我们感谢卡蒂 Andreasson 博士 (斯坦福大学神经学和神经科学系) 使用 qRT PCR 机。这项工作得到了国立卫生研究院资助 K08NS098876 (P.M.G.) 和斯坦福医学院院长的博士后奖学金 (狐臭) 的支持。

所有干细胞和动物程序均经斯坦福干细胞研究监督委员会和斯坦福大学实验室动物护理管理小组 (SCRO-616 和 APLAC-31909) 批准。
1. ITO 玻璃的蚀刻
<ol><li>在玻璃的导电面上放置一个掩蔽剂, 制备铟锡氧化物 (ITO) 覆盖的玻璃滑块。 注: 大多数商用透明胶带可用作掩蔽剂。<ol>
<li>用刀片去掉多余的面具, 只留下玻璃上覆盖的最终 ITO 设计所需的形状。</li>
	</ol>
</li>
	<li>将 5% (v/v) 的硝酸和 45% (v/五) 的 HCl 在一个通风罩内的玻璃烧杯中组合, 准备蚀刻溶液。<ol>
<li>使用热板和温度计, 以确保蚀刻溶液的温度保持在摄氏70摄氏度。</li>
	</ol>
</li>
	<li>一旦蚀刻溶液达到70摄氏度, 将屏蔽 ito 玻璃滑入溶液中2分钟, 以去除多余的 ITO 层。 注: 胶带掩蔽保护 ITO 层从接触酸性溶液。</li>
	<li>从解决方案中取出幻灯片, 并用去离子 (DI) H2O 冲洗它。</li>
	<li>小心地取下面罩, 用万用表测量电阻, 以确保其余的 ITO 完好无损。所需蚀刻区域的读数应约为0Ω。</li>
</ol>2. 吡咯溶液的制备
<ol><li>在1000毫升玻璃瓶中, 将70克 dodecylbenzenesulfonate 钠 (NaDBS) 溶于600毫升的 DI H2O。</li>
	<li>一旦 NaDBS 溶解, 添加14毫升 0.2 M 吡咯和386毫升的 DI H2O 的解决方案。</li>
	<li>用铝箔覆盖溶液, 贮存在4摄氏度。</li>
</ol>3. 在 ITO 玻璃上电镀聚吡咯
<ol><li>将吡咯溶液加热到室温, 直到 NaDBS 完全 redissolved。 注意: 这需要大约15到20分钟。</li>
	<li>将25毫升的吡咯溶液倒入烧杯中。</li>
	<li>将蚀刻 ITO 玻璃与万用表连接, 将滑块浸入吡咯溶液中。<ol>
<li>确保0Ω电阻的一侧朝向铂网参考电极。</li>
	</ol>
</li>
	<li>使用万用表 (电流控制在3毫安/cm2 4 小时) 上, 应用电流启动 ITO 表面上的聚吡咯电镀。</li>
	<li>将 ITO 玻璃从万用表中取出, 并在 DI H2O 中洗涤电镀-聚吡咯, 以除去残留表面活性剂。</li>
	<li>用剃刀刀片轻轻地将电镀聚吡咯板从 ITO 玻璃中分离出来。<ol>
<li>在室温下贮存聚吡咯板。</li>
	</ol>
</li>
</ol>4. 烷的制备
<ol><li>使用刮刀和重量盘, 混合18克的基础剂与2克固化剂, 并倒入两个10厘米 x 8 厘米玻璃模具的混合物。</li>
	<li>使用真空室从模具中取出气泡 15-20 分钟。</li>
	<li>将模具放置在70摄氏度烤箱中3小时。</li>
	<li>冷却后, 使用平铲, 从模具中取出。</li>
</ol>5. 制作体外电刺激室
<ol><li>高压釜的金属板 (WxL, 2.5 厘米 x 12.5 厘米) 和流量阀在120°c 1 小时。</li>
	<li>使用会议厅幻灯片作为模板, 剪切两个片。 注: 在细胞室内有两个相邻正方形的切口, 其中一条为细胞室的周长。另一条是该会议厅外围的轮廓。<ol>
<li>切出内部的细胞室, 使它是方形的形状和匹配的细胞室。确保两片的整体形状是长方形的, 并与会议厅的滑动相匹配。</li>
	</ol>
</li>
	<li>将材料从下至上分层: (1) 金属板、(2) (不带切口)、(3) 聚吡咯板 (垂直于的)、(4) (带切口) 和 (5) 单元格室.  </li>
	<li>在完全对齐后, 将设备夹紧。</li>
	<li>切割两个60厘米的金属线, 用银糊将一根电线连接到聚吡咯板的每一端, 从腔体外凸出。确保电线足够长, 可以从设备延伸到孵化器的外部。</li>
	<li>一旦银糊固化, 加强和密封的电线连接与环氧树脂。<ol>
<li>使用万用表记录电阻, 以确保每个腔内的应用领域相同。</li>
		<li>植入时, 取下导线;松开细胞室和硅烷, 然后将它们与导电支架分开。植入支架的尺寸约为1毫米 x 3 毫米 x 0.25 毫米。</li>
	</ol>
</li>
</ol>6. 聚吡咯上的人神经祖细胞 (hNPCs) 的电镀
<ol><li>在紫外线照射下将组装的房间消毒2小时。<ol>
<li>1小时后, 旋转组装腔90°。</li>
	</ol>
</li>
	<li>灭菌后, 将聚吡咯的表面涂上800µL 的涂层衬底, 并使基体在 5%CO 2 为 1 h 的孵化器中的聚吡咯板上凝固。</li>
	<li>1小时后, 从室中取出上清, 用1毫升的 DPBS + 钙和镁轻轻冲洗每井。 注意: 避免用力清洗聚吡咯表面, 因为这会导致涂层衬底脱落。</li>
	<li>使用新鲜的细胞培养基, 机械分离培养细胞用于与室从组织培养板与温和的吹打。 注: 细胞在离解时应为 90-100% 汇合。</li>
	<li>用离心 8000 x g 收集 hNPC 颗粒5分钟。</li>
	<li>使用 hemocytometer, 在10万个单元格/cm2的卷上计数和并用重悬单元格。</li>
	<li>将10万个单元格放入每个腔室 (10万个单元格/cm2在1毫升介质中)。</li>
	<li>返回室到孵化器在37°c 在 5% CO2.
</li>
</ol>7. 电刺激 hNPCs
<ol><li>在将细胞放进腔室后的某一天, 使用波形发生器将电刺激应用于细胞。</li>
	<li>在刺激之前, 从每个腔室中清除旧介质, 并补充800µL 的新鲜培养基。</li>
	<li>在媒体被改变后, 将分庭放回孵化器, 将导线从孵化器中延伸出来, 并将它们连接到波形发生器上。</li>
	<li>在±400 mV 中使用100赫兹1小时的方波信号对细胞进行刺激。</li>
	<li>在刺激以后, 去除导线和孵化室在37°c 设置的孵化器在 5% CO2的另一天。</li>
	<li>根据制造商的协议, 进行后续的生物分析, 包括细胞活力和定量的实时聚合酶链反应 (qRT PCR)。</li>
</ol>8.体内聚吡咯植入术
<ol><li>对 T 细胞缺损的成年雄性裸鼠 (NIH-RNU 230, 30 克) 进行远端大脑中动脉 (dMCA) 闭塞中风模型, 如前所述2。一周后 dMCA 闭塞进行植入术。</li>
	<li>在手术前的某一天, 将氨苄西林 (1 毫克/毫升) 给笼水中的老鼠。</li>
	<li>麻醉在诱导腔内使用 0.5-3% 毫克/千克的异氟醚进行吸入。</li>
	<li>通过缺乏脚趾捏反射反应来证实大鼠麻醉。<ol>
<li>当动物在麻醉下, 继续监测其膜的颜色, 呼吸模式, 直肠温度每15分钟。</li>
	</ol>
</li>
	<li>一旦麻醉被证实, 在老鼠的眼睛上涂抹人工泪以防止干燥。</li>
	<li>通过使用无菌手套和无菌手术悬垂13确保在本过程中保持无菌技术。在不育的领域内保持无菌手术器械。</li>
	<li>当老鼠被麻醉时, 在左皮层上方钻一个减压。打开硬脑膜。<ol>
<li>用微薄的外科针从大脑中取出硬脑膜。</li>
	</ol>
</li>
	<li>将导电支架从体外系统移植到大鼠皮层。 注: 对于我们的实验, 我们在 hNPC 电镀后3天内将支架植入体外。<ol>
<li>把植入物主要放在半影皮质内侧的中风病灶。</li>
	</ol>
</li>
	<li>脚手架放置后, 放置 surgicel 在植入物上以防止皮肤闭合的运动。</li>
	<li>缝合伤口闭合, 皮下注射丁丙诺啡锶1毫克/千克剂量的大鼠, 以管理与立体定向手术和 dMCA 闭塞有关的疼痛。</li>
	<li>当它恢复知觉时, 把老鼠放在康复笼里。<ol>
<li>不要让动物在无意识的时候无人看管。</li>
		<li>不要把动物和别人放在一起, 直到它完全获得知觉。</li>
	</ol>
</li>
	<li>每天监测动物的疼痛症状, 包括体重下降, 减少食物/水摄入量, 减少梳理/蓬乱大衣, 减少/增加自发活动, 异常姿势, 脱水/皮肤 tenting, 凹陷的眼睛, 隐藏, 自我残害,快速呼吸, 张开嘴呼吸, 抽搐, 颤抖, 震颤和发声。<ol>
<li>每天监视动物, 直到看起来他们正在进食、饮酒、梳洗、发胖;然后每周体重增加。</li>
	</ol>
</li>
	<li>早期 euthanization 通过 co2吸入和二次确认 euthanization (以确保动物不会复活, 由于正常的氧气供应后 CO2吸入) 将发生的任何动物似乎不吃, 饮酒和开始手术后体重增加;出现在痛苦中;或似乎无法完成的行为测试, 由于大于预期的运动皮质赤字。</li>
</ol>

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在这项工作中, 作者没有任何利益冲突要申报。

越来越多的证据表明干细胞作为一种新型中风疗法的前景。这一承诺导致了一项重大的努力, 以推进干细胞治疗到床边至少40正在进行或完成的临床试验。中风病病理学提供了一种独特的神经紊乱, 使自己的干细胞治疗, 因为在急性侮辱后, 没有神经退行性的过程, 以防止恢复。干细胞增强脑卒中恢复的确切机制尚不清楚。血管生成、synaptogenesis 和突触重塑都被证明是重要的。当移除 BDNF 和 THBS-1 等?...

中风是世界上第二大死因, 也是美国第五大死亡原因。尽管有这些高死亡率, 中风康复治疗目前仍然是一个挑战, 目前尚无可行的医疗选项1。目前大约有300项临床试验涉及缺血性中风, 其中只有40的人利用干细胞。以前的研究表明, 干细胞疗法对中风修复有有益的影响2,3。由移植的人神经祖细胞 (hNPCs) 释放的脑源性神经营养因子 (BDNF) 和 thrombospondin-1 (THBS-1) 等分泌因素表明, 通过与增加突触形成, 血管生成, 树突状分支和新的轴突投射, 以及调节免疫系统4,5,6。然而, 干细胞的最佳分娩方法仍然是难以捉摸的。
成功的干细胞传递到大脑仍然是一个挑战。目前, 注射用水凝胶和高分子脚手架系统已被引入, 以提供干细胞。这些分娩方法保护干细胞移植期间, 并提供保护免受严酷的中风后环境, 包括宿主的炎症反应和缺氧条件7,8,9,10. 然而, 最常用的材料是惰性的, 这限制了连续调制 (即, 电刺激) 的细胞11的使用。电刺激是影响分化、离子通道密度和干细胞突起生长的一个提示12。与惰性聚合物相比, 导电聚合物可以携带电流, 允许电刺激和操纵干细胞2。然而, 电刺激调节神经营养因子释放 (即, BDNF 和 THBS-1) 的精确机制仍未得到充分探讨。
在本协议中, 我们描述了构建一个细胞培养系统的步骤, 该体系由导电高分子支架、聚吡咯 (聚吡咯) 组成,允许体外电刺激。由于细胞培养系统的制作方式, 随后将干细胞种子支架植入到周围梗死皮层是可能的。对于这个系统, 我们在植入前的短时间内, 在支架上电先决条件干细胞。继电刺激后, 携带细胞的导电高分子支架成功地植入 intracranially 使用微创方法。

<table border="0" cellpadding="0" cellspacing="0" width="623">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">FGF-Basic</td>
			<td style="width:103px;">Invitrogen</td>
			<td style="width:119px;">CTP0261</td>
			<td style="width:188px;">20 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Matrigel</td>
			<td style="width:103px;">Corning</td>
			<td style="width:119px;">cb40234a</td>
			<td style="width:188px;">1:200 dilution</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:213px;">LIF Protein, Recom. Hum. (10 &#181;g/mL)</td>
			<td style="width:103px;">EMD Millipore</td>
			<td style="width:119px;">LIF1010</td>
			<td style="width:188px;">10 ng/mL for working media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sylgard 184 silicone 3.9 kg</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">NC0162601</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Hydrochloric acid</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA56-4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Nitric Acid Concentrate (Certified) ACS, Fisher Chemical</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">SA95</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">ITO Glass</td>
			<td style="width:103px;">Delta Technologies</td>
			<td style="width:119px;">CG-40IN-0115</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Sodium dodecylbenzenesulfonate</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">289957-1KG</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Pyrrole</td>
			<td style="width:103px;">Sigma</td>
			<td style="width:119px;">131709-500ML</td>
			<td style="width:188px;">Protect pyrrole solution from light and room temperature</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">8 well glass slide chambers</td>
			<td style="width:103px;">Thermo Sci Nuc&#160;</td>
			<td style="width:119px;">125658</td>
			<td style="width:188px;">Detach the cell chamber and keep it under sterilized conditions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Flat-Surface Bracket, 3&#34;x1&#34;</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">1030A4</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">TWO PART SILVER PAINT 14G</td>
			<td style="width:103px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:119px;">1264214</td>
			<td style="width:188px;">Mix two parts (1:1) in plastic plate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">DPBS, 1x, with Ca and Mg, No Phenol Red</td>
			<td style="width:103px;">Genesse&#160;</td>
			<td style="width:119px;">25-508C</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">AB2 ArunA Neural Cell Culture Media Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">ABNS7013.2</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">hNP1 Human Neural Progenitor Expansion Kit</td>
			<td style="width:103px;">Aruna Biomedical&#160;</td>
			<td style="width:119px;">&#160;hNP7013.1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:213px;">Noncontact Flow-Adjustment Valve, Nickel-Plated Brass, for 3/32&#34; to 5/8&#34; Tube OD</td>
			<td style="width:103px;">McMaster-Carr&#160;</td>
			<td style="width:119px;">5330K22</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Multimeter</td>
			<td style="width:103px;">Keysight&#160;</td>
			<td style="width:119px;">E3641A</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Wavefoam generator</td>
			<td style="width:103px;">Keysight</td>
			<td style="width:119px;">33210A-10MHz</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Pt meshes</td>
			<td style="width:103px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">298107-425MG</td>
			<td style="width:188px;">Reference electrode with dimensions, 1x1 cm</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">L3224</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">BDNF</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02718934_s1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">THBS1</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs00962908_m1</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">GAPDH</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">Hs02758991_g1</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNeasy Mini Kit (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">74106</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">QIAshredder (250)</td>
			<td style="width:103px;">Qiagen</td>
			<td style="width:119px;">79656</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">RNase-Free DNase Set (50)</td>
			<td style="width:103px;">QIAGEN</td>
			<td style="width:119px;">79254</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">iScript cDNA Synthesis Kit, 100 x 20 &#181;L rxns</td>
			<td style="width:103px;">BIORAD</td>
			<td style="width:119px;">1708891</td>
			<td style="width:188px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:103px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">4369510</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">7-8 Week Old, male, RNU Rats</td>
			<td style="width:103px;">NCI-Frederick</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">4-0 Ethicon Silk Suture</td>
			<td style="width:103px;">eSutures.com</td>
			<td style="width:119px;">683G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Isoflurane</td>
			<td style="width:103px;">Henry Schein</td>
			<td style="width:119px;">29405</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">V-1 Tabletop Laboratory Animal Anesthesia System</td>
			<td style="width:103px;">VetEquip</td>
			<td style="width:119px;">901806</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Surgicel Original Absorbable Hemostat</td>
			<td style="width:103px;">Ethicon</td>
			<td style="width:119px;">1952</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Lab Standard Stereotaxic Instrument, Rat</td>
			<td style="width:103px;">Stoelting</td>
			<td style="width:119px;">51600</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Kimberly-Clark Professional&#160;Safeskin Purple Nitrile Sterile Exam Gloves</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">19-063-130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:213px;">Sterile Drape</td>
			<td style="width:103px;">Medline</td>
			<td style="width:119px;">DYNJSD1092</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:213px;">Thermo Scientific&#160;Shandon Stainless-Steel Scalpel Blade Handle, Holds No. 20-25 Blades</td>
			<td style="width:103px;">Fisherbrand</td>
			<td style="width:119px;">53-34</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:213px;">Walter Stern Scalpel Blade Series 300</td>
			<td style="width:103px;">Fisherbrand</td>
			<td>17-654-456</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:213px;">QuantStudio 6 Flex Real-Time PCR System</td>
			<td style="width:103px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4484642</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:213px;">Frazier Micro Dissecting Hook</td>
			<td style="width:103px;">Harvard Apparatus</td>
			<td>52-2706</td>
			<td></td>
		</tr>
	</tbody>
</table>

electrically conductive scaffold to modulate and deliver stem cells

干细胞治疗已成为一种令人兴奋的中风治疗, 但最佳分娩方法仍不清楚。虽然显微注射技术已用于数十年来交付干细胞的中风模型, 这项技术是有限的能力, 缺乏操作前的干细胞注射。本文详细介绍了一种利用导电高分子支架进行干细胞输送的方法。利用导电高分子支架对干细胞进行电刺激会改变干细胞的基因, 包括细胞存活、炎症反应和突触重塑。电预处理后, 支架上的干细胞移植 intracranially 在大脑中动脉闭塞大鼠模型中。该协议描述了一个强大的技术, 通过导电高分子支架操作干细胞, 并创造了新的工具, 以进一步发展干细胞为基础的治疗。

图 1中显示的示意图代表了 hNPCs 和潜在下游应用程序的电刺激的总体工作流。目前干细胞治疗的一个局限是干细胞暴露在一个严酷的后移植环境中, 包括炎症和缺血性疾病。这些困难的条件可能限制其治疗效果14,15。使用导电支架保护 hNPCs 免受这种环境的侵害, 可以通过电预处理增加 hNPCs 治疗的益处。这种...

Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

本协议描述了细胞培养系统的制作, 允许在导电高分子支架上播种干细胞, 用于体外电刺激和随后的体内植入干细胞种子支架, 使用微创技术。

导电支架调节和传递干细胞

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

electrically-conductive-scaffold-to-modulate-and-deliver-stem-cells

Research

JoVE Journal

Bioengineering

12.3K 次观看.  Stanford University School of Medicine. 本协议描述了细胞培养系统的制作, 允许在导电高分子支架上播种干细胞, 用于体外电刺激和随后的体内植入干细胞种子支架, 使用微创技术。

视频: Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

平面和立体印刷的导电油墨

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

科研

生物工程

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

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

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

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

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

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

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

订货单细胞和单胚胎的三维约束:高内涵筛选新设备

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

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

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal 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 ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

MicroRNA 移植成人骨髓造血干细胞促进细胞工程与磁性靶向结合的协议

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

人类诱导多能干细胞衍生心肌细胞中的单细胞光学作用潜在测量

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

维持静止人类造血干细胞的文化方法

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...