马丁 Danner 在准备样品和建立细胞培养程序方面得到了认可。作者要感谢 Biesterfeld Spezialchemie GmbH (德国汉堡), 特别是罗伯特 Radsziwill 为连续的支持和讨论。导致这些结果的研究得到了欧洲研究理事会根据欧洲联盟第七框架方案 (FP/2007-2013)/紧急救济协定340929的资助。

警告: 使用前请查阅所有相关的材料安全数据表 (MSDS)。本议定书中使用的一些化学品具有刺激性、剧毒和/或致癌性。在处理这些化学品时, 请使用所有适当的安全措施。这包括使用工程 (化学柜) 和个人防护设备 (安全眼镜, 手套, 实验室大衣, 全长长裤和闭合脚趾鞋)。下列程序的一部分涉及动物细胞系的培养。因此, 请遵守具体的生物安全条例。化学和生物废物需要根据具体的国家和机构规则和建议加以处理。
1. 硅弹性薄膜复合结构的制备
<ol><li>
聚合物的制备
	<ol>
<li>在比值10:1 中制备1.1 克的二甲基硅烷, 混合1.0 克的复方 a 与0.1 克复合 B。</li>
		<li>混合和 degase 前聚合物的速度搅拌机在 2350 rpm 下真空3分钟。</li>
		<li>将复合 a 和复合 B 之间的质量比率改变为45:1 和70:1。准备类似于1.1.2 中描述的方法。</li>
		<li>准备1克的软皮肤胶粘剂 (SSA) 的比例为50:50。因此, 混合0.5 克化合物 a 和0.5 克化合物 B, 如1.1.2 所述。</li>
	</ol>
</li>
	<li>
聚 (乙烯醇) (PVA) 包覆聚酯箔的制备
	<ol>
<li>在水中加入聚乙烯醇, 用磁性搅拌器在一夜之间混合, 制备 18% (w/w) pva 溶液。将此解决方案存储在摄氏4摄氏度。</li>
		<li>用刀片应用机制作15µm 有效厚度的薄膜, 使用100µm 缺口, 速度约2.0 毫米/秒。</li>
		<li>将影片放在烤箱中95摄氏度, 15 分钟。</li>
	</ol>
</li>
	<li>
用刮刀技术制备10:1 混合比的支撑层
	<ol>
<li>使用自动控制的医生刀片应用机器为薄膜的准备。</li>
		<li>用100% 异丙醇清洗 PET 箔, 并将其放在医生刀片应用区域的表面。</li>
		<li>将医生刀片放在箔顶部, 用微定位螺钉调整厚度。对于湿层的制造, 应用厚度为60µm, 100 µm, 200 µm 和500µm。</li>
		<li>用注射器将步骤1.1 中制备的10:1 聚合物填充在医生刀片的库中。开始移动的刀片速度约2.0 毫米/秒。</li>
		<li>从机器上取下10:1 涂层的 PET 膜, 将其放在烤箱中, 1 小时95摄氏度, 位于一个房间, 在40% 和65% 之间显示湿度。</li>
		<li>用异丙醇和纸巾清洁医生刀片。</li>
		<li>对所有要求的厚度重复此过程。</li>
	</ol>
</li>
	<li>
用刀片技术制备不同配比的聚硅烷顶层
	<ol>
<li>用手术刀或剃刀刀片去除底层薄膜的长度两侧的薄条纹, 以允许在 PET 箔上放置和滑动医生刀片。</li>
		<li>按照协议步骤1.3.3 到1.3.6。涂膜湿厚度为160µm。</li>
		<li>重复这一过程, 生产两个独立的电影, 每一个与其他混合比的新一组的组件 (45:1 和 70:1)。在室温下贮存薄膜 (约22摄氏度, 湿度介于40和65% 之间), 以防止污染和灰尘。</li>
	</ol>
</li>
	<li>
不同厚度的 SSA 50:50 层薄复合膜的制备
	<ol>
<li>按照步骤1.3 前的描述, 将10:1 电影作为支持层进行准备。</li>
		<li>按照协议步骤1.4.1 和1.4.2 生成这些影片。使用 SSA 的混合比例为 50:50, 并制造一个膜的湿厚度为40µm。</li>
		<li>重复该过程的额外湿厚度: 120 µm, 300 µm, 500 µm。</li>
	</ol>
</li>
</ol>2. 使用不同表面粗糙度的基体进行正常粘接测量
<ol><li>
不同表面粗糙度玻璃基片的制备与表征
	<ol>
<li>使用2毫米直径的玻璃圆筒作为 ' 光滑的基体 '。</li>
		<li>用玻璃切割机制造 "粗糙的衬底", 从磨砂的玻璃滑梯上将一件与有关 4x4 mm 的尺寸。使用研磨金刚石手垫获得约3毫米直径的圆形区域。</li>
		<li>用 uv 胶水将玻璃连接到铝锥上, 在紫外线照射室中照射3分钟。</li>
		<li>用光学显微镜确定基体表面的半径。根据公式 A = πr2计算面积。</li>
		<li>确定粗糙度参数 ra和 rz (根据: DIN EN ISO 4287, ASME B46.1) 与手写笔轮廓。</li>
		<li>在轮廓的样品台上贴上基体, 并将尖端 (钻石, 标准: 2 µm/) 与样品接触。</li>
		<li>记录粗糙度剖面, 速度为0.3 毫米/秒, 长度为1毫米。</li>
		<li>要分析表面形貌, 用手写笔轮廓测量一个精确 1 mm2的面积, 由相关软件操作。 注意: 轮廓由外部计算机操作。当1毫米的位移在 x 方向到达时, 支架在 y 方向被转移0.001 毫米。记录在案。RS3 文件是导入 Surfcom 地图专家软件, 以创建3D 图像。</li>
	</ol>
</li>
	<li>
硅烷或 SSA 制造薄膜的正常粘附测量
	<ol>
<li>使用剃刀刀片将 PET 箔上的胶片切成小块, 面积约为4.0 厘米2 , 然后将它们放在带有 UV 胶水的玻璃滑梯上。用 UV 光照射3分钟。</li>
		<li>在样品架上安装高分子样品。</li>
		<li>用乙醇轻轻地清洁基体表面, 用氮气干燥。</li>
		<li>将玻璃基板安装到铝锥上, 再连接到负载单元。</li>
		<li>使用 tiltable 表 (量角器), 通过调整靠近聚合膜的基底倾角来精确地对齐曲面。为了做到这一点, 把承印物手动与胶片接触。改变倾斜角度, 直到两个曲面的完全平行对齐, 由相机图像可视化, 得到。 注意: 负载单元格连接到 tiltable 表。玻璃棱镜位于样品下面, 如图 4所示, 允许用两个摄像头可视化接触区域, 并使基板对聚合物薄膜进行对准。</li>
		<li>将基板移动到聚合物薄膜表面, 直到达到13到5帕的预紧应力 (图 4)。</li>
		<li>启动在 LabView 编写的定制程序软件包, 以控制所需的测量参数, 如保持时间和接近/撤回速度。保持时间 t保持为1秒, 方法和分离速度分别为30µm 和10µm/秒。</li>
		<li>对三个独立制造的样品和每片表面的六个不同位置执行粘附测量。</li>
	</ol>
</li>
	<li>
关键机械因素的数据分析和计算: 拉应力和分离工作。
	<ol>
<li>通过将记录<img alt="Equation 1" src="/files/ftp_upload/57573/57573eq1.jpg">的力除以基底面积 A 来计算应力. <img alt="Equation 2" src="/files/ftp_upload/57573/57573eq2.jpg">
</li>
		<li>确定拉断应力, 被描述为正常应力的最大值。 <img alt="Equation 3" src="/files/ftp_upload/57573/57573eq3.jpg">
</li>
		<li>通过将拉伸状态的起始位置减去0的位移Δs, 从试样位置结束, 完成了脱粘。将拉伸机制的开始定义为 s0 = 0。 <img alt="Equation 4" src="/files/ftp_upload/57573/57573eq4.jpg">
</li>
		<li>根据下面的公式, 按照系统符合性 C 更正采样位置的测量值: <img alt="Equation 5" src="/files/ftp_upload/57573/57573eq5.jpg">
</li>
		<li>为了计算分离的工作, 将 s0和 s端之间的应力位移曲线进行积分。 <img alt="Equation 6" src="/files/ftp_upload/57573/57573eq6.jpg">
</li>
	</ol>
</li>
	<li>
利用数学计算软件来源计算关键机械因素。
	<ol>
<li>从源表中的单个粘附度量中导入记录的. dat 文件。记录的参数为时间、样本位置和力。在列 A (时间)、B (样本位置) 和 C (强制) 中插入这些参数。</li>
		<li>为了确定空值, 在接触聚合物膜之前, 平均大约有20的测量值。命名此平均值 F偏移量并将其粘贴到 D 列中。</li>
		<li>根据以下公式计算背景修正力 F * <img alt="Equation 7" src="/files/ftp_upload/57573/57573eq7.jpg"> 并将此公式插入到 E 列中, 如下所示。 <img alt="Equation 8" src="/files/ftp_upload/57573/57573eq8.jpg">
</li>
		<li>将拉伸机制的开始定义为零位移,即s0 = 0。因此, 确定 s0并将其从 B 列中的位移中减去, 并将其保存在 F 列中: <img alt="Equation 9" src="/files/ftp_upload/57573/57573eq9.jpg">
</li>
		<li>此外, 通过机器的符合性来更正样品位置。此更正在列 g 中执行. 将以下公式插入 g 列中 <img alt="Equation 10" src="/files/ftp_upload/57573/57573eq10.jpg">
</li>
		<li>计算下一列 H 中的应力。因此, 将力除以基底区域。插入以下公式 <img alt="Equation 11" src="/files/ftp_upload/57573/57573eq11.jpg"> 其中 A 是玻璃基体的表面区域在毫米2 (确定在 2.1)。</li>
		<li>计算从应力和位移值的分离工作。因此, 沿 x 轴和沿 y 轴的应力绘制位移。将该图从 s0到 s端, 将 s末端定义为拉伸应力返回到零的位移, 即完全脱离发生。要集成图形, 请选择集成函数。将计算值添加到列 I 和 J 中。</li>
	</ol>
</li>
</ol>3. 用扫描电子显微镜 (SEM) 和光学显微镜对薄膜进行表征
<ol><li>
光学显微镜
	<ol>
<li>用剃刀刀片将聚合物薄膜切成小块 (约0.25 厘米2), 并将其附着在玻璃滑梯的边缘。将玻璃滑动垂直放置在直立显微镜下, 测量胶片横断面的厚度。 注: 使用20X 目标 (NA = 0.45, 理论分辨率为 800 nm 1.1 µm), 以测量大约≤20µm 的胶片厚度值。为影片厚度在范围20µm 50 µm 使用10X 目标 (NA = 0.30, 理论决议在800毫微米1.6 µm) 和为影片厚度≥50µm 使用5X 目标 (NA = 0.15, 理论决议在800毫微米3.3 µm)。</li>
	</ol>
</li>
	<li>
扫描电镜调查
	<ol>
<li>切下 PET 箔, 并将约2厘米2的样品贴在玻璃滑轨上, 并将其垂直放置在样品架内的夹紧机构中, 其顶部表面低于支架的顶端。</li>
		<li>选择加速度电压 10 kV, 背散射电子探测器 (BSD) 和低真空条件 (60 Pa)。</li>
		<li>调整图像的焦点、放大倍数、亮度和对比度。</li>
		<li>选择二十八年代的图像采集时间, 分辨率为 1024年 x 2048 像素。</li>
		<li>从 SEM 上取下样品架。</li>
	</ol>
</li>
</ol>4. 生物调查
<ol><li>L929 细胞的常规细胞培养
	<ol>
<li>用小鼠成纤维细胞系 L929 进行调查。培养细胞在伊罗戈罗斯威尔公园纪念学院 (RPMI) 1640 基底培养基, 补充10% 胎牛血清和青霉素和链霉素在37°c, 5% CO2在 T75 细胞培养烧瓶。通过细胞在大约70% 到80% 的汇合处。</li>
		<li>对于细胞传代, 去除培养基的吸入和洗涤钙和镁游离磷酸盐缓冲 (DPBS) 在三十年代下层流内阁。之后孵化细胞与2毫升 Accutase, 酵素解答以蛋白水解和 collagenolytic 活动为5分钟在37°c, 5% CO2。</li>
		<li>用相衬显微镜验证细胞培养瓶表面的脱离。</li>
		<li>在烧瓶中加入8毫升含血清的培养基, 将细胞悬浮液转移到15毫升的反应管中。</li>
		<li>从细胞悬浮液中抽取10µL 样品, 并与10µL 的台盼蓝混合。</li>
		<li>用 Neubauer 室确定细胞数并计算细胞总数。 注意: 台盼蓝是有毒的, 因此咨询 msds, 按照 msds 中规定的强制性程序, 必须穿戴适当的人身安全保护和在化学品柜下处理。收集废物用于化学废物沉积。 注: 台盼蓝阳性细胞呈彩色蓝色, 显示非完整的细胞膜。</li>
		<li>对于下一个段落, 培养 5 x 105细胞在一个新的无菌细胞培养瓶与10毫升新的培养基。为实验条件, 培养 3 x 105细胞在6个井板和 6 x 104个细胞在24个井板的每个井, 包含聚合物样品 (协议步骤 4.2)。</li>
	</ol></li>
	<li>细胞培养实验用复合膜的制备。
	<ol>
<li>将在协议步骤1.4 和1.5 中制造的所需尺寸的单片胶片从 PET 支撑层中制成, 用手术刀和将镊子放置在玻璃盖板的表面上, 显示直径为12毫米. 将样品放在油井的24井板。</li>
		<li>对于细胞毒性测定和计数, 请勿从 PET 箔中取出胶片。切圆面积约9.4 厘米2, 整齐地装入6井板的单井, 从1.4 和1.5 生产的影片并且安置他们入细胞培养板材的井。</li>
		<li>将高分子样品浸泡在去离子 H2O 上以≥30分钟。 注: 聚合物样品可能被热处理灭菌。因此, 从细胞培养皿中除去所有含有样品的聚合物, 并将其放入玻璃培养皿内。灭菌在2.05 巴的高压釜中进行, 温度为121摄氏度时为20分钟。</li>
	</ol></li>
	<li>聚合物等离子体处理
	<ol>
<li>将附着在 PET 箔上的薄膜或在等离子装置的反应室内的圆形玻璃盖板上 (在4.2.1 中制造)。</li>
		<li>关闭盖子和疏散, 直到 1.6 x 10-2毫巴的压力达到。</li>
		<li>进行血浆治疗3分钟。</li>
		<li>通风反应室和放置样品在24井或6井菜为进一步细胞培养调查。</li>
		<li>使用一个量角器的水接触角测定样品。因此, 将注射器靠近聚合物表面, 使用软件包, 将3µL 的水放在表面上。用量角器软件计算静水接触角。</li>
	</ol></li>
	<li>染色和显微术
	<ol>
<li>按照步骤4.1.7 和文化中的描述, 在37摄氏度和 5% CO2处准备单元格. 3 d。</li>
		<li>在固定前的三天内, 在原始的和等离子处理的胶片上捕获细胞的相对比图片。</li>
		<li>准备 PBS 辅以 0.2% Triton-X-100。慢慢地将200µL 的溶液注入100毫升的 pbs (pbs t)。</li>
		<li>准备一个4% 多聚甲醛/pbs 解决方案 (粉煤灰/pbs)。 警告: 多聚甲醛是有毒的, 因此咨询 msds, 遵循在 msds 中描述的强制性程序, 并佩戴适当的人身安全保护和处理在一个化学品柜是必需的。</li>
		<li>准备5% 的 BSA/PBS 解决方案。</li>
		<li>在叶片流柜下吸入培养基。将 PBS 添加到井中以去除介质残留物。</li>
		<li>将该板块转移到化学柜中, 用400µL 的粉煤灰/PBS 溶液在室温下更换 pbs 25 分钟。</li>
		<li>从单井中取出粉煤灰/PBS 溶液, 用 PBS 仔细清洗四次。在每个洗涤步骤之间等待3分钟, 并收集化学废料处理的解决方案。直接使用盘子, 或储存在4摄氏度。</li>
		<li>添加5% 牛血清白蛋白 (BSA)/PBS t 的水井和孵化60分钟的 RT, 以阻止不特异的结合点。</li>
		<li>吸入溶液, 并用罗丹明共轭 Alexa-488 (1:160 稀释)/PBS T 溶液补充 0.2% Triton-X-100。 警告: Phalloidin-488 是有毒的, 因此咨询 msds, 遵循在 msds 中描述的强制性程序, 并佩戴适当的人身安全保护和处理在一个化学品柜是必需的。</li>
		<li>盖板与铝箔和孵化它为3小时在 RT 或隔夜在4摄氏度。</li>
		<li>吸入溶液, 用 PBS 清洗三次。在每个洗涤步骤之间等待3分钟。收集化学废物处理的解决方案。</li>
		<li>准备1µL 赫斯特染料 33342 (库存溶液1毫克/毫升) 的溶液。1:1000 稀释吸管1µL 赫斯特染料3334到1毫升的 PBS 和混合良好。添加300µL 的赫斯特染料33342溶液的水井和孵化10分钟在 RT 在黑暗中。 注意: 赫斯特染料33342是一种 DNA 锂试剂, 因此有可能致突变, 因此咨询 msds, 遵循 msds 中描述的强制性程序, 并佩戴适当的人身安全保护和处理, 根据需要化学柜。</li>
		<li>吸入溶液, 用 PBS 清洗样品四次。在每个洗涤步骤之间等待3分钟。收集化学废物处理的解决方案。</li>
		<li>为嵌入, 小心地删除的影片从文化表面, 并把它们放在显微镜玻璃幻灯片。将20至40µL 水溶性嵌入介质添加到薄膜中, 并在顶部附加一个新的圆形玻璃盖板, 使用轻微的压力。</li>
		<li>用荧光显微镜进行成像。照明所需的过滤器: Alexa-488 的激发最大值为 496 nm, 最大发射量发生在 519 nm。因此, 发射颜色为绿色。赫斯特染料 33342 trihydrochlorid 与 dna 复合, 其激发最大值为 355 nm, 最大 DNA 复发射发生在 465 nm。</li>
	</ol></li>
	<li>细胞毒性测定及其数量的测定
	<ol>
<li>在步骤4.3 中制备的6井板中生长的细胞和步骤4.1.7 中制备的细胞进行实验。培养细胞3天在37°c 和 5% CO2。对于正控制, 使用在细胞培养中生长的细胞, 处理过聚苯乙烯表面, 不含聚合膜。为背景确定 (阴性情况), 获得媒介从井没有细胞。 注: 培养基也可取自培养在细胞培养的聚苯表面上的含孔细胞。</li>
		<li>根据实验样品的数量标注15毫升管。</li>
		<li>添加40µL 0.9% 海卫 X-100 包含 PBS 解决方案的积极控制和搅拌与1000µL 提示。等待约3分钟。</li>
		<li>不去除附着于表面的细胞, 从所有样品中吸取培养基, 包括在4.5.3 中制备的样品, 并将培养基转移至15毫升管。添加3毫升的 DPBS-/-到单井, 并存储在层流柜下的板块, 以确定细胞数, 如4.5.9 所述。</li>
		<li>离心机在 200 x g 3 分钟, 并删除1毫升的上清的乳酸脱氢酶活性测定。在层流柜下储存含有细胞和剩余介质的15毫升管。</li>
		<li>为化验一个黑色96井板材以平底被使用。添加50µL 的 CytoTox-一试剂到50µL 的样品培养基和混合良好三十年代。</li>
		<li>用铝箔盖住盘子, 在 RT 上贮存10分钟。</li>
		<li>在每个井中添加25µL 的停止解, 并用荧光板读取器记录荧光强度。摇动盘十年代和检测荧光信号与激发波长的520毫微米和发射波长560毫微米。避免气泡。</li>
		<li>对于细胞数的测定, 从4.5.4 中的培养板井中吸取 DPBS, 并将解决方案添加到包含协议步骤编号4.5.5 中所收集的上清液的15毫升反应管中。</li>
		<li>离心15毫升反应管在 200 x g 3 分钟和吸入上清。加入0.5 毫升的胰蛋白酶/EDTA, 孵育10分钟37摄氏度。</li>
		<li>加入2毫升的胰蛋白酶/EDTA 在该板块的水井和孵化约10分钟37摄氏度, 以分离细胞从聚合物薄膜。</li>
		<li>细胞悬浮被转移到15毫升反应管从步数4.6.10。另外, 用含有培养基的血清大力冲洗盘子。</li>
		<li>离心样品在 200 x g 3 分钟, 吸出上清, 并添加血清含培养基的管。</li>
		<li>确定步骤4.1.5 和4.1.6 中所述的细胞数。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

复合结构的设计使材料性能的简单调整, 如杨氏模量或试样的厚度。采用不同的硅胶弹性体30、31, 通过改变两组分的混合比或制造混合料, 可以有效地改变杨氏模量。所描述的方法并不局限于目前的研究中使用的, 但特别是胶粘剂的性能取决于所使用的特定类型。该协议中的一个关键步骤是复合薄膜的制造过程 (图 1)。结果表明, 薄膜厚度对不同基质 (包括皮肤) 的粘附性能有显著影响 (图 5和图 6)。除薄膜厚度外, 固化过程中的时间和温度影响材料性能32。因此, 需要对聚合物层厚度的参数进行仔细的调整和验证。
用两个不同表面粗糙度的玻璃基片对0.338 µm (图 3) 进行了常规力粘附测量, 对薄膜的粘接性能进行了分析。一般而言, 粗糙度对表面的附着力有显著影响, 特别是弹性材料33、34。玻璃的粗糙度可以很容易地由不同的粗糙度的砂纸研磨, 因此允许制造的衬底表现出更高的糙率值21。另外, 其他材料, 例如环氧树脂可以使用为基体15,35的生产。这可能是提出的协议的一个重要修改策略。例如, 如果需要提供不同表面自由能量的基板, 或者需要特定的地形。在此基础上, 用自定义的设置 (宏观粘附测量装置 (MAD,图 4) 分析了该薄膜的拉拔应力和分离工作。36压的光学对准是测量结果分析的关键步骤。因此, 倾斜角的调整需要与量角器, 尽可能精确。这可以达到足够的精度, 手动使基板接触到薄膜表面, 直到达到水平接触。
在当前协议中, 保持时间在一秒钟内保持不变 (图 5 和图 7)。特别是对弹性薄膜在粗糙衬底表面的粘接性能的研究, 保留时间的延长提供了额外的信息。例如, 增加了拉断应力, 增加了保持时间已报告8。除了在当前协议中进行的测量外, 还可以执行其他方法, 例如剥离测试, 允许对附着力性能进行更全面的调查37。
测定了不同膜厚的复合膜在软皮肤胶粘剂中的粘结性能 (图 7)。我们的结果与公布的数据一致, 表明薄膜厚度的降低导致拉断应力的增加,即衬底直径与膜厚度的比值, 增加38,39.根据这些结果和图 7中描述的数据, 我们得出的结论是, 复合薄膜的总厚度约为100µm (大约60µm 的 SSA 层的厚度适用于大约40µm 的一部以上的一片薄膜), 具有良好的附着力。roperties 在粗糙的表面上。
其次, 在原始复合膜和等离子处理复合膜上进行了与生物特性相关的实验 (图 8)。等离子处理硅弹性体是一种常用的, 多功能的技术, 以提高表面亲水性的性质和促进细胞附着和细胞传播40,41。有机硅是众所周知的低毒性和高 biostability, 但可能含有残留单体或催化剂, 可能影响生理过程, 也导致细胞毒性42,43。在进行的实验中, 我们观察到的细胞毒性小于 5%, 以 LDH 释放为指标和台盼蓝排斥试验。在所提出的协议中, 对整个细胞群, 包括从表面分离出来的细胞团聚体进行了分析, 以进行扩散分析 (图 9B)。对议定书的修改可以产生更有差别的结果。对于每个样品, 含有分离细胞集料的上清液可以转移到一个单独的反应管, 而不与酶从聚合物表面移除的细胞结合。这将允许对附着在表面的细胞进行精确的评估, 并最终揭示出聚合物对细胞黏附过程的影响的更详细的测定。除了这里提出的免疫细胞化学方法外, 还可以利用应用免疫印迹的方法来采集干细胞, 从而对蛋白质表达进行详细的定量评估。
总之, 我们已经建立了生产条件的薄弹性复合膜的应用, 在先进的细胞培养研究。此外, 这些薄膜具有很高的适应性, 皮肤粗糙度, 使复杂的皮肤胶粘剂设计。

在该协议中, 提出了制备薄弹性体薄膜的详细方法。该技术广泛应用于薄层复合膜的生产中。在 polyethylenterephtalate (PET) 箔上进行了制造技术, 使这些薄膜能够在很大程度上得到后续生产。本协议的重点是对重复性的评估, 对复合膜的不同层的精确制造, 以及最终复合片的生物和粘附性能的测定。有机硅弹性体 (甲基) 广泛应用于生物医学技术, 包括皮肤胶粘剂的生产、微流体应用和其他研究领域1、2、3 ,4。最近, 一个新的子类, 即所谓的软皮肤胶粘剂 (SSAs) 已经被介绍, 特别是为温和的皮肤粘合和脱粘。
硅胶 ssa 是乙烯基功能弹性体, 不同于类似的聚合物, 没有加强二氧化硅5。类似于其他的, SSA 的杨氏模量可以通过调节交联器浓度或固化时间6,7,8来适应广泛的范围。硅弹性体杨氏模量的这种变化对材料的粘附性能有显著影响, 对9、10表面培养的原核和真核细胞也有深远的影响。,11. 在细胞生物学水平上, 它显示, 真核细胞在信号转导水平上响应, 以调制的基体弹性或厚度的表面9,10,12 ,13,14。因此, 广泛的兴趣在细胞培养应用的聚合物具有可调谐的机械性能存在。重要的是, 硅基弹性体的固有低表面能为真核细胞细胞培养提供了最佳条件。氧等离子体处理是一种应用广泛的技术, 可以暂时增加低表面能量, 从而提高其拉断强度, 减少分子表面吸附, 同时促进附着、扩散和真核细胞增殖15,16,17,18。
除材料性质外, 表面形貌显著影响细胞黏附力和两种材料之间的粘接作用19、20、21、22。表面粗糙度对两个表面的接触形成有若干影响: 接触面积的减少, 粗糙周围的高储能弹性能量, 以及对裂纹扩展的影响可以改变粘结强度23, 24。自粘膜对人体皮肤的粘附是一个新兴的应用领域,如伤口敷料, 心电图电极固定或其他可穿戴电子设备25,26,27, 28。为测量自粘胶在表面形貌方面的粘结性能, 不同粗糙度的玻璃基板可用于正常附着力测量8、21。在这里, 选择了两个玻璃基板来研究聚合物薄膜的粘结性能。首先, 以不同掺量比的10至1重零件的混合比为基材的复合膜。在第二个步骤中, 制备了一个胶粘剂 SSA 层, 其两个组分的重量相等, 并在支持的二甲基硅胶片上有不同的膜厚。

<table>
	<tbody>
		<tr>
			<td>2-Propanol, 97%</td>
			<td>Stockmeier Chemie</td>
			<td>1000452610000</td>
			<td>Isopropanol</td>
		</tr>
		<tr>
			<td>Abrasive diamnod hand pad</td>
			<td>Bohle</td>
			<td>MO 5007522</td>
			<td>Grit: 220</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Capricorn Scientific</td>
			<td>ACC-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraktion V</td>
			<td>Roth</td>
			<td>0163.2</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>ThermoFischer Scientific</td>
			<td>A12379</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Aquamount</td>
			<td>Polysciences</td>
			<td>18606-20</td>
			<td>water soluble mounting medium</td>
		</tr>
		<tr>
			<td>CytoTox-ONE Homogeneous Membrane Integrity Assay</td>
			<td>Promega</td>
			<td>G7890</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, without Ca2+, Mg2+</td>
			<td>ThermoFischer Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum gold</td>
			<td>GE Health Care Life Science</td>
			<td>A15-151</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Goniometer OCA35</td>
			<td>Dataphysics</td>
			<td></td>
			<td>for the determination of the static water contact angle</td>
		</tr>
		<tr>
			<td>Hoechst Dye 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B1155-100MG</td>
			<td>bisBenzimide H 33342 trihydrochloride, highly toxic</td>
		</tr>
		<tr>
			<td>Microscope Axiovert 25</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope used for cell culture documentation</td>
		</tr>
		<tr>
			<td>Microscope Eclipse LV100ND</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope used for film thickness determination</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, aqueous solution 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
			<td>electron microscopy grade</td>
		</tr>
		<tr>
			<td>penicillin und streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenom XL Scanning Electron Microscope (SEM)</td>
			<td>Phenom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-(vinyl alcohol) 4-88, MW 31000</td>
			<td>Sigma-Aldrich</td>
			<td>81381-1KG</td>
			<td>Mowiol 4-88</td>
		</tr>
		<tr>
			<td>Poly-dimethyl siloxanes, Sylgard 184</td>
			<td>Dow Corning</td>
			<td>(400)000108351397</td>
			<td>PDMS</td>
		</tr>
		<tr>
			<td>RPMI 1640 basal medium</td>
			<td>ThermoFischer Scientific</td>
			<td>21875034</td>
			<td></td>
		</tr>
		<tr>
			<td>soft skin adhesive (SSA)</td>
			<td>Dow Corning</td>
			<td>(400)000108251792</td>
			<td>MG 7-9800 Soft Skin Adhesive (SSA)</td>
		</tr>
		<tr>
			<td>speed mixer DAC 600.2 VAC-P</td>
			<td>Hauschild</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stylus profilomter</td>
			<td>Zeiss</td>
			<td></td>
			<td>Model: SURFCOM 1500SD3</td>
		</tr>
		<tr>
			<td>Tecan Infinite M200 pro</td>
			<td>Tecan</td>
			<td></td>
			<td>fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Calbiochem</td>
			<td>648466</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154-100ML</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA solution</td>
			<td>PAN-Biotech</td>
			<td>P10-023500</td>
			<td>0.05% Trypsin, 0.02% EDTA in PBS</td>
		</tr>
		<tr>
			<td>UV glue</td>
			<td>Bohle</td>
			<td>BO MV76002</td>
			<td>medium viscosity</td>
		</tr>
	</tbody>
</table>

thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization

在本协议中, 我们提出了制备薄弹性体复合膜的方法, 用于先进的细胞培养应用和皮肤胶粘剂的发展。两种不同的聚 (二甲基硅氧烷) (中和软皮肤胶粘剂 (SSA)), 已被用于深入调查的生物效应和胶粘剂的特点。复合膜由柔性支撑层和粘接顶涂层组成。这两个层都是由医生刀片应用技术制造的。在本研究中, 对复合膜的粘接性能进行了研究, 作为层厚度的函数或顶层杨氏模量的变化。通过改变基向交联剂混合比, 使其杨氏模量发生了变化。此外, SSA 薄膜的厚度从大约16µm 到大约320µm. 扫描电镜 (SEM) 和光学显微镜已被用于厚度测量。弹性体膜的粘结性能强烈依赖于薄膜厚度、聚合物杨氏模量和表面特性。因此, 研究了这些薄膜在表面光滑粗糙的玻璃基片上的正常附着力。脱扣应力和分离工作取决于有机硅弹性体的配比。
此外, 软皮肤胶粘剂的厚度放置在支持层的顶部已经变化, 以产生补丁皮肤应用。对 L929 小鼠成纤维细胞的细胞毒性、增殖和细胞黏附力进行了研究。我们在这里首次展示了两种聚合物制备的薄层复合薄膜的并排比较, 并对它们的生物和胶粘剂性能进行了研究。

Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

提出了一种具有不同杨氏模量或厚度的高分子薄膜复合结构的制造工艺规程。影片为先进的细胞培养研究或作为皮肤胶粘剂被生产。

用于细胞培养和皮肤应用的薄膜复合硅弹性体: 制造和表征

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

thin-film-composite-silicon-elastomers-for-cell-culture-skin

Research

JoVE Journal

Developmental Biology

10.6K 次观看.  INM - Leibniz Institute for New Materials. 提出了一种具有不同杨氏模量或厚度的高分子薄膜复合结构的制造工艺规程。影片为先进的细胞培养研究或作为皮肤胶粘剂被生产。

视频: Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

咽弓的体外培养研究心脏和肌肉祖细胞和自己的优势

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

培养人多能性和神经干细胞的基本和临床前研究一个封闭的细胞培养系统

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

从人多能干细胞使用高度可重复的小分子为基础的分化协议心肌细胞规模化生产

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...