这项研究得到了部分支持的科技部, 台湾根据赠款编号 106-2119-m-008-003, 105-2119-m-008-006, 和 104-2221-e--008-107-MY3。这项研究也得到了台湾 Landseed 医院项目 (NCU-LSH-105-A-001) 的支持。还承认日本教育、文化、体育、科学和技术部的科研资助 (数字 15K06591)。a. 口想感谢沙特阿拉伯王国利雅得11451沙特国王大学国际科学伙伴计划 (ISPP-0062) 的研究生研究和研究副校长。

本研究的实验得到了台湾 Landseed 医院 (IRB-13-05) 和国立中央大学伦理委员会的批准。在本研究中, 所有的实验都是按照所有相关的和适用的政府和机构的指导方针和规定进行的。
1. 解决方案和媒体准备
<ol><li>
高分子提纯
	<ol>
<li>用乙醇洗涤 p ia 水解 > 96.5% 的羧酸基团净化 p ia。在500毫升的圆锥烧杯中将20克的 P IA 放入200毫升的乙醇中, 在磁力搅拌器上搅拌 24-30 h. 每8-10 小时用新鲜乙醇交换乙醇。</li>
		<li>用 chner 漏斗滤除乙醇中的 P IA。</li>
		<li>室温下真空干燥24小时干燥 P IA。 注: 建议在真空干燥系统 (通过去除乙醇) 经常清洗陷阱, 特别是在最初的几个小时内, 因为在从 P IA 去除大量乙醇后, 陷阱往往会堵塞。</li>
	</ol>
</li>
	<li>
p-IA 溶液的制备 注: 将聚合物慢慢加入溶剂 (水)。建议在溶剂中加入 P IA 至少15分钟。如果在聚合物中加入溶剂, 聚合物就不会完全溶解。小心不要产生爆炸性沸腾的 P IA 解决方案。在制备 P IA 溶液时使用防护眼镜。p ia 溶液的加热过程是溶解结晶 p-ia 的关键。建议 (最好) 在一个相对干净的实验室中, 如果可能的话, 准备 P IA 溶液。<ol>
<li>溶解在纯净水中的 p ia 0.050 weight% 浓度为细胞培养实验或 0.50 weight% 浓度的流变仪测量: 例如, 溶解50毫克的 p ia 在100毫升的纯净水细胞培养和500毫克 p ia 在100毫升的 de电离 (DI) 水的流变仪测量。</li>
		<li>在热板上搅动 1 h 的 P IA 溶液。 注意: 为避免爆沸, 不要加热溶液超过95° c。在高温下, 聚合物溶液的爆炸性沸腾可能会产生皮肤灼伤。因此, 在加热过程中, 要非常仔细地进行 p ia 的加热, 并监测 p ia 溶液的温度。</li>
		<li>在室温下对 p ia 溶液进行冷却后, 在室温下激发 p ia 溶液为 48 h. 将热的 p ia 溶液留在热板上不加热, 然后在室温下不搅动20-24 小时, 以确保气泡在 P IA 解决方案中不存在。</li>
	</ol>
</li>
	<li>
交联溶液的制备
	<ol>
<li>使交联溶液的成分 1.0 weight% 戊二醛从25% 水戊二醛溶液, 20.0 weight% Na2, 使4使用 99% > 纯度, 和 1.0 weight% H2所以4。例如, 对于一个6或12良好细胞培养板, 增加100µL 戊二醛溶液, 2 克硫酸钠, 和100µL 的硫酸成10毫升的纯净水。</li>
	</ol>
</li>
	<li>
人类 ES/PS 细胞培养培养基 注意: 使用 DMEM/F12 媒体, 必要的6媒体, 和必要的8媒体的细胞文化。<ol>
<li>返回冷冻50X 必要8缓慢补充到解决方案过夜通过解冻在一个4° c 冰箱。</li>
		<li>添加一瓶50X 必要的8补充到一瓶基本的8基础媒体。在100毫升离心管中将介质分离成小等分 (每50毫升), 然后将介质存储在-20 ° c。</li>
	</ol>
</li>
</ol>2. P IA 水凝胶盘的制备
<ol><li>
P IA 薄膜制备
	<ol>
<li>在一个35毫米的 TCP 盘中注入一个1毫升的 p ia 溶液的分, 并在一个45° c 的烤箱中烘干2天, 在干净的长凳上产生 p ia 膜。</li>
	</ol>
</li>
	<li>
P IA 水凝胶菜肴的交联
	<ol>
<li>将 P IA 薄膜浸入0.5、1、2、4、6、12、24和 48 h 的水交联溶液中。 注: "p ia-X" (例如, P-IA-12 h) 是指对 p ia 水凝胶交联为X h (如, 12 h)。</li>
		<li>交联后, 在常温下用纯水冲洗 P IA 水凝胶, 在常温下将水凝胶保持在纯净的清水中, 在干净的长凳上。</li>
		<li>在75.0 体积/volume% 乙醇溶液中浸泡1分钟, 对 p ia 水凝胶进行消毒, 在纯水中清洗六次, 然后将 p ia 水凝胶保存在纯水中, 直至用于细胞培养。</li>
	</ol>
</li>
	<li>
肽或 ECM 接枝的 P IA 水凝胶菜肴的制备
	<ol>
<li>通过浸入含10毫克/毫升n的水溶液中的1毫升活化 P IA 凝胶-(3-Dimethylaminopropyl)-n "-ethylcarbodiimide 盐酸盐和10毫克/毫升N-琥珀酰亚胺 (NHS) 在37° c 或 4 h 处为1小时在4° c。</li>
		<li>用1毫升磷酸缓冲盐水冲洗 p ia 水凝胶 (pbs, pH 7.2) 3 次, 并将 p ia 凝胶浸入含有1毫升肽 (100-1, 500 µg/毫升) 或 ECM (10-100 µg/毫升) 的 PBS 溶液中, 24 ° c 时为4小时。 注意: 在表 1中总结了用于干细胞培养的肽序列和 ECMs。</li>
		<li>在接肽或 ECM 后, 用清水洗涤3次的 P IA 水凝胶。 注: 与Y µg/毫升的肽或 ECM (Z) 接枝的 p ia 水凝胶以后被称为 p ia-xh-z或p ia xh-z-y, 其中, X指的是交联时间 (h),Y指示肽或 ecm 的浓度, 而Z表示不同的肽或 ecm。</li>
	</ol>
</li>
</ol>3. 人类 ES/iPS 细胞培养
<ol><li>
未分化人 ES 和 iPS 细胞的通道和维持方法
	<ol>
<li>使用标准的人类 es/ips 细胞培养协议28,32, 在 6 cm 菜肴中, 维护人类的 es (如、WA09 或 H9) 细胞或人类 iPS (如如、HS0077) 细胞基质的8介质。</li>
		<li>孵育近汇合的人类 es/ips 细胞与2.0 毫克/毫升中性 II 在 DMEM/F-12 介质在37° c 为8-10 分钟, 然后冲洗人类 es/ips 细胞两次 DMEM/F12 媒体。</li>
		<li>在2毫升的 DMEM/F-12 培养基加入人类 ES/iPS 细胞培养皿后, 用细胞刮刀或移分离弱附着的菌落。</li>
		<li>将人类 es/ips 细胞收集到15毫升离心管中, 并将人类 es/ips 细胞在 160 x g 上离心, 在37° c 5 分钟。</li>
		<li>离心后, 丢弃 DMEM/F12, 在1毫升的 E8 介质中暂停人类的 ES/iPS 细胞, 然后使用细胞计数器计算细胞密度。</li>
		<li>在适当的密度调整后接种 ES/iPS 细胞 (1-5 x 104细胞, 每 cm2用于传代或指示) 进入新的培养皿 (肽或 ECM 接枝的 P IA 水凝胶)。</li>
	</ol>
</li>
</ol>4. 人体 ES/iPS 细胞特性表征
<ol><li>碱性磷酸酶 (AP) 活性的评价
	<ol>
<li>用标准碱性磷酸酶活染色法测定人 ES/iPS 细胞的碱性磷酸酶 (AP) 活性。</li>
	</ol></li>
	<li>免疫
	<ol>
<li>执行免疫的 Tra-1-81, SSEA-4, Sox2 和 Oct3/4 在他和臀部细胞, 以调查多遵循常规的协议28,32。</li>
		<li>将0.5 毫升体积的 4% (体积/体积) 甲醛到每24井道, 其中人类 ES/iPS 细胞的培养, 然后, 孵化皿15分钟在4° c 固定细胞。</li>
		<li>从每个井中吸取甲醛溶液。然后, 增加1毫升的 pbs 每井和吸气 pbs 冲洗细胞。执行冲洗过程3次。</li>
		<li>Permeabilize 细胞膜通过在 PBS 中加入0.5 毫升 0.3% (体积/体积) 海卫一 x 100 溶液, 室温下为30分钟。</li>
		<li>吸入海卫一 x 100 溶液, 并在 PBS (µL) 中加入300个 2% (重量/体积) 牛血清白蛋白 (BSA), 并在室温下孵育30分钟。</li>
		<li>通过移移除阻塞缓冲区, 然后添加所需的主抗体 (Oct3/4 (1:200)、Sox2 (1:200)、SSEA-4 (1:200) 和 1-81 (1:200) 吸管, 请参见表 2) 到每个井中, 其中1:200 表示抗体被稀释与 PBS 200 倍和孵化一天在4° c。</li>
		<li>在 PBS 溶液中, 用300µL 0.05% 吐温20的细胞在室温下, 以3倍的方式将原抗体溶液抽吸。</li>
		<li>添加300µL 的二次抗体 (1:200, 见表 2), 这是特定的主要 IgG 亚型, 在 2% (重量/体积) BSA 溶液入每井和孵化为 1 h 在室温下在黑暗中。</li>
		<li>在 PBS 溶液中, 用300µL 0.05% 吐温20的电池在室温下洗涤3次。</li>
		<li>用荧光显微镜或共聚焦显微镜对染色细胞进行分析。</li>
	</ol></li>
	<li>胚体形成
	<ol>
<li>通过10和20段的胚体 (EB) 形成研究人类 ES/iPS 细胞的多。 注: 近80% 汇合在 6-井板是充足的为电子束形成的准备。</li>
		<li>用2毫升 DMEM/F-12 介质冲洗人类 ES 或 iPS 细胞两次, 然后将细胞浸泡在1.5 毫升的基本6介质中。</li>
		<li>用200µL 的技巧将近80% 汇合的人类 ES 或 iPS 细胞切割成大约32块。接下来, 用刮板机将细胞与盘子分离。</li>
		<li>收集人类的 ES/iPS 细胞和转移到一个6井超低的附件盘。</li>
		<li>交换必要的6媒介由移老媒介和增加新鲜的媒介每2天。 注: 培养细胞悬浮2周在37° c 与 5% CO2。</li>
		<li>在 ebs 被 homogenously 悬浮在必要的6媒介之后, 转移 ebs 到文化在24个井 TCP 盘子涂上与 0.1 weight% 明胶, 并且培养细胞在必要的6媒介为另外的星期。</li>
		<li>用抗体对细胞进行染色, 对来自三胚胎系层的细胞标记物 (AFP (内胚层)、建筑面积 (外胚层)、β III-蛋白 (外胚层) 和 SMA (胚层)) 进行检测, 并通过上述免疫方法对细胞进行评价。</li>
	</ol></li>
	<li>畸胎瘤形成
	<ol>
<li>通过10和20段的畸胎瘤形成研究人类 ES/iPS 细胞的多。 注: 5 盘近80% 汇合在 6-井板是足够为畸胎瘤形成 (至少 3 x 106细胞为畸胎瘤形成实验是必要的)。</li>
		<li>用2毫升 DMEM/F-12 介质冲洗两次细胞, 然后在细胞培养皿中加入1.5 毫升 DMEM/F-12 介质。</li>
		<li>用200µL 的技巧将近80% 汇合的人类 ES 或 iPS 细胞切成大约32块。接下来, 用刮板机将细胞与盘子分离。</li>
		<li>将人类 ES/iPS 细胞收集到15毫升的离心管中, 并在 160 x g 离心分离细胞, 在37° c 5 分钟。</li>
		<li>离心后, 悬浮在100µL DMEM/F12 细胞小球, 然后混合细胞小球与100µL 基质 (1:1 体积比)。</li>
		<li>将细胞悬浮液转化为-20 ° c 前冷却的注射器。注射, 总共至少 3 x 106细胞皮下注射到男性 nod 免疫缺陷 (点头。CB17-Prkda无缺陷/JNarl) 小鼠 (5-8 周).</li>
		<li>5-8 周后, 解剖畸胎瘤, 修复 4.0% (重量/体积) 甲醛溶液, 然后存储在4° c。</li>
		<li>用石蜡修复畸胎瘤, 切片石蜡嵌入畸胎瘤, 用苏木精和曙红 (H & E) 染色, 使用标准的协议28,32。 注意: 研究人员可以向公司发送固定的畸胎瘤组织, 用石蜡修复畸胎瘤, 切片石蜡包埋的畸胎瘤, 并用 H & E 染色样品, 这通常在医院病理科使用。</li>
	</ol></li>
	<li>人脂肪源性干细胞 (ADS) 细胞培养
	<ol>
<li>温培养基 (DMEM 含有1% 抗抗生素和10% 胎牛血清 (FBS)), 0.25% 的胰蛋白酶-EDTA, PBS 到37° c 在水浴前使用。</li>
		<li>通过移10毫升的 PBS, 将人体广告细胞洗净到每 6 cm 培养皿中的细胞。</li>
		<li>将1毫升的胰蛋白酶-EDTA 溶液 (0.25%) 加入到 6 cm 培养皿中, 在37° c 下孵育溶液5分钟。</li>
		<li>显微镜下观察 6 cm 培养皿中的细胞, 确认细胞分离。</li>
		<li>将细胞收集到15毫升的离心管中, 并在离心管中加入等量的培养基 (DMEM 含有1% 抗抗生素和 10% FBS), 以中和胰蛋白酶-EDTA。</li>
		<li>离心细胞在 250 x g 为5分钟在37° c。</li>
		<li>离心后, 移小心丢弃上清液, 不干扰细胞颗粒。</li>
		<li>重在培养基中的细胞, 然后在适当的密度 (5-10 x 103细胞每 cm2的传代或如所示) 进入新的培养皿 (P IA 水凝胶接枝和没有肽和 ECM)。</li>
	</ol></li>
	<li>人羊膜液干 (AFS) 细胞培养
	<ol>
<li>暖培养培养基 (DMEM/MCDB 201 (2:3), 含有20% 胎牛血清 (FBS), 5 ng/毫升 bFGF 和1% 抗抗生素), 0.25% 的胰蛋白酶-EDTA, PBS 到37° c 在水浴前使用。</li>
		<li>在每 6 cm 培养皿中, 用移10毫升的 PBS 洗涤人类 AFS 细胞。</li>
		<li>将1毫升的胰蛋白酶-EDTA 溶液 (0.25%) 放入6厘米培养皿中, 并在37° c 下孵育5分钟的细胞。</li>
		<li>显微镜下观察 6 cm 培养皿中的细胞, 确认细胞分离。</li>
		<li>将细胞收集到一个15毫升的离心管中, 并加入等量的培养基 (DMEM/MCDB 201 (2:3), 含有20% 胎牛血清 (FBS)、5 ng/mL bFGF 和1% 抗抗生素) 进入离心管中, 以中和胰蛋白酶-EDTA。</li>
		<li>离心细胞在 250 x g 为5分钟在37° c。</li>
		<li>离心后, 在不干扰细胞颗粒的情况下, 小心丢弃上清移。</li>
		<li>重培养培养基中的细胞, 然后在适当的密度下种子细胞 (5-10x103细胞每 cm2为传代或被表明) 入新的文化菜 (P IA 水凝胶嫁接与和没有肽和 ECM)。</li>
	</ol></li>
</ol>

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作者没有什么可透露的。

为了长期扩展人类 ES 和 iPS 细胞, 在异自由条件下维持其多超过十通道, 以及对人类 AFS 细胞、广告细胞的培养, 建立了具有不同刚度的 p-ia-oligoECM 和 p-ia 型 ECM 水凝胶,和造血干细胞25,28,32。oligoECM 固定化的 p-IA 水凝胶是细胞培养材料研究细胞培养材料对不同类型的干细胞分化和增殖的命运以及原代细胞的影响的绝佳候选物。癌?...

干细胞分化为特定细胞谱系的命运和干细胞的长期增殖, 特别是人类诱导的多能 (iPS) 细胞和人类胚胎干细胞, 已知受抑制剂、生长因子和/或培养培养基中的小生物活性分子。最近, 生物材料的物理线索, 特别是细胞培养生物材料的硬度, 已被认为是一个重要的因素, 引导干细胞增殖和分化的命运1,2, 3、4、5、6。因此, 一些研究人员已经开始研究干细胞的命运, 它们在水凝胶上培养, 分化, 主要是采用不同刚度的聚丙烯酰胺水凝胶。
生物材料的硬度可以控制局灶粘连, 细胞形态学, 细胞表型和干细胞黏附, 特别是在二维 (2 维) 培养1,2,3,5。干细胞对生物材料的机械传感通常是通过整合素受体的黏着性信号来控制的。NMMIIA, nonmuscle 肌球蛋白 IIA 与肌动蛋白细胞骨架的依赖性收缩作用在2维细胞培养系统中的干细胞 mechanosensing 过程中起着至关重要的角色3,4,5, 7,8,9,10,11。
恩和他的同事们提出了一个有趣的观点, 即成体干细胞, 如骨髓干细胞培养的细胞培养生物材料具有类似的刚性与特定组织, 往往分化成细胞起源于特定的组织5。在膨胀介质中, 培养的2维软质聚丙烯酰胺水凝胶 (与脑组织的硬度可比) 被自发诱导分化为早期神经元谱系, 而 bms 细胞在在2维聚丙烯酰胺水凝胶3,5中, 与肌肉或胶原骨组织相似的水凝胶分别被发现诱导分化为心肌细胞和成骨细胞的早期谱系。许多研究者研究了胶原蛋白固定化培养的聚丙烯酰胺水凝胶的干细胞命运. i12,13,14,15,16,17,18,19,20,21. 但是, 应该提到一些相互矛盾的报告1,18,22,23,24为恩建议的知名的想法存在et al.5这是因为恩的想法5仅是在聚丙烯酰胺水凝胶上开发的, 其结果来源于生物材料 (聚丙烯酰胺) 的特定特性, 而不仅仅是物理提示 (刚度)生物材料因此, 开发另一种类型的水凝胶是很重要的, 其中的刚性可以通过交联凝胶来控制。为此, 研制了 bioinert 水凝胶, 由聚乙烯醇-共衣康酸 (P IA) 与不同的刚度, 这是由交联度控制与改变交联时间25,26,27,28,29,30,31,32. 干细胞可以在 nonmodified 的 p-ia 水凝胶上培养, 以及与胞外基质 (ECMs) 和肽接枝的 p ia 水凝胶。在先前的研究25中, 从脐带血中培养出的人造血干细胞 (hHSCs) 在不同刚度值的 P IA 水凝胶中进行, 从3帕斯卡到30帕斯卡的储存模数, 其中纤维连接蛋白或肽来自纤维连接蛋白 (CS1, EILDVPST) 被嫁接到 P IA 水凝胶。在 CS1 或纤维连接蛋白接枝的 P IA 水凝胶中观察到 hHSCs 的高体外折叠扩张, 显示了从12帕斯卡到30帕的中间刚度不等的 25。
人类 ips 和 ES 细胞不能在传统的组织培养聚苯乙烯 (TCP) 菜肴上培养33,34 , 因为人类 es 和 ips 细胞需要对 ECMs (如受精或层粘连蛋白) 进行特定的绑定, 以保持其多在长期文化。因此, 设计并制备了具有最佳刚度特性的肽接枝型 P IA 水凝胶结构, 并在单链、单链、联合段、双链、节段、分支式链32。从 ECMs 的整合素和多糖结合域中选择了肽序列。受精肽与双链或关节段连接的 P IA 水凝胶, 其储存模量在大约25人民军, 支持人类 ES 和 iPS 细胞的长期培养, 超过12个通道在无异和化学定义的条件32。在水凝胶上的关节段和双链细胞黏附分子促进了人类 ES 和 iPS 细胞的增殖和多32。这里, 一个关于制备 P IA 水凝胶的协议 (以储存模数从10人民军到30帕, 这是测量在空气湿条件下) 接枝和没有肽或 ECMs 描述。如何培养和通过几个干细胞 (包括羊水干细胞, 脂肪来源干细胞, 人 ES 细胞, 人类 iPS 细胞) 显示。

<table>
	<tbody>
		<tr>
			<td>GTPGPQGIAGQRGVV</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: Cyclic RGD, cRGD</td>
		</tr>
		<tr>
			<td>GACRGDCLGA</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: FN1</td>
		</tr>
		<tr>
			<td>KGGAVTGRGDSPASS</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: CS1</td>
		</tr>
		<tr>
			<td>EILDVPST</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1</td>
		</tr>
		<tr>
			<td>KGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP1</td>
		</tr>
		<tr>
			<td>GKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: HBP2C</td>
		</tr>
		<tr>
			<td>CGGGKKQRFRHRNRKG</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN1G</td>
		</tr>
		<tr>
			<td>GGGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Oligopeptide 
			Abbreviation: VN2C</td>
		</tr>
		<tr>
			<td>GCGGKGGPQVTRGDVFTMP</td>
			<td>PH Japan</td>
			<td>none</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: rVN</td>
		</tr>
		<tr>
			<td>Vitronectin</td>
			<td>Thermo Fisher scientific</td>
			<td>A14700</td>
			<td>Specification: Extracellular matrix 
			Abbreviation: FN</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F2006</td>
			<td>Specification: Commercially available coating material 
			Abbreviation: Synthemax II</td>
		</tr>
		<tr>
			<td>Synthemax II</td>
			<td>Corning</td>
			<td>3535</td>
			<td>Specification: Polymer</td>
		</tr>
		<tr>
			<td>Polyvinylalcohol-co-itaconic acid</td>
			<td>Japan VAM &#38; Poval</td>
			<td>AF-17</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Na2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>H2SO4</td>
			<td>Sigma-Aldrich</td>
			<td>339741</td>
			<td>Specification: Chemical 
			Abbreviation: EDC</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#39;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>E7750</td>
			<td>Specification: Chemical 
			Abbreviation: NHS</td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide</td>
			<td>Sigma-Aldrich</td>
			<td>56480</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>Specification: Chemical</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Corning</td>
			<td>3008</td>
			<td>Specification: Cell culture consumable</td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D4693</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 6</td>
			<td>Thermo Fisher scientific</td>
			<td>A1516401</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>Essential 8</td>
			<td>Thermo Fisher scientific</td>
			<td>A1517001</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo Fisher scientific</td>
			<td>11330-032</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher scientific</td>
			<td>12800-017</td>
			<td>Specification: Cell culture medium</td>
		</tr>
		<tr>
			<td>MCDB 201</td>
			<td>Sigma-Aldrich</td>
			<td>M6770</td>
			<td>Specification: ES cell</td>
		</tr>
		<tr>
			<td>Human ES cell</td>
			<td>WiCell Research Institute, Inc..</td>
			<td>WA09</td>
			<td>Specification: iPS cell</td>
		</tr>
		<tr>
			<td>Human iPS cell</td>
			<td>Riken Cell Bank</td>
			<td>HS0077</td>
			<td>Specification: 35 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Specification: 60 mm 
			Abbreviation: TCP</td>
		</tr>
		<tr>
			<td>TCP dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Specification: 24 well dish</td>
		</tr>
		<tr>
			<td>24 well dish</td>
			<td>Corning</td>
			<td>353047</td>
			<td>Specification: Blocking agent</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Alkaline phosphatase live stain</td>
			<td>Thermo Fisher scientific</td>
			<td>A14353</td>
			<td>Specification: Detection reagent</td>
		</tr>
		<tr>
			<td>Hematoxylin &#38; eosin</td>
			<td>Sigma-Aldrich</td>
			<td>1.05175</td>
			<td>Specification: EB formation dish</td>
		</tr>
		<tr>
			<td>6-well ultralow attachment dish</td>
			<td>Corning</td>
			<td>3471</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G9391</td>
			<td>Specification: Coating material</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Specification: Mice</td>
		</tr>
		<tr>
			<td>NOD-SCID mice</td>
			<td>National Applied Research Laboratories</td>
			<td>None</td>
			<td>Specification: Serum</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Biological Industries</td>
			<td>04-001-1A</td>
			<td>Specification: Antibiotic</td>
		</tr>
		<tr>
			<td>antimycotic antibiotic</td>
			<td>Thermo Fisher scientific</td>
			<td>15240-062</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Nanog</td>
			<td>Invitrogen</td>
			<td>MA1-017</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for SSEA4</td>
			<td>Abcam</td>
			<td>ab16287</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for OCT3/4</td>
			<td>Invitrogen</td>
			<td>PA5-27438</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Sox2</td>
			<td>Invitrogen</td>
			<td>48-1400</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for Smooth Muscle Actin</td>
			<td>Invitrogen</td>
			<td>PA5-19465</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody for AFP</td>
			<td>Invitrogen</td>
			<td>PA5-21004</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Antibody GFAP</td>
			<td>Invitrogen</td>
			<td>MA5-15086</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 &#8211; conjugated Goat anti-Mouse antibody</td>
			<td>Invitrogen</td>
			<td>A-21422</td>
			<td>Specification: Antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 &#8211; conjugated Goat anti-Rabbit antibody</td>
			<td>Invitrogen</td>
			<td>A-11008</td>
			<td>Specification: Antibody</td>
		</tr>
	</tbody>
</table>

human pluripotent stem cell culture on polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness under xeno-free conditions

一些研究人员研究了物理线索, 如生物材料的硬度对干细胞增殖和分化的影响。然而, 这些研究人员大多用聚丙烯酰胺水凝胶进行干细胞培养。因此, 它们的结果是有争议的, 因为这些结果可能来源于聚丙烯酰胺的具体特性, 而不是生物材料的物理提示 (刚度)。在这里, 我们描述了一个用于制备水凝胶的协议, 它不是以聚丙烯酰胺为基础的, 它可以培养各种干细胞, 包括人类胚胎干细胞和人类诱导的多能干细胞 (iPS) 细胞。从 bioinert 聚乙烯醇-共衣酸 (P IA) 制备了不同刚度的水凝胶, 通过改变交联时间来控制交联度的刚度。以细胞外基质为基础, 研究了肽的 P IA 水凝胶作为干细胞培养和分化的未来平台。本文详细介绍了羊水干细胞、脂肪源性干细胞、人 ES 细胞和人类 iPS 细胞的培养和传代。肽的 P IA 水凝胶表现出优异的性能, 这是由它们的刚度特性引起的。该协议报告了生物材料的合成, 他们的表面操作, 以及控制的刚度性质, 最后, 它们对干细胞的命运的影响, 利用异的自由文化条件。根据最近的研究, 这种改良的基板可以作为未来的平台, 支持和指导各种干细胞线的命运, 以不同的联系;进一步, 再生和恢复失去的器官或组织的功能。

采用肽 (oligoECM) 或不同弹性的 ecm 接枝的 P IA 水凝胶是通过以下的反应方案来制备的, 如图 1A所示, 使用不同类型的 oligoECM (图 1B)。水凝胶的弹性由应用的交联强度 (时间) (图 1C) 调节。采用受精衍生肽接枝的 P IA 水凝胶, 具有25.3 帕 (24 h 交联时间) 的贮存模量, 支持人类 iPS 和 ES 细胞长期培养超过10-20 通道。...

Watch this Scientific Journal Video about Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions at JoVE.com

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

提出了一种不同刚度的聚乙烯醇-共衣酸凝胶的制备方法, 并与无肽接枝, 研究了生物材料的硬度对其分化和增殖的影响。干细胞。水凝胶的刚度受交联时间的控制。

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

human-pluripotent-stem-cell-culture-on-polyvinyl-alcohol-co-itaconic

Research

JoVE Journal

Bioengineering

9.2K 次观看.  National Central University. 提出了一种不同刚度的聚乙烯醇-共衣酸凝胶的制备方法, 并与无肽接枝, 研究了生物材料的硬度对其分化和增殖的影响。干细胞。水凝胶的刚度受交联时间的控制。

视频: Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

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 ...

科研

生物工程

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

用于细胞培养应用光介导的形成和构图的水凝胶

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

可调水凝胶由肺细胞外基质的三维细胞培养

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney disease and eventual end-stage renal failure are often caused by the damage to the glomerular podocytes, which are the highly specialized epithelial cells that function together with endothelial cells and the glomerular basement membrane to form the kidney&#8217;s filtration barrier. Advances in renal medicine have been hindered by the limited availability of primary tissues and the lack of robust methods for the derivation of functional human kidney cells, such as podocytes. The ability to derive podocytes from renewable sources, such as stem cells, could help advance current understanding of the mechanisms of human kidney development and disease, as well as provide new tools for therapeutic discovery. The goal of this protocol was to develop a method to derive mature, post-mitotic podocytes from human induced pluripotent stem (hiPS) cells with high efficiency and specificity, and under chemically defined conditions. The hiPS cell-derived podocytes produced by this method express lineage-specific markers (including nephrin, podocin, and Wilm&#8217;s Tumor 1) and exhibit the specialized morphological characteristics (including primary and secondary foot processes) associated with mature and functional podocytes. Intriguingly, these specialized features are notably absent in the immortalized podocyte cell line widely used in the field, which suggests that the protocol described herein produces human kidney podocytes that have a developmentally more mature phenotype than the existing podocyte cell lines typically used to study human kidney biology.

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease modeling.

在化学定义条件下，成熟肾细胞与人类诱导多能干细胞的引导分化

Kidney disease affects more than 10% of the global population and costs billions of dollars in federal expenditures. The most severe forms of kidney ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

实时显微镜成像下的 3D 水凝胶控制应变

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

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, ...

Microfluidic Fabrication of Core-Shell Microcapsules carrying Human Pluripotent Stem Cell Spheroids

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 &#181;m diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with &gt;95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had &lt;50% viability 3 days after encapsulation. Spheroid formation of hPSCs inside core-shell microcapsules occurred within 48 h after encapsulation, with the spheroid diameter being a function of cell inoculation density. Overall, the microfluidic encapsulation technology described in this protocol was well-suited for hPSCs encapsulation and spheroid formation.

This article describes encapsulation of human pluripotent stem cells (hPSCs) using a co-axial flow focusing device. We demonstrate that this microfluidic encapsulation technology enables efficient formation of hPSC spheroids.

携带人多能干细胞球体的核壳微胶囊的微流体制造

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and ...

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

异条件下具有不同刚度的聚乙烯醇-钴-衣康酸水凝胶的人多能干细胞培养

摘要

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