作者要感谢费德里卡Zanzottera博士对促进执行流式细胞分析研究。

伦理陈述:自协议的临床应用程序需要使用患者的皮肤自体组织，其在体外表征同源皮肤组织在艾米利亚-罗马涅地区的皮肤银行下列国家规定的采伐，加工指引临床使用之前进行和分发用于移植的组织（CNT 2013）。 1.临床应用生物综合楼注:该协议是基于临床的使用Rigeneracons（组织干扰物）和Rigenera机（组织干扰物系统）（ 图1A）。组织破坏剂是能够扰乱小片使用由六角形刀片和过滤的细胞和细胞外基质的成分具有约50微米的截止提供一网格组织的人体组织的生物医学干扰物。 <ol><li>通过双向收集患者的skinsamplesOPSY冲头（ 图1B）和分解加入1ml生理盐水，每件以获得自体微移植物6,7,9（或见步骤2.1）。 </li><li>放置1毫升胶原海绵的微移植物的（ 图1C）toform生物复合物用于临床应用。 </li><li>培养另一1毫升上在6ml补充有10％胎牛血清的DMEM培养基中的存在胶原海绵的微移植物的在37℃在5％CO 2的潮湿气氛中。 </li><li>以下培养3天后，固定生物复合物用0.3％多聚甲醛在室温10分钟。倾直接特定分配器到样品的石蜡。得到的切片用厚度为5μm的切片，并在玻璃载玻片放直接。 </li><li>浸泡5微米石蜡切片中用于组织学分析的玻璃，含有15 - 20毫升二甲苯（市售 - 间 - 二甲苯（40混合 - 65％），对二甲苯（20％），邻二甲苯（20％ ）和ETH基苯（6 - 20％）和甲苯，三甲基苯，苯酚，噻吩，吡啶和硫化氢）的每个3分钟的痕迹。 </li><li>沉浸在降低等级的乙醇的切片（100％至70％）（100％乙醇1小时，95％的乙醇1小时后，80％的乙醇1小时，70％的乙醇进行1小时），然后去离子水进行去paraffinizing和再水化的章节。 </li><li>染色切片与1 - 2毫升1克/ L Ematoxylin为1 - 2分钟，随后在水中漂洗，以除去任何Ematoxylin盈余。 </li><li>染色为4的部分 - 与曙红Y醇溶液5分钟，在浓度与70％乙醇混合的1％，而在用水稀释。 </li><li>使用1 - 2毫升曙红Y为每张幻灯片部分和流动自来水下冲洗。 </li><li>浸入部分中增加的乙醇等级（见步骤1.6），最后，在二甲苯中的通道1小时，盖玻片与基于安装mediumand放大100倍（ 图1D）在光学显微镜下观察之后。 </li> &lt;/ OL&gt; 2.收集，解体并在组织的体外分析<ol><li>分别为0.6毫米1毫米或2毫米的4种不同的多器官和/或多的行李箱区域的厚度用皮，走自主皮肤组织，乳头去epidermized真皮（DED）或真皮网状层（真皮）从40到55年的范围内组织的捐助者，下面就采收，加工和销售用于移植组织国家规则（CNT 2013年）。 <ol><li>轻轻冲洗在0.9％NaCl溶液的所有样品将它们在培养皿中在轨道摇动器5分钟。 </li><li>使用5毫米活检穿孔，创建样品它是均匀的，从皮肤组织，扣减和真皮直径和分解之前称重所有组织标本。 </li><li>插入皮肤组织，扣减或分别真皮，在组织破坏剂八个，三个或四个均匀样品，加入1.5毫升的解聚盐溶液。 </li><li>执行不同对于所有组织样品解聚的时间在表1中所指示的。 </li><li>使用从完整的组织样本作为对照衍生冲床活检的相应数字。 </li><li>机械解聚后，吸含有微移植物和地点分别在一个12孔板的单个孔的每个样品的盐溶液。为进行完整控制冲床活检相同的协议。 </li><li>添加补充有10％胎牛血清（FBS）和抗生素，以每个样品的RPMI 1640培养基1ml中。 </li><li>立即评估细胞生存力。到每个孔中含有微移植物（分别由皮肤组织，扣减或真皮八个，三个或四个均匀样品同时分解获得）添加含有0.5毫克/毫升的MTT（3- [介质1毫升4,5-二甲基吡啶-2-基] -2,5-二苯基溴化）溶液，并在5％CO 2 /空气的气氛中，在37℃孵育3小时。为进行完整控制相同的协议冲床活检。 </li><li>温育后，除去所有含培养基的MTT，并添加到每个样品1毫升二甲基亚砜（DMSO）中10分钟。 </li><li>转移的每个样品和DMSO在反应杯并使用分光光度计在570纳米处光密度（OD）读取。计算细胞存活率作为吸光度在570nm和该比在解聚之前使用组织的克数（克）的重量。为进行完整控制冲床活检相同的协议。 </li></ol></li><li>机械分解后，吸出含有皮肤组织，单个供体的扣减和真皮样品衍生微移植盐溶液。 <ol><li>分别将每个样品中的12孔平板的单个孔或在用于分别细胞活力测试和形态分析的培养烧瓶中。 </li><li>培养微移植物加入1ml（12孔板）或5毫升（培养瓶）的RPMI补充有10％FBS的1640培养基和抗生素在37℃在5％CO 2 / Al的气氛r表示24小时或7天。 </li><li>后24小时或7天（重复步骤2.1.8-2.1.10）评估细胞活力。 </li><li> 24小时后，并在烧瓶中培养7天后用光学显微镜进行形态分析评估细胞悬浮液的存在。 </li><li>分析真皮的样品阳性的间质和造血细胞标志物，包括CD146，CD34和CD45抗原通过FACS分析6。 </li><li>在层流罩种子每个微接枝和各组织样本的相应小片段不完全分解的（由皮肤组织，分别扣减或真皮八个，三个或四个均匀样品同时分解获得）（&gt; 50微米的尺寸后在含有5％羊血肉汤100μl的哥伦比亚琼脂平板分解处理）。 </li><li>孵育在37℃的板三天，以评估不育上哥伦比亚琼脂板上进行微生物分析11。 </li></ol></li></ol>

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作者安东尼奥·格拉齐亚诺是人类脑电波SRL，生产和市场Rigenera系统的科学主任。笔者莱蒂齐亚已找到是人类脑电波SRL科学分部的合作者

这一初步研究表明，通过该协议得到微移植物可与胶原海绵结合，如已经报道了其它临床应用中，以优化微移植物的植入物9的效力，10，特别是，本研究中报道的生物的能力-complexes，通过微移植物和胶原海绵构成的，之后，从临床应用30天佐剂的腿病变的伤口愈​​合。此外， 在体外结果提供的证据有关此协议的有效性来分解三个不同的皮肤组织，保持可观的细胞存活率都机械解聚后，立即并24小时后和7天培养的。 细胞生存力的这种均化后的维持允许获得仅在一个步骤无菌自体微移植物，避免了大量的操纵。这个证据是一致最近其他文件中，该协议在体外的功效也测试了其他类型的组织，包括骨膜，心脏心房和眼球5直肌的活检。特别是，我们扣减和真皮样品中观察到增加的细胞存活率培养7天之后，可能是由于使用补充有10％胎牛血清的培养基。 除了维持细胞活力的，该系统是安全的，容易使用，并在几分钟内能够以机械地分解不同类型的组织防止细胞结构的破坏，对于细胞分离的传统的方法，如酶需要执行较长的时间消化。此外，该系统能够选择具有50微米的截止细胞和这方面是在考虑可注射微移植物的成功的非常重要的，因为该范围包含祖细胞能够在许多细胞类型的分化，然后改善病变的修复。该协议允许我们不仅获得可行的，但也自体微移植物，因为施主主题也是这些微移植物的受体。这种系统的获得自体微移植物的能力在它已经在非显示出的自体牙髓干细胞的该移植（牙髓干细胞）的牙科领域进行了特别报告所载infrabony缺陷造成牙周修理和萎缩的再生颌骨5,6。这些微移植物的改善的伤口愈合过程的能力也被预先在复杂的伤口手术干预或并发症4后的管理报告。 关于使用自体，并准备使用的微移植物的另一个优点当然是在无菌条件鉴于上皮损它们随后植入物的保存。 总之，本文描述的协议表明创建人微移植组织准备在临床干预用来改善急性/慢性皮肤伤口，例如那些在本研究表明愈合的能力。 在体外结果的可能性，以建立可行的微移植物也有报道并且该参数是肯定显著以增加组织修复成功的百分比。两者合计，该数据表明，这种新的系统，可考虑临床医生谁需要在皮肤伤口的管理的快速和安全仪器的一个有希望的工具。 此刻，没有特别的变更或限制为在此特定的应用的协议，因为皮肤表示的组织很容易使用和在其它研究中，我们报道了不同皮损6,7-相同的协议的功效</SUP&gt;。我们希望在未来使用该系统上有大量的科目来验证其临床应用在其他临床区，（例如骨科骨再生或血管药的溃疡治疗）。

在过去的几年中，一些新的方法，已经开发了生物技术在手术过程中，得到的自体细胞的悬浮液的领域中，以治疗急性和慢性皮肤损伤提供一步法治疗。而且，从创伤，糖尿病，感染和其他原因造成的急性，但主要是慢性伤口的管理，仍然具有挑战性。有越来越多的证据表明，慢性伤口已成为严重的全球性健康问题，引起全世界对1的医疗保健系统巨大的财政负担。 为了增加在皮肤损伤的治疗成功率，缺乏广泛操纵（包括蜂窝增强）和无菌条件下的维护是必要的，以创建可以在的受损区域被立即施加一个细胞悬浮液患者中，从而避免在洁净室较长的处理，如细胞工厂。圣从小皮肤活检，研磨，离心等分离方法（ 例如，酶或机械）arting，经常用于获得一个细胞悬浮液，其可在生长培养基中培养。所有的这些方法通常需要执行的很长一段时间，强调细胞结构，并导致降低细胞生存力的。另一个显著方面是获得准备好由临床医师可使用自体的细胞悬浮液，例如，以修复受损的区域。此外，众所周知，自体组织移植物存活的过户手续于通过感应和传导2,3的原则接收站点，最终生存下来。理想的移植组织应该随时可用并具有低抗原性和供区的发病率4。 这些证据的基础上，本研究的第一个目的是创建适合自体生物复合物在组织修复的临床应用。为此，我们描述了一个新的方法，以从被这个协议分类的皮肤组织开始自体微移植。案例呈现在本文中也描述为临床应用该协议结合胶原蛋白海绵获得自体微移植。这种方法已被报道为在人组织5的机械分解效率，并已用于临床移植物和真皮组织6,7的再生以及用于在口腔颌面外科8-10结缔组织再生疗法。 此外，本研究的第二个目的是皮肤组织的本协议其分解后的生物学特性。为此目的，皮肤组织的不同的同源样品来自不同的多器官的躯干区衍生和/或多组织捐赠者进行了处理上的收获，加工下列国家的规则和分配在艾米利亚 - 罗马涅地区的皮肤银行移植组织（CNT 2013年）。 病例报告: 显示由于车祸复杂外伤35岁的女病人被送往医院安科纳的重症监护病房。病人表现的腿感染，由于一个开放的伤口，并用外固定稳定复合骨折。两个病灶清除，实施时间与伤口成为负压治疗（VAC疗法）后的清洁和骨膜出现了健康，我们就从恢复两个月后，应用的协议。这个系统分解后，得到了微移植物被用来创建生物复合物胶原海绵这些建议后来为了植入，调查其对病灶修复功效。

<table border="0" cellpadding="0" cellspacing="0" width="978">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Rigenera Machine</td>
			<td style="width:257px;">Human Brain Wave</td>
			<td style="width:217px;">79210R</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Rigeneracons</td>
			<td style="width:257px;">Human Brain Wave</td>
			<td style="width:217px;">79450S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">MTT</td>
			<td style="width:257px;">Roche Diagnostic GmbH</td>
			<td style="width:217px;">11465007001</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:245px;">RPMI medium</td>
			<td style="width:257px;">PBI International</td>
			<td style="width:217px;">733-2292</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:245px;">DMEM medium</td>
			<td style="width:257px;">PBI International</td>
			<td style="width:217px;">F 0415</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Antibiotics</td>
			<td style="width:257px;">Biological Industries PBI</td>
			<td style="width:217px;">03-038-1 &#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:245px;">Antibodies for FACS Analysis</td>
			<td style="width:257px;">eBiosciences</td>
			<td style="width:217px;">code 12-1469 for CD146-P; code 17-0349&#160; for CD34-APC&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Columbia agar</td>
			<td style="width:257px;">BioMerieux Company</td>
			<td style="width:217px;">43041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Condress&#174;</td>
			<td style="width:257px;">Abiogen Pharma</td>
			<td style="width:217px;"></td>
			<td>collagen sponge used for bio-complexes</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">DMSO&#160;</td>
			<td style="width:257px;">Bioniche Pharma USA LLC, Lake Forest, IL</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl solution</td>
			<td>Fresenius Kabi, Bad Homburg, Germany</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene&#160;</td>
			<td style="width:257px;">Carlo Erba</td>
			<td style="width:217px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue characterization after a new disaggregation method for skin micro-grafts generation

几种新的方法，已经开发了生物技术在手术过程中，得到的自体细胞的悬浮液的领域中，为了对急性和慢性皮肤损害提供一个步骤的治疗方法。而且，从创伤，糖尿病，感染和其他原因引起的慢性而且急性伤口的管理，仍然具有挑战性。在这项研究中，我们描述了一个新的方法来创建从单个患者的皮肤组织及其临床应用自体微移植。此外，还进行体外皮肤，去epidermized真皮（DED）和多器官和/或多组织捐助者的真皮源性皮肤组织的生物学特性。所有组织被这个新协议分类的，使我们能够得到可行的微移植物。特别是，我们报道了这一创新协议是能够创建自体微移植物和胶原海绵准备上皮损要施加由生物复合物。在克里尼上的腿病变自体生物复合物的校准应用也有报告，表示再epitalization过程和病变的柔软性两者的改进。此外，我们的体外模型表明，该系统机械解聚后的细胞活力保持在一段时间内培养可达七（7）天。我们还观察到，通过流式细胞术分析，从解聚获得的细胞的池是由几个细胞类型，包括间质干细胞，即施加在组织再生和修复的方法中的一个关键的作用，对它们的高的再生潜力。最后，我们表现出体外 ，在琼脂培养的菜时，此过程中保持微移植的不育。总之，我们的结论是这种新的再生方法可以是一个有希望的工具，临床医师在一个步骤上可行，无菌的，并且准备使用，可以单独或组合使用最常见的生物scaffo施加微移植物获得LDS。

在这个初步研究，第一个目的是探讨人类自体微移植与生物支持相结合，如胶原蛋白的生产生物复合物准备使用的能力。这些生物复合物在患者中植入 引起的车祸中（ 图2A）腿部病变和30天后（ 图2B）的组织修复相关的完整的再上皮中观察到。此外，临床随访上升5个月（ 图2C）后显示出已损坏的区域的良好的质地和柔软度，在平行于临床应用，还进行体外研究，以评估不同的皮肤组织的细胞生存力，如皮肤组织的，扣减和真皮前，由该系统分解后。特别是，考虑到每个类型的组织的不同的厚度，八阈值，可以在对皮肤组织，扣减和真皮单一步骤进行处理四加三活检，分别成立。活检的既定数目然后在四个不同的时间分列如表1所示，根据以查明解聚的最佳条件，以维持良好的细胞生存力的生物学特性。 虽然组织处理本身必然诱导相比完整的组织细胞活力的损害，与此系统进行机械解聚似乎皮肤，扣减和真皮的所有样品中，以保持30％的细胞存活率的平均值 在不同的时间（ 图3A），相对于完整的组织（ 图3B）评价（平均92 OD /克为完整皮肤组织和29的OD /克分解后的;从22 OD /克至7 OD /克FOř完好DED相对于分解并最终从16 OD /克至2​​.7的OD /克用于相对于分解完整真皮）。 因此，这些初步结果表明，该系统能够立即分解后维持细胞活力的可观的水平。解聚对细胞存活率的影响也被研究了24小时或7天及可变结果培养观察组织样本。特别是，细胞活力的皮肤组织样品中没有实质性的变化是由比较起始时间（T 0）（ 图4A）均质化和培养的时间独立地观察到。另一方面，扣减样本的降低存活率后培养24小时相比，起始时间（T 0）被观察到，但令人惊讶的细胞存活率后7天培养的（ 图4B）恢复。类似的结果也被用于真皮的样品观察到的（ 图4C）。每个样品一式两份，并在表2中报道的值进行评价。 在这些结果的基础上，我们确定了分解维持三种皮肤组织的细胞生存力的合适的时间如报道于表3除了细胞活力，培养的细胞悬浮液的形态方面也评价，识别单个细胞之后，从组织分解24小时，同时观察7天有两种皮肤组织和扣减/真皮样品纤维残渣后（ 图5 AB）。另外，通过流式细胞术分析细胞表征真皮的样品中其均化后进行的:由几个细胞类型，包括内皮细胞和间质干细胞组成的细胞的异质池被鉴定（ 图6）。此外，由于没有细菌生长的鉴定我<html> n的所有样本分类巧接种于琼脂菜，证明实验步骤（ 图7）的无菌。 <img alt="图1" src="/files/ftp_upload/53579/53579fig1.jpg" /> 图1.生物复合 组织 建设 扰乱系统（A）和收集从患者的皮损区超凡的一小块，这是与组织干扰物随后进行分类。获得胶原蛋白海绵结合微移植物的生物复合物以获得人的补丁直接用于病变的修复（C）（D）苏木伊红（H＆E）染色的代表性生物复杂，在DMEM培养基中培养3天，观察到镜检<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig1large.jpg" target="_blank">请点击这里查看大图这个数字。</a> 内容"FO:保together.within页="1"&gt; <img alt="图2" src="/files/ftp_upload/53579/53579fig2.jpg" /> 被应用在腿病变（A）的 图上腿病变由胶原和从患者获得的微移植物组成的生物复合2.生物复合物的应用和后30天（B）和5个月伤口进行评价（℃ ）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53579/53579fig3.jpg" /> 图3. 有 丝可行性立即在三个类型皮肤组织（A）的不同时间相对完整的皮肤组织（B），皮肤解体后 ，从四个不同的供体的扣减和真皮组织与组织disru分类ptors如表示在文本中的相应部分。细胞活力，一式两份每个组织样本进行MTT检测评估。该图是代表以双份对于各样品进行四种不同的实验。该结果表示为光密度（OD）和组织的克数（克）之间的比率;标准偏差计算对组织的光密度（OD）和克（GR）之间的比率。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53579/53579fig4.jpg" /> 皮肤组织（A）的分列样品 图4. 有 丝生存能力水平，扣减（B）和真皮（三）开始时间（T 0）和继代培养24小时，7天之后，该组织与组织匀浆作为干扰籼TE在文本中的相应部分。在分类样品随后培养解聚24小时后和7天，并保持在RPMI 1640培养基中，在5％CO 2 /空气的气氛中补充有10％胎牛血清和抗生素的，在37℃。如前所述细胞活力通过MTT评估。该图是代表一式两份对皮肤组织，扣减和真皮从单个供体衍生的各样品进行的实验的。结果表现为组织的光密度（OD）和克（GR）之间的比值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53579/53579fig5.jpg" /> 图5. 分门别类的皮肤组织形态分析（A）和后扣减/真皮（B）24小时和邪教的7天<html> URE。使用后24小时，在RPMI介质对皮肤组织和扣减/真皮组织中存在培养7天后光镜下进行形态分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53579/53579fig6.jpg" /> 图6.通过流式细胞仪分析细胞特征。机械解体后，获得的真皮微移植物放在文化和后7天进行分析，积极为间质细胞和造血细胞标记物，包括CD146，CD34和CD45抗原。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> LES / ftp_upload / 53579 / 53579fig7.jpg"/&gt; 分门别类皮肤组织，扣减和真皮样品的分类组织样品的小片段图7. 微生物分析。接种于哥伦比亚琼脂加入下层流罩5％羊血（生物梅里埃公司），在37℃下培养三天进行微生物分析和评估过程的无菌。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53579/53579fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td> 组织样本 </td><td> 分解时间（秒/分） </td></tr><tr><td>皮肤</td><td> 30秒</td></tr><tr><td></td><td> 1分钟</td></tr><tr><td></td><td> 2分钟</td></tr><tr><td></td><td> 5分钟</td></tr><tr><td>扣减</td><td> 1分钟</td></tr><tr><td></td><td> 2分钟</td></tr><tr><td></td><td> 5分钟</td></tr><tr><td></td><td> 10分钟</td></tr><tr><td>真皮</td><td> 1分钟</td></tr><tr><td></td><td> 2分钟</td></tr><tr><td></td><td> 5分钟</td></tr><tr><td></td><td> 10分钟</td></tr></tbody></table> 表1.用于皮肤的解体四个不同时代的示意图，扣减和真皮八 ，四，三皮打孔活检，扣减和真皮分别在四个不同的时间分类，根据其生物学特性。 <table fo:keep-together.within-page="1"><tbody><tr><td colspan="6"> 皮肤组织 </td></tr><tr><td colspan="2"> 至 </td><td colspan="2"> 24小时 </td><td colspan="2"> 7天 </td></tr><tr><td> 2 </s仲&gt;" </td><td> 5&#39; </td><td> 2&#39; </td><td> 5&#39; </td><td> 2&#39; </td><td> 5&#39; </td></tr><tr><td> 37.1 </td><td> 41.07143 </td><td> 41.07143 </td><td> 65.78571 </td><td> 41.14286 </td><td> 37.42857 </td></tr><tr><td> 35.8 </td><td> 43.82143 </td><td> 36.60714 </td><td> 66.5 </td><td> 41.46429 </td><td> 38.67857 </td></tr><tr><td colspan="6"> 扣减 </td></tr><tr><td colspan="2"> T 0的 </td><td colspan="2"> 24小时 </td><td colspan="2"> 7天 </td></tr><tr><td> 5&#39; </td><td> 10&#39; </td><td> 5&#39; </td><td> 10&#39; </td><td> 5&#39; </tD&gt; <td> 10&#39; </td></tr><tr><td> 6,649485 </td><td> 8.237113 </td><td> 5.463918 </td><td> 5.731959 </td><td> 7.835052 </td><td> 7.85567 </td></tr><tr><td> 6,845361 </td><td> 8,360825 </td><td> 5.257732 </td><td> 5.989691 </td><td> 8 </td><td> 7.938144 </td></tr><tr><td colspan="6"> 真皮 </td></tr><tr><td colspan="2"> 至 </td><td colspan="2"> 24小时 </td><td colspan="2"> 7天 </td></tr><tr><td> 10&#39; </td><td> 5&#39; </td><td> 10&#39; </td><td> 5&#39; </td><td> 10&#39; </td><td></td></tr><tr><td> 1.690909 </td><td> 2.77193 </td><td> 0.293898 </td><td> 0.786704 </td><td> 2.880952 </td><td> 2.869048 </td></tr><tr><td> 1.736364 </td><td> 2.830409 </td><td> 0.306351 </td><td> 0.814404 </td><td> 2.940476 </td&gt; <td> 2.928571 </td></tr></tbody></table> 表2.在范围OD /克表达了一种生物复制价值观。从皮肤组织细胞活力的价值观，扣减和真皮评估，起始时间和机械分解到指定的时间后。结果代表一式两份进行，并表示为组织的光密度（OD）和克（克）之比一个实验的。 <table fo:keep-together.within-page="1"><tbody><tr><td> 标本 </td><td> 解体时间 </td></tr><tr><td>皮肤组织</td><td> 5分钟</td></tr><tr><td>扣减</td><td> 1分钟</td></tr><tr><td>真皮</td><td> 2分钟</td></tr></tbody></table> 表3.解体的最佳时机的示意图，以保持各色中一个良好的细胞活力nt的组织样本。之后分别分解的5,1和2分钟，皮肤，扣减和真皮组织样本显示的最佳细胞活力。

Watch this Scientific Journal Video about Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation at JoVE.com

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

该协议描述了一种新的方法来分解人体组织和​​创建自体微移植，与胶原蛋白海绵相结合，引起人体生物复合物准备皮损的治疗使用。另外，该系统保持在机械解聚后不同时间的微移植物的细胞生存力。

后皮肤微移植生成一个新的方法解聚表征的组织

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

tissue-characterization-after-new-disaggregation-method-for-skin

Nanyang Technological University

Research

JoVE Journal

Medicine

21.5K 次观看.  Burns Centre and Emilia Romagna Regional Skin Bank. 该协议描述了一种新的方法来分解人体组织和​​创建自体微移植，与胶原蛋白海绵相结合，引起人体生物复合物准备皮损的治疗使用。另外，该系统保持在机械解聚后不同时间的微移植物的细胞生存力。 

视频: Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

危重病人中的应用手册肌力测试:下肢肌力测量方法

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

科研

医学

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

A Next-generation Tissue Microarray (ngTMA) Protocol for Biomarker Studies

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into a &#8216;recipient&#8217; block and then used for a variety of biomarker applications. The construction of conventional TMAs is labor intensive, imprecise, and time-consuming. Here, a protocol using next-generation Tissue Microarrays (ngTMA) is outlined. ngTMA is based on TMA planning and design, digital pathology, and automated tissue microarraying. The protocol is illustrated using an example of 134 metastatic colorectal cancer patients. Histological, statistical and logistical aspects are considered, such as the tissue type, specific histological regions, and cell types for inclusion in the TMA, the number of tissue spots, sample size, statistical analysis, and number of TMA copies. Histological slides for each patient are scanned and uploaded onto a web-based digital platform. There, they are viewed and annotated (marked) using a 0.6-2.0 mm diameter tool, multiple times using various colors to distinguish tissue areas. Donor blocks and 12 &#8216;recipient&#8217; blocks are loaded into the instrument. Digital slides are retrieved and matched to donor block images. Repeated arraying of annotated regions is automatically performed resulting in an ngTMA. In this example, six ngTMAs are planned containing six different tissue types/histological zones. Two copies of the ngTMAs are desired. Three to four slides for each patient are scanned; 3 scan runs are necessary and performed overnight. All slides are annotated; different colors are used to represent the different tissues/zones, namely tumor center, invasion front, tumor/stroma, lymph node metastases, liver metastases, and normal tissue. 17 annotations/case are made; time for annotation is 2-3 min/case. 12 ngTMAs are produced containing 4,556 spots. Arraying time is 15-20 hr. Due to its precision, flexibility and speed, ngTMA is a powerful tool to further improve the quality of TMAs used in clinical and translational research.

A Next-generation Tissue Microarray ngTMA Protocol for Biomarker Studies

The protocol aims at optimizing the construction and quality of tissue microarrays for biomarker research. It includes aspects of planning and design, digital pathology, virtual slide annotation, and automated tissue arraying.

生物标志物的研究下一代组织微阵列（ngTMA）协议

Biomarker research relies on tissue microarrays (TMA). TMAs are produced by repeated transfer of small tissue cores from a &#8216;donor&#8217; block into ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

在背皮褶商会鼠标的缺血性组织损伤:一个皮瓣模型，探讨急性缺血持续

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

一个<em&gt;在体外</em&gt;对雌激素敏感的乳腺癌休眠模式在骨髓中:分子机理研究与假设生成的一个工具

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮肤去神经外科手术:一种用于皮肤病的小鼠模型测试要求的神经方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

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

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

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

经非透法皮下腹脂肪组织活检技术

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

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

用于疾病建模的人三维肺组织培养 (3D-LTCs) 的生成

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

一种分离和扩增人类脂肪衍生的美源性干细胞的无酶方法

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...