作者感谢莱纳Schultheiss和沃尔夫冈沙登他们的灵感为这个模型。我们也感谢基督教Dorfmüller他所有的时间了巨大的努力，以支持我们的研究。 非常感谢罗伯特Göschl和汉斯Hohenegger仔细的技术实现我们的想法！

道德许可取得患者的书面知情同意书后，脐带从在妇产科学系剖腹产的人脐静脉内皮细胞（HUVEC）中分离获得。权限是从因斯布鲁克医科大学（编号：UN4435）伦理委员会给出。 1，准备IVSWT水浴<ol><li>制备3.5升的自来水在适当的容器。水需要被加热到37°C（见协议6）。 </li><li>水填充入水浴，直到水位大约为3厘米以下的优势。心灵完全覆盖水的膜。这将需要大约准备3.5 L。 </li><li>将温度传感器连接到电源。 </li><li>把传感器放到预期的拟合置水浴上的后墙。 </li><li>连接加热至温度传感器的电源。没有不要这样做水在浴中的加热器将熔融树脂玻璃。 </li><li>等到水温达到37°C的温度稳定与某种棍定期搅水，以保证整个水浴恒温。 </li></ol> 2，准备和细胞培养瓶<ol><li>种子细胞在晚上直接，直到他们达到所需的汇合在T25培养瓶或培养的细胞在培养瓶所需的密度。 </li><li>在实验前，垂直对齐的烧瓶中。 </li><li>填补了培养瓶用培养液，直到下方脖子。不要填写太多的介质进入接触到颈部或封闭盖的瓶为介质将受到污染。 </li><li>用螺丝固上限瓶无过滤器，以防止污染的水。记住，水在浴室和浴缸本身不是无菌的。 </li><li>插入水中洗澡前用封口膜密封的瓶盖。 </li> &lt;李&gt;修复密封培养瓶在所提供的立场。 </li><li>将固定烧瓶放入水浴中。注意使细胞培养烧瓶的中间是在同一高度上的冲击波施加器的膜的中央。 A线在洗澡的侧面引导你。 </li></ol> 3，定义处理参数<ol><li>定义冲击波源和样品之间的距离。通过进行该参数的发现导频实验如在图6中所述确定理想的治疗参数。 </li><li>选择冲击波设备上正确的治疗参数（能量密度，频率）。再次，参考图6。 </li></ol> 4，冲击波的应用<ol><li>把市售的超声波传输胶量充足的冲击波敷贴以及置水浴上的膜。这是为了保证耦合。没有空气或气泡应该施加器和膜之间的它会吸收冲击波。 </li><li>连接敷贴到膜上，并保持稳定的膜的中央。请注意，这是水平对齐。 </li><li>确保水浴内探头的正确的垂直位置与所述指示标记在恒温槽的侧线。 </li><li>保持在一个水平行中的敷贴器，将烧瓶稳定的中心。 </li><li>激活冲击波装置和应用该脉冲，同时保持培养瓶中，以及在整个手术过程中稳定的位置的敷贴。 </li></ol> 5，治疗后<ol><li>就拿培养瓶中出来的水洗澡。 </li><li>与普通纸巾擦干。 </li><li>准确地擦拭量瓶中，用消毒剂。 </li><li>除去封口膜密封帽。 </li><li>吸管中的烧瓶内到离心管中。 </li><li>离心机适当。离心参数取决于所用的细胞类型， <em&gt;如。对脐静脉内皮细胞，用290克在室温下。 </li><li>加细胞培养基5ml的烧瓶中。重悬离心细胞沉淀用2ml细胞培养基。移液器的悬浮液加入到烧瓶中。因此，可能在治疗过程中已脱落细胞碎片或细胞不迷路。 </li></ol>陷阱<ol><li>制剂以及治疗应进行层流内的无菌条件下，以避免细胞培养物污染。 </li><li>良好的细胞培养实践建议，以避免探针的污染。特别是瓶外的消毒，强烈建议把他们带回孵化器之前。水浴，以及里面的水，是没有出息！ </li><li>不要将加热器连接到电源，除非水浴是装满了水，以免损坏内置浴有机玻璃。 </li><li>使用超声波凝胶的数额慷慨的冲击波APPLicator以保证正确的耦合和波的传播进入浴缸。空气，甚至小气泡做吸收冲击波！ </li><li>检查水浴相对于敷贴器内的探针的位置，以确保治疗的整个生长区域。 </li><li>不要扣篮探头插入过深的水，以避免污染。 </li></ol>

<ol>
	<li>Haupt, G., Haupt, A., Ekkernkamp, A., Gerety, B., Chvapil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shock+waves+on+fracture+healing.">Influence of shock waves on fracture healing.</a> Urology. 39, 529-532 (1992).</li><li>Schaden, W., Fischer, A., Sailler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+of+nonunion+or+delayed+osseous+union.">Extracorporeal shock wave therapy of nonunion or delayed osseous union.</a> Clin. Orthop. Relat. Res. 387, 90-94 (2001).</li><li>Schaden, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+therapy+for+acute+and+chronic+soft+tissue+wounds:+a+feasibility+study.">Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study.</a> J. Surg. Res. 143, 1-12 (2007).</li><li>Tepek&#246;yl&#252;, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+treatment+induces+angiogenesis+and+mobilizes+endogenous+CD31/CD34-positive+endothelial+cells+in+a+hindlimb+ischemia+model:+Implications+for+angiogenesis+and+vasculogenesis.">Shock wave treatment induces angiogenesis and mobilizes endogenous CD31/CD34-positive endothelial cells in a hindlimb ischemia model: Implications for angiogenesis and vasculogenesis.</a> J. Thorac. Cardiovasc. Surg. 146, 971-978 (2013).</li><li>Nishida, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+cardiac+shock+wave+therapy+markedly+ameliorates+ischemia-induced+myocardial+dysfunction+in+pigs+in+vivo.">Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo.</a> Circulation. 110, 3055-3061 (2004).</li><li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+cardiac+shock+wave+therapy+ameliorates+myocardial+ischemia+in+patients+with+severe+coronary+artery+disease.">Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease.</a> Coron. Artery Dis. 17, 63-70 (2006).</li><li>Gotte, G., Amelio, E., Russo, S., Marlinghaus, E., Musci, G., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-time+non-enzymatic+nitric+oxide+synthesis+from+L-arginine+and+hydrogen+peroxide+induced+by+shock+waves+treatment.">Short-time non-enzymatic nitric oxide synthesis from L-arginine and hydrogen peroxide induced by shock waves treatment.</a> FEBS Lett. 520, 153-155 (2002).</li><li>Wang, F. S., Wang, C. J., Huang, H. J., Chung, H., Chen, R. F., Yang, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+shock+wave+mediates+membrane+hyperpolarization+and+Ras+activation+for+osteogenesis+in+human+bone+marrow+stromal+cells.">Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.</a> Biochem. Biophys. Res. Commun. 287, 648-655 (2001).</li><li>Mittermayr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+(ESWT)+minimizes+ischemic+tissue+necrosis+irrespective+of+application+time+and+promotes+tissue+revascularization+by+stimulating+angiogenesis.">Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis.</a> Ann. Surg. 253, 1024-1032 (2011).</li><li>Baker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat. Protoc. 22, 89-104 (2011).</li></ol>

作者什么都没有透露。

该模型用于体外冲击波治疗的意义是，波可以通过细胞培养而相比之下，现有的模型后传播。由此，能够避免干扰物质的影响，如拉力。该模型由他人直接涂布波到其细胞培养瓶更接近于体内条件比。 一个附加的优点是不同冲击波源和细胞之间的距离的可能性。这将是不可能的，如果直接处理的培养烧瓶中。然而， 体内存在的涂抹器与目标区域之间的某?...

冲击波是从能源， 如突然释放所产生的声压波。如打雷时，闪电。在医学上的冲击波已经使用了30多年的碎石治疗肾结石的解体。由于骨骼增厚偶然发现在碎石患者在20世纪80年代初，第一次进行了研究，以评估骨愈合1冲击波治疗（SWT）的效果。长骨骨不连愈合改善令人印象深刻的结果可以观察到2。随后，适应症扩大到软组织伤口3。基础研究的成果表明，冲击波也引起生物刺激靶组织没有任何后续的损害。的血管生长因子（VEGF 例如 ，的PlGF，FGF）发行其次是显著的血管生成。这导致适应症对缺血性病变进一步扩大。本集团与其他人表现的积极EFF抽搐SWT对缺血性心脏疾病的动物模型中以及在临床试验4-6。 然而，如何SWT的物理刺激被翻译成的生物信号（机械传导）的确切机制仍然知之甚少。从医学不断增加几个字段在SWT的利益，追求的机制越来越激烈。因此， 体外冲击波实验是越来越重要。除了 ​​减少的动物实验和成本效益， 体外冲击波治疗（IVSWT）的最大优势可能是学习某一细胞类型的特定行为的可能性。在冲击波介导的组织再生最有可能被治疗组织的所有细胞都参与其中，甚至全身作用进行了讨论。然而，不同的细胞类型中起着特殊的作用，有其自身固有的功能。 IVSWT使我们能够检测到这种特殊功能的ND从而使我们更好地理解复杂的基本流程。 今天的关于细胞培养冲击波的影响的认识，包括增加增殖，改变细胞膜受体，增加和细胞分化的加速，生长因子和化学引诱剂释放以及增加细胞迁移7-9。 在体外模型施加冲击波到细胞培养物中的各种方法中最令人分心的物理效应进行了描述。这一事实导致了它是非常难以比较的结果，如细胞刺激身体条件这些模型之间完全不同的问题。一般来说，所有现有车型集中在如何最好地运用冲击波到细胞。 但是，这个问题仍然是：通过细胞培养后发生了什么浪？主要的问题在于，所述声的差细胞培养基和环境空气的阻抗是高的，即99％以上的冲击波被反射图1。 由于在两种介质的声阻抗的差异波不仅反映但相移为180°时产生强的拉力向细胞图2。 声阻抗被定义为材料的密度和它的产品声速Z =ρX C。对于水的声阻抗是ZWater = 1440000 Ns个/ m 3时，对空气，只有420纳秒/米3。这两个值的巨大差异导致了冲击波的反射和相移。相移变成正压脉冲成拉伸波。 即使这样的拉伸力是不会伤害到细胞内，它干扰了模仿在体外体内冲击波影响的想法。 体内这些拉力很难由于大身体结构发生。 此外，行波后面甚至可以打扰传入的。这可能会引起干扰。两种类型的干扰是已知的。建设性的干扰是指两个波被添加，从而导致了一倍振幅图3。如果波相遇截然相反的发生破坏性干扰。它会导致波的取消（ 图3）。因此，IVSWT需要一个模型，使冲击波通过细胞培养后进行传播。 IVSWT水浴按照上面提到的顾虑考虑导致我们设计的水浴中，以避免所描述的问题的图4。基本上，它包括一个有机玻璃的内置容器用膜来连接各种冲击波施加器。对于这种膜和涂敷超声传输格之间的耦合升已被使用。水浴填充脱气的水，以避免气蚀，如果气体中的水固溶会发生的。加热器在底部有温度传感器连接到控制单元使得能够调节温度为模仿体内条件和避免细胞培养的过程中，以进行冷却。温度可维持在37℃以下，因为它是在培养箱中进行。为细胞样品持有人允许任何浸入一种文化烧瓶或试管的。由此，试样容器需要被完全装满培养基，因为气泡会阻止冲击波！甲楔形吸收体在沐浴的后壁解构，为了不被反射并运行回，以避免干扰波。 进一步的优点与其他IVSWT模型是不同的涂抹器和培养瓶之间的距离的可能性。谁使用这种模式显然是我们小组的研究结果和其他怎么每个细胞类型的反应非常明确不同的治疗参数。此外，限定了波的源和样品之间的距离是至关重要的，因为它使我们能够控制细胞是在相对于冲击波施加器的焦点的特定位置。

<table border="0" cellpadding="0" cellspacing="0" width="2002">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Orthogold shock wave device</td>
			<td colspan="2" style="width:756px;">Tissue Regeneration Technologies, Woodstock, GA &#8211; manufactured by MTS-Europe GmbH, Konstanz, Germany</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">IVSWT Water Bath V2.0</td>
			<td style="width:537px;">Johann Hohenegger - Technical Products</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EBM-2 Basal Medium 500 m +EGM-2 SingleQuot Suppl.&#38;Growth Factors</td>
			<td style="width:537px;">Lonza</td>
			<td style="width:219px;">CC-3156 &#38; CC-4176</td>
			<td>This medium was used for the shown experiments with HUVECs to fill the cell culture flask. For other cell types, use the recommended medium.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"><a href="http://www.labplanet.com/pechiney-parafilm-m-pm996.html">Pechiney&#160;Parafilm M PM996</a></td>
			<td style="width:537px;">Pechiney Plastic Packaging</td>
			<td>PH-LF-PM996-EA at labplanet.com</td>
			<td>for sealing flasks</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Falcon Serological pipets 25ml</td>
			<td style="width:537px;">Becton Dickinson Labware</td>
			<td style="width:219px;">357525</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">CellMate II Serological Pipette&#160;</td>
			<td style="width:537px;">Matrix Technologies</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Skintact Ultrasonic Gel</td>
			<td style="width:537px;">Skintact</td>
			<td style="width:219px;">UL-01 250 ml</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">T25 cell culture flasks</td>
			<td style="width:537px;">COSTAR</td>
			<td style="width:219px;">3056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">mikrozid disinfectant</td>
			<td style="width:537px;">Sch&#252;lke</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">3,5l degassed water</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">paper towels</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

shock wave application to cell cultures

冲击波现在是众所周知的再生效果。基础研究的成果表明，冲击波也引起生物刺激靶细胞或组织没有任何后续的损害。因此， 在体外实验中面临着越来越大的兴趣。施加冲击波到细胞培养物中的各种方法进行了描述。一般来说，所有现有车型集中在如何最好地运用冲击波到细胞。 但是，这个问题仍然是：通过细胞培养后发生了什么浪？细胞培养基中的声阻抗与空气的区别在于高，即99％以上的冲击波被反射！因此，我们开发了一个模型，主要由树脂玻璃建造的集装箱，使波通过细胞培养后，在水中传播的。这就避免了空化效应，以及，否则将扰乱即将到来的那些波的反射。无线日这个模型，我们能够模拟体内条件，从而获得关于如何冲击波的物理刺激被转换为生物细胞信号（“机械传导”）越来越多的知识。

使用我们应用的冲击波，以人脐静脉内皮细胞所描述的方法（内皮细胞），我们前述隔离脐带。脐带从选修剖腹产获得。 脐静脉内皮细胞均在90％的汇合在一个T25细胞培养瓶中用液电冲击波治疗系统治疗。治疗参数为0.1毫焦耳/ 平方毫米和5 Hz的频率的能量通量密度。 300冲动被应用从5厘米的冲击波源的距离的细胞层。 的Tie-2的基因表达（免疫球蛋?...

Watch this Scientific Journal Video about Shock Wave Application to Cell Cultures at JoVE.com

Shock Wave Application to Cell Cultures

冲击波现在是众所周知的再生效果。因此， 在体外实验中面临着越来越大的兴趣。因此，我们开发了一个模型体外冲击波试验（IVSWT），使我们能够在体内条件，从而避免分散注意力的物理效应模拟。

冲击波应用细胞培养

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

Research

Education

JoVE Journal

JoVE Core

Physics

Bioengineering

Unit 1

Waves

SCIENTISTS IN ACTION

12.6K 次观看.  Innsbruck Medical University. 冲击波现在是众所周知的再生效果。因此， 在体外实验中面临着越来越大的兴趣。因此，我们开发了一个模型体外冲击波试验（IVSWT），使我们能够在体内条件，从而避免分散注意力的物理效应模拟。 

视频: Shock Wave Application to Cell Cultures

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 ...

科研

生物工程

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

肌成纤维细胞和约束的应用工程纤维蛋白的​​组织结构和应变诱导细胞和胶原蛋白（RE）组织

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

生物相容的液晶弹性体泡沫的合成中用作细胞支架的三维空间细胞培养

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of nanometer-scale features. Historically, a major drawback of scanning probe lithography has been the intrinsically low throughput of single probe systems. This has been tackled by the use of arrays of multiple probes to enable increased nanolithography throughput. In order to implement such parallelized nanolithography, the accurate alignment of probe arrays with the substrate surface is vital, so that all probes make contact with the surface simultaneously when lithographic patterning begins. This protocol describes the utilization of polymer pen lithography to produce nanometer-scale features over centimeter-sized areas, facilitated by the use of an algorithm for the rapid, accurate, and automated alignment of probe arrays. Here, nanolithography of thiols on gold substrates demonstrates the generation of features with high uniformity. These patterns are then functionalized with fibronectin for use in the context of surface-directed cell morphology studies.

Here we present a protocol for wide-area scanning probe nanolithography enabled by the iterative alignment of probe arrays, as well as the utilization of lithographic patterns for cell-surface interaction studies.

基于自动对准的大面积扫描探针光刻及其在细胞培养研究中的应用

Scanning probe microscopy has enabled the creation of a variety of methods for the constructive (&#39;additive&#39;) top-down fabrication of ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture.
Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices.
This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Dissociated hippocampal cell culture is a pivotal experimental tool in neuroscience. Neural cell survival and function in culture is enhanced when coralline skeletons are used as matrices, due to their neuroprotective and neuromodulative roles. Hence, neural cells grown on coralline matrix show higher durability, and thereby are more adequate for culturing.

使用生物活性珊瑚基质提高解离神经细胞培养物的耐久性

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Application of Atomic Force Microscopy to Detect Early Osteoarthritis

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in elasticity can propagate or trigger the onset of major diseases like cancer or osteoarthritis (OA). Atomic force microscopy (AFM) has emerged as a strong tool to qualitatively and quantitatively characterize the biomechanical properties of specific biological target structures on a microscopic scale, measuring forces in a range from as small as the piconewton to the micronewton. Biomechanical properties are of special importance in musculoskeletal tissues, which are subjected to high levels of strain. OA as a degenerative disease of the cartilage results in the disruption of the pericellular matrix (PCM) and the spatial rearrangement of the chondrocytes embedded in their extracellular matrix (ECM). Disruption in PCM and ECM has been associated with changes in the biomechanical properties of cartilage. In the present study we used AFM to quantify these changes in relation to the specific spatial pattern changes of the chondrocytes. With each pattern change, significant changes in elasticity were observed for both the PCM and ECM. Measuring the local elasticity thus allows for drawing direct conclusions about the degree of local tissue degeneration in OA.

We present a method to investigate early osteoarthritic changes at the cellular level in articular cartilage by using atomic force microscopy (AFM).

原子力显微镜在早期骨关节炎检测中的应用

Biomechanical properties of cells and tissues not only regulate their shape and function but are also crucial for maintaining their vitality. Changes in ...