sample, overcoming limitations associated with the relative size of the microscope imaging window (540 &#181;m by 540 &#181;m) and the length of a typical trabecula (&#62;2000 &#181;m). Platinum electrodes at either end of the muscle chamber stimulate the trabecula at a user-defined rate. We exploit the stimulation signal as a trigger for synchronizing the data from each imaging window to reconstruct the entire sample twitching under steady-state conditions. Applying image-processing techniques to these brightfield imaging data provides tissue displacement and sarcomere length maps. Such a collection of data, when incorporated into an experiment-modeling pipeline, will provide a deeper understanding of muscle contractile homogeneity and heterogeneity in physiology and pathophysiology.

Simultaneous Brightfield, Fluorescence, and Optical Coherence Tomographic Imaging of Contracting Cardiac Trabeculae Ex Vivo

This protocol presents a collection of sarcomere, calcium, and macroscopic geometric&#160;data from an actively contracting cardiac trabecula ex vivo. These simultaneous measurements are made possible by the integration of three imaging modalities.

同时布莱特菲尔德， 荧光， 和光学相干断层成像收缩心脏特拉贝库莱 Ex Vivo

In cardiac muscle, intracellular Ca2+ transients activate contractile myofilaments, causing contraction, macroscopic shortening, and ...

All-optical Mechanobiology Interrogation of Yes-associated Protein in Human Cancer and Normal Cells using a Multi-functional System

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. However, manual control of various equipment produced by different manufacturers is labor-intensive and time-consuming, potentially decreasing the accuracy, reproducibility, and quality of acquired data. Therefore, an all-in-one and user-programmable system that enables automatic, multi-functional, and long-term image acquisition and is compatible with most fluorescent microscopy platforms can benefit the scientific community. This paper introduces the complete operating protocols of utilizing a novel integrated software system that consists of (1) a home-built software program, titled "Automatic Multi-functional Integration Program (AMFIP)," which enables automatic multi-channel imaging acquisition, and (2) a suite of quantitative imaging analysis and cell traction computation packages. 
This integrated system is applied to reveal the previously unknown relationship between the spatial-temporal distribution of mechano-sensitive Yes-associated protein (YAP) and the cell mechanics, including cell spreading and traction, in CRISPR/Cas9-engineered human normal cells (B2B) and lung cancer cells (PC9). Leveraging this system's capability of multi-channel control and readout, the result shows: (1) B2B normal cells and PC9 cancer cells show a distinct relationship between YAP expression, traction, and cell dynamics during cell spreading and migration processes; and (2) PC9 cancer cells apply noticeable peri-nuclear forces on substrates. In summary, this paper presents a detailed stepwise protocol on how to utilize an integrated user-programmable system that enables automatic multi-functional imaging and analysis to elucidate YAP mechano-sensitivity. These tools open the possibility for detailed explorations of multifaceted signaling dynamics in the context of cell physiology and pathology.

This paper presents a detailed stepwise protocol on how to utilize an integrated multi-functional and user-programmable system that enables automatic multi-channel imaging and mechanobiological analysis to elucidate the mechano-sensitivity of Yes-associated protein (YAP).

使用多功能系统对人类癌症和正常细胞中Yes相关蛋白的全光学机械生物学询问

Long-term multi-functional imaging and analysis of live cells require streamlined, functional coordination of various hardware and software platforms. ...

用于灌注血管离体培养的新型 3D 打印生物反应器

Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users&#39; operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

作者聚焦：EasyFlow - 一种适用于大型血管培养的经济且适应性强的灌流生物反应器 

This protocol presents the setup and operation of a newly developed, 3D printed bioreactor for the ex vivo culture of blood vessels in perfusion. The system is designed to be easily adopted by other users, practical, affordable, and adaptable to different experimental applications, such as basic biology and pharmacological studies.

离体 在3D打印生物反应器中对大血管进行灌注培养

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious ...