Name: electrically conductive scaffold to modulate and deliver stem cells
Uploaded: 2018-04-13T13:30:00.000+00:00
Duration: 5 min 49 s
Description: Watch this Scientific Journal Video about Electrically Conductive Scaffold to Modulate and Deliver Stem Cells at JoVE.com

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

12.4K 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

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

人类血管周围干细胞用于骨再生

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

科研

生物工程

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程：建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

优化人骨髓间充质干细胞附着在聚（ε己内酯）静电纺纱线

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Electrospun Nanofiber Scaffolds with Gradations in Fiber Organization

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible applications in controlling cell morphology/orientation. Nanofiber organization is controlled with a new fabrication apparatus that enables the gradual decrease of fiber organization in a scaffold. Changing the alignment of fibers is achieved through decreasing deposition time of random electrospun fibers on a uniaxially aligned fiber mat. By covering the collector with a moving barrier/mask, along the same axis as fiber deposition, the organizational structure is easily controlled. For tissue engineering purposes, adipose-derived stem cells can be seeded to these scaffolds. Stem cells undergo morphological changes as a result of their position on the varied organizational structure, and can potentially differentiate into different cell types depending on their locations. Additionally, the graded organization of fibers enhances the biomimicry of nanofiber scaffolds so they more closely resemble the natural orientations of collagen nanofibers at tendon-to-bone insertion site compared to traditional scaffolds. Through nanoencapsulation, the gradated fibers also afford the possibility to construct chemical gradients in fiber scaffolds, and thereby further strengthen their potential applications in fast screening of cell-materials interaction and interfacial tissue regeneration. This technique enables the production of continuous gradient scaffolds, but it also can potentially produce fibers in discrete steps by controlling the movement of the moving barrier/mask in a discrete fashion.

Here, we present a protocol to fabricate electrospun nanofiber scaffolds with gradated organization of fibers and explore their applications in regulating cell morphology/orientation. Gradients with regard to physical and chemical properties of the nanofiber scaffolds offer a wide variety of applications in the biomedical field.

静电纳米纤维支架与渐变的纤维组织

The goal of this protocol is to report a simple method for generating nanofiber scaffolds with gradations in fiber organization and test their possible ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

同时进行机电刺激增强细胞心肌成功能的潜能

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

电刺激室的构建与应用, 促进间充质茎间质细胞体外成骨分化

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

细胞电微环境的有限元素建模

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Optimizing Extracellular Vesicle Delivery Using a Core-Sheath 3D-Bioprinted Scaffold for Chronic Wound Management

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

This study presents a protocol for fabricating core-sheath 3D-bio-printed scaffolds for chronic wound healing. Extracellular vesicles are isolated from mesenchymal stem cells, and loaded into the core (alginate) with the sheath made from carboxymethyl cellulose and alginate lyase. This design allows controlled scaffold degradation and efficient EVs release.

使用核心鞘 3D 生物打印支架优化细胞外囊泡递送用于慢性伤口管理

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol ...