Name: apparatus for harvesting tissue microcolumns
Uploaded: 2018-10-25T13:30:00
Description: Watch this Scientific Journal Video about Apparatus for Harvesting Tissue Microcolumns at JoVE.com

这项工作部分由陆军、海军、NIH、空军、VA 和卫生事务部支持, 以支持一家 II 的努力, 根据奖号。W81XWH-13-2-0054。美国陆军医学研究收购活动, 820 钱德勒街, 德特里克堡, MD 21702-5014 是授予和管理收购办公室。意见、解释、结论和建议是作者的观点, 并不一定得到国防部的认可。

所有涉及活体动物和动物组织样本的工作均已获得马萨诸塞州总医院机构动物护理和使用委员会 (IACUC) 的批准。
1. 收获针的生产
<ol><li>生产阶段的设置<ol>
<li>将一个母鲁尔锁连接器固定在柱子上, 然后将开机自检安装到旋转台上, 以便鲁尔锁位于舞台的中心 (图 1a)。</li>
		<li>垂直放置第一个旋转阶段, 并垂直安装到第二个水平旋转阶段 (<stro...

<ol>
	<li>Sun, B. K., Siprashvili, Z., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+skin+grafting+and+treatment+of+cutaneous+wounds.">Advances in skin grafting and treatment of cutaneous wounds.</a> Science. 346 (6212), 941-945 (2014).</li><li>Singh, 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=Challenging+the+Conventional+Therapy:+Emerging+Skin+Graft+Techniques+for+Wound+Healing.">Challenging the Conventional Therapy: Emerging Skin Graft Techniques for Wound Healing.</a> Plastic and Reconstructive Surgery. 136 (4), 524-530 (2015).</li><li>Tam, J., 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=Fractional+Skin+Harvesting:+Autologous+Skin+Grafting+without+Donor-site+Morbidity.">Fractional Skin Harvesting: Autologous Skin Grafting without Donor-site Morbidity.</a> Plastic and Reconstructive Surgery. Global Open. 1 (6), 47 (2013).</li><li>Tam, J., 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=Reconstitution+of+full-thickness+skin+by+microcolumn+grafting.">Reconstitution of full-thickness skin by microcolumn grafting.</a> Journal of Tissue Engineering and Regenerative Medicine. 11 (10), 2796-2805 (2017).</li><li>Huang, 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=Regeneration+of+hair+and+other+skin+appendages:+A+microenvironment-centric+view.">Regeneration of hair and other skin appendages: A microenvironment-centric view.</a> Wound Repair and Regeneration. 24 (5), 759-766 (2016).</li><li>Fernandes, J. 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=Micro-mechanical+fractional+skin+rejuvenation.">Micro-mechanical fractional skin rejuvenation.</a> Plastic and Reconstructive Surgery. 131 (2), 216-223 (2013).</li><li>Rettinger, C. L., Fletcher, J. L., Carlsson, A. H., Chan, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+epithelialization+and+improved+wound+healing+metrics+in+porcine+full-thickness+wounds+transplanted+with+full-thickness+skin+micrografts.">Accelerated epithelialization and improved wound healing metrics in porcine full-thickness wounds transplanted with full-thickness skin micrografts.</a> Wound Repair and Regeneration. 25 (5), 816-827 (2017).</li><li>Franco, 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=Fractional+skin+harvesting:+device+operational+principles+and+deployment+evaluation.">Fractional skin harvesting: device operational principles and deployment evaluation.</a> Journal of Medical Devices. 8 (4), 041005 (2014).</li><li>Rasmussen, C. A., 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=Chimeric+autologous/allogeneic+constructs+for+skin+regeneration.">Chimeric autologous/allogeneic constructs for skin regeneration.</a> Military Medicine. 179, 71-78 (2014).</li><li>Ter Horst, B., Chouhan, G., Moiemen, N. S., Grover, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+keratinocyte+delivery+in+burn+wound+care.">Advances in keratinocyte delivery in burn wound care.</a> Advanced Drug Delivery Reviews. 123, 18-32 (2018).</li><li>Wong, V. W., Levi, B., Rajadas, J., Longaker, M. T., Gurtner, G. C. <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+niches+for+skin+regeneration.">Stem cell niches for skin regeneration.</a> International Journal of Biomaterials. 2012, 926059 (2012).</li><li>Manstein, D., Herron, G. S., Sink, R. K., Tanner, H., Anderson, R. 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这里描述的方法旨在使组织水样的收集足够数量的体内大型动物研究, 使用工具从商业上可用的实验室用品。本仪器以前在收割组织从被切除的人皮肤4,9以及活的猪皮肤3。所描述的具体参数是那些被发现最适合在猪中使用。预计同样的仪器可以修改和适应收集从啮齿动物和其他小动物的组织, 但这在我们的实验室没有测试。
<...

自体皮肤移植是创面修复的主要支柱, 但由于供血部位缺乏和发病率的限制, 导致近几十年来共同努力开发替代传统皮肤移植的新治疗方案1,2.我们最近开发了一种替代方法来收获皮肤, 利用全厚度的皮肤移植的好处, 同时尽量减少捐献者的部位发病率。通过收集全厚度的皮肤以小 (约0.5 毫米直径) "水样", 捐助者的网站能够在正常情况下快速愈合和没有疤痕 (对于潜在的例外, 请参阅下面的讨论部分)3。水样可直接应用于伤口床上, 以加速伤口闭合, 减少收缩3, 恢复各种表皮和真皮细胞类型和功能性附件结构4, 其中许多缺乏传统的剥离厚度皮肤移植或当前生物工程皮肤替代5。水样的能力, 以增加愈合和他们的捐助者的网站愈合没有疤痕都已独立验证的其他研究组6,7。
我们以前开发了一个实验室收割系统, 以使收集的水样在规模8;但是, 该系统由许多未广泛使用的自定义组件组成。在这里, 我们详细描述了生产收获针的过程, 以及简单的收集系统, 由大多数现成的组件制成, 可用于实现大容量收割。本手稿中描述的仪器适合于体外和动物工作, 但不适用于人类。在人类中应用这种技术的临床设备具有 FDA 许可, 但在这里将不会详细讨论。

<table border="0" cellpadding="0" cellspacing="0" style="width:688px;" width="687">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Diamond wheel</td>
			<td style="width:135px;">Dremel</td>
			<td style="width:119px;">545</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Hypodermic needle (19G)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">14-840-98</td>
			<td style="width:219px;">Other needle sizes could be used, depending on experimental needs</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">Stome wheel</td>
			<td style="width:135px;">Dremel</td>
			<td style="width:119px;">540</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Syringe (20mL with luer lock)</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">22-124-967</td>
			<td style="width:219px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Suction adapter</td>
			<td style="width:135px;">Tulip Medical</td>
			<td style="width:119px;">PA20BD</td>
			<td style="width:219px;">Optional, for high volume harvesting</td>
		</tr>
		<tr height="70">
			<td height="70" style="height:71px;width:216px;">Suction canister</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">19-898-212</td>
			<td style="width:219px;">Optional, for high volume harvesting. Sterilize before use.</td>
		</tr>
		<tr height="58">
			<td height="58" style="height:59px;width:216px;">Suction tubing</td>
			<td style="width:135px;">Medline</td>
			<td style="width:119px;">DYND50216H</td>
			<td style="width:219px;">Optional, for high volume harvesting</td>
		</tr>
	</tbody>
</table>

apparatus for harvesting tissue microcolumns

本手稿描述了由现成组件制成的实验室设备的生产过程, 可用于收集水样的全厚度皮肤组织。小尺寸的水样允许供体部位快速愈合, 而不会造成供体部位瘢痕, 而采集全厚度组织则可将皮肤组织的所有细胞和细胞外成分包括在内。有更深的真皮区域和附件皮肤结构, 尚未成功再现使用传统的组织工程技术。水样可直接应用于皮肤伤口, 以增加愈合, 也可用作其他组织工程方法的自体细胞/组织来源。收集针是通过修改标准皮下注射针, 它们可以单独用于收割少量的组织或与一个简单的吸基于收集系统 (也由通常可用的实验室用品)大容量收割, 以促进大型动物模型的研究。

收获针应该能够收集水样的全厚度皮肤组织与大约80-90% 的成功率, 每个微柱应包含表皮, 真皮, 和一些皮下脂肪 (图 4)。如果收获的成功率很低, 或者如果在组织中插入针头变得困难, 那么可能需要新的针头。如果收获的成功率一直很低, 即使用新的针头, 那么针头可能太短。
如果在体内使用, 捐助者?...

Watch this Scientific Journal Video about Apparatus for Harvesting Tissue Microcolumns at JoVE.com

Apparatus for Harvesting Tissue Microcolumns

在这里, 我们描述了一个生产收集针的协议, 可用于收集全厚度的皮肤组织, 而不会造成捐赠部位疤痕。针头可以与一个简单的收集系统结合, 以实现大容量收割。

水样组织的采集装置

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

此方法可以帮助回答皮肤组织再生领域的基本问题，即如何取代自然皮肤中所含的各种细胞类型和结构。该技术的主要优点是，可以收集自体全厚皮肤，以最小的供体站点发病率。演示这个程序的将是比尔·法里内利，他是我们实验室的研究员。从生产阶段的设置开始此过程，如文本协议中所述。将两个切断轮同心地安装到旋转工具上。在较大的较高栅格石切割轮上使用较小的下格金刚石切割轮。将可调节臂放在旋转工具上，将灯对着切割轮。为了增强可视化效果，在生产设置上放置解剖显微镜，以便目镜聚焦在切割盘上。重塑针尖时，请佩戴防护眼镜和手术面罩，以防止细金属颗粒进入眼睛或气道。根据实验要求选择适当仪表尺寸的皮下针。标记每个收获针上的预期长度。将针头垂直于旋转工具，并打开电源，使用外部切割盘的边缘切断针在标记点上的多余的长度。将缩短的钝针连接到生产阶段的母 Luer 锁连接器。调整水平旋转阶段，使指针与旋转工具上的切割盘平行 12 度角。打开头顶灯并调整其位置，同时在放大下观察针头，直到光线从针头的中线反射出来。打开旋转工具的电源。然后使用平移阶段将针头推进到内部切割盘。继续缓慢地将针对着切割盘，直到切割盘到达针头的中线。缓慢地将切割针表面从内金刚石轮移动到外石轮上，以更精细的抛光完成切割针表面。将针头从切割盘上收回。使用垂直旋转阶段，将针旋转 180 度。重复这些步骤以重塑针头的另一侧。现在，将针头从生产阶段取出。用略小于针头内径的金属丝清洁内孔。使用锋利的木棍，去除可能仍附着在新成型针头边缘的任何毛刺。当需要大量微柱时，从 20 毫升注射器中取出柱塞。然后将注射器连接到吸气适配器上。通过将收获针连接到注射器来完成组装。使用一块无菌吸管将吸气适配器连接到无菌吸气罐。确保收割装置连接到罐式输入，使液体畅通无阻地流入罐体。现在将设备连接到负压源。通过将收获针插入皮肤来收获微层。在收获过程中，间歇性地将收获针浸入无菌盐水容器中，以冲洗系统。手放在一根金属线或略小于收获针内径的针。如果针头堵塞，可以通过将金属丝插入针孔来清除。当所需的微量在罐中积聚时，断开设备与吸力的连接。然后将吸气罐中的内容物倒入过滤器中，收集微镜。此处显示的是从正面和侧面查看的成品收获针。请注意尖端的两个切割点。收获针应该能够从全厚皮肤组织中收集微球，成功率约为80%至90%。在此图中，检查标记表示一毫米。每个微镜应包含表皮、真皮和一些皮下脂肪。微哥伦布可以直接应用于伤口愈合，也可以与不同的基质材料结合，产生组合结构。微哥伦布也可以在培养中保持用于体外研究。在执行大容量收获时，重要的是要记住加入间歇性盐水冲洗。虽然这种方法是为皮肤伤口修复而设计的，但类似的原理可以应用于其他器官系统，如需要自体组织移植的肌肉。

Research

JoVE Journal

Bioengineering

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

静电基础：优化解决方案和设备参数

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

科研

生物工程

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus (SCUVA)

Quantitatively Measuring In situ Flows using a Self-Contained Underwater Velocimetry Apparatus SCUVA

定量测量原位流动，使用一个自包含的水下测速仪（SCUVA）

The ability to directly measure velocity fields in a fluid environment is necessary to provide empirical data for studies in fields as diverse as ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

非接触，无标签使用拉曼光谱研究细胞和细胞外基质的监测

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

血浆或尿液的水凝胶纳米粒子收获检测低丰度蛋白

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Custom-designed Laser-based Heating Apparatus for Triggered Release of Cisplatin from Thermosensitive Liposomes with Magnetic Resonance Image Guidance

定制设计的基于激光的加热装置顺铂触发释放的热敏脂质体与磁共振成像制导

Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

三明治般的微环境来驾驭细胞/材料相互作用

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...