NIH 机构博士后培训补助金, NIH-HL-007260

所有的动物外科手术程序都是在南卡罗来纳州医科大学的机构动物护理和使用委员会 (IACUC) 批准的, 然后再进行任何动物手术或任何动物组织的使用。
1. 小鼠肾切除术
<ol><li>不要用手术面罩和蓬松帽来减少污染的风险。在手术区的设置中保持不孕。<ol>
<li>将非 fenestrated 的手术窗帘放在手术台上。</li>
		<li>在无菌窗帘上打开一包蒸压仪器。肾切除术所需的器械包括3小止血剂、牙精钳、无牙精钳、直虹膜剪刀。</li>
		<li>将另一种非 fenestrated 的手术悬垂放在手术台的中心。</li>
		<li>用钙和镁制备5毫升的 Dulbecco 磷酸盐缓冲盐水 (DPBS), 在15毫升无菌圆锥管中补充1% 青霉素/链霉素。将此解决方案放在操作表旁边的冰上。</li>
	</ol>
</li>
	<li>从动物住房综合体获得 8-12 周大的 C57BL/6 鼠 (男性或女性)。</li>
	<li>将鼠标放在麻醉感应腔内, 并诱导3.5% 吸入异氟醚。在鼠标上放置一个鼻锥, 使用2% 异氟醚进行维护麻醉。<ol>
<li>通过评估踏板反射 (牢固的脚趾捏), 在整个手术过程中周期性地监测足够的麻醉深度。</li>
	</ol>
</li>
	<li>使用脱毛膏去除腹侧躯干的所有毛发。用蒸馏水冲洗这个区域, 以确保所有的毛发都已从腹部取出。</li>
	<li>在顶部非 fenestrated 悬垂下, 将鼠标转移到仰卧位的无菌手术台上。切出一个 2 x 2 厘米2开窗, 在刚刚覆盖鼠标腹部的褶皱。</li>
	<li>应用聚维酮碘后, 70% 乙醇的腹部使用无菌纱布或棉花球。</li>
	<li>使用牙和虹膜剪刀精钳, 使 3-4 厘米垂直腹部皮肤切口平行的中线, 0.5 厘米到左中线 注意:如果试图删除右肾, 这将是一个切口0.5 厘米右侧的中线。<ol>
<li>用细钳无牙抓腹膜, 用虹膜剪刀切割进入腹腔。将止血剂应用于皮肤和腹膜的上、下侧边缘 , 以横向放置张力 , 增露。</li>
		<li>将肠道内容微妙地移到腹部右侧, 以暴露左肾。轻轻地把牵引放在肾脏上, 把它从腹部抬高。</li>
		<li>在左肾的脐处放置一个止血。用虹膜剪刀对输尿管和肾血管进行横断面。</li>
		<li>在冰上放置左肾1% 青霉素/链霉素 DPBS 溶液。</li>
		<li>将含有肾脏的管转移到无菌组织培养罩中。将肾脏从15毫升锥形管转移到60毫米培养皿 (图 1)。用5毫升 DPBS 与钙和镁冲洗两次。保持肾脏悬浮在5毫升 DPBS 在60毫米菜。<ol>
<li>准备一个额外的60毫米培养皿与5毫升 DPBS 将用于在显微解剖协议。</li>
		</ol>
</li>
		<li>根据批准的 IACUC 动物协议, 弄死通过开胸和放血后的肾脏的采购。</li>
	</ol>
</li>
</ol>2. 小鼠肾脏显微切割分离肾段
<ol><li>继续使用无菌技术的这一部分的协议, 包括无菌手套, 手术面罩, 蓬松, 以减少污染风险。<ol>
<li>将无菌窗帘放在立体声显微镜平台上, 以及在显微镜的左侧和右侧的区域有一个无菌的领域为仪器。将塑料粘合剂片放在显微镜聚焦旋钮上, 喷雾70% 乙醇。打开双鹅颈灯照明舞台。</li>
		<li>开放式消毒器械 (无牙精钳, 手术刀柄, #15 手术刀刀片, 两个直止血剂, 两个30½口径空心孔针) 到无菌窗帘。现在将 #15 刀片放到手术刀手柄上, 并将一个止血连接到每个针适配器/轮毂, 以创建用于以后使用的针式解剖仪器, 如图 1所示。</li>
	</ol>
</li>
	<li>移动60毫米培养皿, 一个包含肾脏在 DPBS 和其他仅 DPBS, 从组织培养敞篷到显微镜区域。<ol>
<li>将60毫米培养皿置于目标下, 取出盖子露出肾脏。将含有 DPBS 的盘子留在不育的田地旁, 供日后使用。</li>
		<li>移动的菜, 以便只有前或后表面的肾脏是在视图 (即, 大平脸的肾脏, 与另一张脸接触底部的菜)。缩放到 3.2 4X 的过程的这一部分。使用非显性手, 使用无牙的细钳刺穿和针在肾脏的下和上两极附近的盘子。</li>
		<li>保持不占支配地位的手固定肾脏。用主导的手, 使用 #15 刀片从暴露的表面去除半透明纤维囊层。从肾脏外露表面刮出最浅的0.5 毫米, 除去胶囊的其余部分。</li>
		<li>使用 #15 刀片从 decapsulated 区域解剖组织的倒置金字塔, 大小约为2毫米3 。检索60毫米的菜只含有 DPBS, 并将组织金字塔放到干净的盘子里。丢弃肾脏的其余部分。</li>
		<li>在解剖的其余部分减少 1.5-2.0X 的放大倍数。使用两个止血针仪器作为切割仪器, 切片的组织片段成逐渐小的片断, 直到组织段小于或等于针尖的直径。肾脏组织2毫米3的大小将产生超过50段, 一旦完全解剖。</li>
		<li>将60毫米的菜与肾段移至组织培养罩。用1000µL 吸管去除 DPBS。添加5毫升 DMEM, 辅以10% 胎牛血清 (血清) 和1% 青霉素/链霉素。</li>
		<li>培养3天在37°c 在孵化器或植入细胞结构立即。</li>
	</ol>
</li>
</ol>3. 肾段细胞结构制作
<ol><li>培养正常的人真皮成纤维细胞 (NHDF) 和人脂肪微血管内皮细胞 (HAMEC) 几个星期, 以获得 7.2 x 105成纤维细胞和 1.8 x 105内皮细胞每个结构所需的。在组织培养罩中, 将成纤维细胞的适当比例 (4:1) 放入琼脂糖模中的棒状井中, 形成无支架的前血管内皮-成纤维细胞结构 (规格), 如前文所述 Czajka et 19。</li>
	<li>获得灭菌止血, 30½ G 针, 和无菌铲与平端。<ol>
<li>从含有肾脏片段的60毫米盘子中取出大部分培养基, 小心不要吸入并取出肾部分。</li>
		<li>配备手术放大镜, 以可视化的片段植入。</li>
		<li>在60毫米的盘子底部轻轻地刮擦刮刀的末端, 以获得 10-15 段, 沿铲片的尖端均匀分布。把刮刀的尖端尽可能靠近规格的种子的唇口。</li>
		<li>另一方面, 使用 hemostat-30½的 G 针工具, 将每个部分从铲头移动到井的边缘。用针尖轻轻地将每个段向下移动到井中, 直到在先前播种的细胞悬浮液中略微淹没。</li>
		<li>培养肾段-规格在 2:1: 1 FGM-2/EGM-2/DMEM 培养基的比例 (2.5 毫升体积总数)。每24小时更改文化媒体3天。从模具中取出 72 h 的构造, 然后进行修复或闪速冻结处理。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

用于设计活体肾脏组织结构的方法在所使用的细胞类型和生物材料方面差别很大, 在许多情况下, 在文献7中已经过时或没有很好的特征。虽然许多人使用干细胞方法或综述独立的肾脏结构的个别成分, 但从细胞悬浮中人工再造26多个不同分化细胞类型的整个器官的前景是压倒性地考虑21。这很可能是其他人在组织工程应用中使用肾组织片段的方法。如上所述, 以前...

慢性肾脏疾病 (CKD) 是当前全球主要的公共卫生挑战之一1。在美国, CKD 患病率超过总人口的 14%, 超过60万的美国人患有最严重的疾病, 终末期肾病 (ESRD)2。目前的治疗方案可供那些 ESRD 包括透析和肾移植。虽然每年约有2.5万名患者接受肾移植, 但每年有大量患者被增加, 导致等待救生器官和接受移植的人之间的巨大差距3。除了对长寿和生活质量造成严重的负面影响外, 透析还带来了惊人的经济负担。在 2014年, 医疗保险支付的索赔总额超过300亿美元的 ESRD 患者2。由于器官供应有限, 需要透析的患者没有明显的下降趋势, 因此, 旨在确定透析和移植的替代解决方案的研究工作一直是重要的。即使是相对较短的延迟, 透析的需要增加了病人的质量调整生活年限和生产力的大量, 而推迟透析相关费用4,5,6。
在组织工程和再生医学实验室中, ESRD 的功能性组织丧失的解决方案目前正在研究中, 从脚手架为基础的 organoid 制造到整个器官工程都有广泛多样的方法。瓣膜细胞植入的器官结构7,8,9,10,11。综述复杂的肾脏结构从边缘或被放弃的肾脏仅部份地被调查了。事实上, 为移植而采购的肾脏的近20% 因各种原因被丢弃,12,13。这些假定的移植物的功能性肾脏组织可以被利用并纳入一个或多个组织工程结构。先前的研究表明, 与这些被丢弃的器官合作的可行性, 利用肾脏为组织工程目的的额外细胞矩阵14,15。然而, 很少有使用健康肾脏的主要 nephronal 组织为组织工程目的16,17,18。
Kim et以前描述的一种方法是将肾脏 "段" 从健康的大鼠肾脏中分离出来, 然后在羟基酸 (PGA) 支架上播种, 用于构造制造16。然而, 关于精确解剖方法的信息很少, 从精细切碎和过滤的结合中得到了部分。我们描述了这个协议的修改, 这同样产生了离散的肾段与完整的 nephronal 体系结构, 而是依靠显微切割技术。切除是对活体成年小鼠进行的, 然后将肾脏转移到切除肾囊的解剖显微镜, 并进一步解剖组织。小孔 30½ G 针作为切割仪器, 也作为辅助解剖辅助, 因为针直径等于肾段的靶直径。这种孤立的, 在这种情况下, 小鼠, 肾部分保持在培养的生存能力, 并纳入无支架内皮细胞成纤维细胞结构19。这些构造以前被用来设计其他器官, 包括生物人工胰腺20。

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			<td height="42" style="height:42px;width:173px;">Non-fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">696</td>
			<td style="width:199px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fenestrated Sterile Field</td>
			<td style="width:181px;">Busse Hospital Disposables</td>
			<td style="width:153px;">697</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Halsted Mosquito Forceps 5 Curved</td>
			<td style="width:181px;">Miltex</td>
			<td style="width:153px;">Mil-7-4</td>
			<td>&#34;Hemostat&#34; in manuscript</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Extra Fine Graefe Forceps, Curved with teeth</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">11155-10</td>
			<td>Fine forceps with teeth</td>
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		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Extra Fine Graefe Forceps, Serrated (without teeth)</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">11152-10</td>
			<td>Fine forceps without teeth</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fine Scissors - Tungsten Carbide</td>
			<td style="width:181px;">Fine Science Tools</td>
			<td style="width:153px;">14568-09</td>
			<td>Iris Scissors</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Betadine Surgical Scrub with Pump, Povidone-iodine 7.5%</td>
			<td style="width:181px;">Purdue Products L.P.</td>
			<td style="width:153px;">67618-151-17</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Sterile Cotton Gauze Pad (4&#34; x 4&#34;)</td>
			<td style="width:181px;">Fisher Healthcare</td>
			<td style="width:153px;">22-415-469</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Dulbecco&#39;s Phosphate Buffered Solution</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">21-030-CV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Penicillin/Streptomycin Solution, 100X</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">30-002-Cl</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Isoflurane, USP</td>
			<td style="width:181px;">Manufacturer: Piramal, Distributor: McKesson</td>
			<td style="width:153px;">2254845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Nair Hair Remover</td>
			<td style="width:181px;">Nair</td>
			<td style="width:153px;">22600-23307</td>
			<td>Hair Removal Cream in text</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">200 Proof Ethanol</td>
			<td style="width:181px;">Decon Laboratories</td>
			<td style="width:153px;">2705</td>
			<td>Diluted to 70% Ethanol Solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">BioLite 60mm Tissue Culture Dish</td>
			<td style="width:181px;">Themo-Scientific</td>
			<td style="width:153px;">130181</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Press&#39;n Seal</td>
			<td style="width:181px;">Glad</td>
			<td style="width:153px;">12587-70441</td>
			<td>Applied to Stereoscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">SZX16 Stereo Microscope</td>
			<td style="width:181px;">Olympus</td>
			<td style="width:153px;">SZX16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Fiber Optic Illuminator</td>
			<td style="width:181px;">Cole Parmer</td>
			<td style="width:153px;">41720-20</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Self-Supporting Dual-Light Pipe, 23&#34; L Gooseneck</td>
			<td style="width:181px;">Cole Parmer</td>
			<td style="width:153px;">EW-41720-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Scalpel Handle #3</td>
			<td style="width:181px;">Miltex</td>
			<td style="width:153px;">Mil-4-7</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Sterile Rib-Back Carbon Steel Blade, Blade Size 15</td>
			<td style="width:181px;">Bard-Parker</td>
			<td style="width:153px;">371115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">31 1/2 Gauge Needle</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">14-826F</td>
			<td style="width:199px;">Becton Dickinson 305106</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:181px;">Corning</td>
			<td style="width:153px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Fetal Select 100% Bovine Serum</td>
			<td style="width:181px;">Atlas Biologicals</td>
			<td style="width:153px;">FS-0500-AD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Normal Human Dermal Fibroblasts</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-2511</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Human Adipose Microvascular Endothelial Cells</td>
			<td style="width:181px;">Sciencell Research Laboratories</td>
			<td style="width:153px;">7200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Surgical Loupes (2.5x)</td>
			<td style="width:181px;">Orascoptic</td>
			<td style="width:153px;">(N/A) Custom Order</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">FGM-2 (Fibroblast Basal Medium with FGM-2 SingleQuots Added)</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3131, CC-4126</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">EGM-2 (Endothelial Basal Medium with EGM-2 SingleQuots Added)</td>
			<td style="width:181px;">Lonza</td>
			<td style="width:153px;">CC-3156, CC-4176</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">L3224</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Anti-Cytokeratin-18 Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Goat anti-Mouse IgG, Alexa Fluor 633</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-21052</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Goat anti-Rabbit IgG, Alexa Fluor 546</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:153px;">A-11010</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:173px;">Anti-Von Willebrand Factor Antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:153px;">ab6994</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:173px;">Albumin, Fluorescein isothiocyanate Conjugate</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:153px;">A9771-50MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Hoescht 33342</td>
			<td style="width:181px;">BD Pharmingen</td>
			<td style="width:153px;">561908</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:173px;">Background Buster</td>
			<td style="width:181px;">Innovex Biosciences</td>
			<td style="width:153px;">NB306</td>
			<td></td>
		</tr>
	</tbody>
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microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology

肾脏移植现在是治疗终末期肾脏疾病的主流疗法。然而, 约有9.6万人在等待名单上, 只有1/4 的患者实现移植, 迫切需要为那些器官衰竭者提供替代品。为了减少透析带来的有害后果, 以及它所招致的整体医疗费用, 正在积极调查寻找移植器官的替代办法。植入性组织工程的肾细胞结构是一种替代失去的肾脏功能的可行方法。这里, 第一次被描述, 是小鼠肾脏的显微解剖为分离生活皮质髓质肾脏片断。这些片段能够迅速纳入无支架的内皮纤维细胞结构, 这可能使与宿主血管的快速连接一旦植入。成年小鼠肾脏是从活体捐献者那里获得的, 其次是立体镜显微解剖, 得到 200-300 µm 直径的肾段。多肾结构是用从一肾中收获的原发性肾段制作而成。该方法证明了一种可以从器官中抢救功能性肾组织的程序, 否则将被丢弃。

所描述的协议产生大约50个肾脏片段每锥体2毫米3段肾脏组织。已处理和成像的肾段有不同比例的管状和肾小球成分 (请参见图 2)。完整的片段进行了化验, 以确定每24小时的不同片段的生存时间三天。绿色荧光 calcein 是存在的细胞内酯酶活性, 指示的活细胞。红荧光溴 homodimer-1 被认为是由于等离子膜完整性的丧失。虽然这些片段是完整的, 三?...

Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

组织工程的肾脏结构提供了解决的器官短缺和有害影响的透析。在这里, 我们描述了一个小鼠肾脏的显微解剖的协议, 以隔离皮质髓质段。这些片段植入无支架细胞结构, 形成肾 organoids。

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

microdissection-primary-renal-tissue-segments-incorporation-with

Research

JoVE Journal

Bioengineering

7.3K 次观看.  Medical University of South Carolina. 组织工程的肾脏结构提供了解决的器官短缺和有害影响的透析。在这里, 我们描述了一个小鼠肾脏的显微解剖的协议, 以隔离皮质髓质段。这些片段植入无支架细胞结构, 形成肾 organoids。

视频: Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

磁共振弹性成像方法评价组织工程构造增长

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

科研

生物工程

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

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, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

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, ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

在细胞和组织力学生物学应用一种新型的拉伸平台

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Antimicrobial Peptides Produced by Selective Pressure Incorporation of Non-canonical Amino Acids

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all variations are based on the 20 canonical amino acids (cAAs). As a way to introduce additional physicochemical properties into polypeptides, the incorporation of non-canonical amino acids (ncAAs) is increasingly used in protein engineering. Due to their relatively short length, the modification of ribosomally synthesized and post-translationally modified peptides by ncAAs is particularly attractive. New functionalities and chemical handles can be generated by specific modifications of individual residues. The selective pressure incorporation (SPI) method utilizes auxotrophic host strains that are deprived of an essential amino acid in chemically defined growth media. Several structurally and chemically similar amino acid analogs can then be activated by the corresponding aminoacyl-tRNA synthetase and provide residue-specific cAA(s) &#8594; ncAA(s) substitutions in the target peptide or protein sequence. Although, in the context of the SPI method, ncAAs are also incorporated into the host proteome during the phase of recombinant gene expression, the majority of the cell&#39;s resources are assigned to the expression of the target gene. This enables efficient residue-specific incorporation of ncAAs often accompanied with high amounts of modified target. The presented work describes the in vivo incorporation of six proline analogs into the antimicrobial peptide nisin, a lantibiotic naturally produced by Lactococcus lactis. Antimicrobial properties of nisin can be changed and further expanded during its fermentation and expression in auxotrophic Escherichia coli strains in defined growth media. Thereby, the effects of residue-specific replacement of cAAs with ncAAs can deliver changes in antimicrobial activity and specificity. Antimicrobial activity assays and fluorescence microscopy are used to test the new nisin variants for growth inhibition of a Gram-positive Lactococcus lactis indicator strain. Mass spectroscopy is used to confirm ncAA incorporation in bioactive nisin variants.

The protocol presents the Escherichia coli-based selective pressure incorporation of non-canonical amino acids (ncAAs) into the lactococcal antimicrobial peptide nisin. Its properties can be changed during recombinant expression via substitution with desired ncAAs in defined growth media. Resulting changes in bioactivity are mapped by growth inhibition assays and fluorescence microscopy.

非规范氨基酸选择性压力合成抗菌肽的研究

Nature has a variety of possibilities to create new protein functions by modifying the sequence of the individual amino acid building blocks. However, all ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.