作者感谢沙特国王大学医学院实验性手术和动物实验室的工作人员的合作与支持, 特别是侯赛因 Almukhayzim 和 Hisham Aloudah。作者还要感谢沙特国王-阿齐兹大学的媒体小组准备了视觉材料, 特别是 Muath Ghannam 和 Abdulwahab Alsulami。

该议定书遵循沙特国王大学伦理委员会的动物保育准则。在动物身上使用的外科手术、麻醉剂和其他药物都是由沙特国王大学的伦理委员会批准的。所有实验工作都由受过适当训练的人员完成。
1. 细胞板结构
<ol><li>用未稀释的胎牛血清 (血清) 涂上培养皿 (3.5 厘米温度敏感的培养皿), 确保覆盖所有的盘子表面。 注意: 这一步对细胞表的分离很重要, 因为它支持单层细胞的生长, 并防止细胞聚集。</li>
	<li>在含有 5% CO2和95% 空气的湿润气氛中, 在37摄氏度处孵化出24小时的菜肴。</li>
	<li>从涂装的盘子中除去多余的血清, 让盘子在室温下 (RT) 干燥至少1小时。</li>
	<li>准备三细胞悬浮: 1 x 106 HepG2 细胞, 1 x 106 HepG2 细胞与 BMMSCs, 1 x 106 HepG2 细胞与 UCMSCs (在 BMMSCs 和 UCMSCs 的比率 4:1)。</li>
	<li>种子适当的细胞悬浮到每个标记的盘子。</li>
	<li>悬浮 DMEM 培养基中的所有细胞, 辅以10% 血清和1% 青霉素/链霉素。</li>
	<li>在含有 5% CO2和95% 空气的湿润气氛中孵化37摄氏度的细胞。</li>
</ol>2. 细胞板脱离
<ol><li>在100% 细胞汇合处, 把盘子从37摄氏度孵化器中取出。</li>
	<li>孵育 HepG2 细胞片1小时在 RT, 而孵化 HepG2/BMMSC 和 HepG2/UCMSC 细胞板菜 30-40 分钟在20°c 在湿润的气氛中包含 5% CO2和95% 空气。 注: 仅由 HepG2 制成的细胞片是易碎的;将温度降低到20摄氏度会导致纸张结构损坏。</li>
</ol>3. 手术过程的表现
注: 在孵化时间为细胞片分离, 启动动物程序。
<ol><li>
手术区准备说明
	<ol>
<li>在实验室内不育的单独区域内进行所有外科手术, 如遮光罩、层流柜或无菌手术室。确保手术区域的所有表面都是无孔、密封、耐用和 sanitizable 的。</li>
		<li>佩戴适当的防护用具, 包括磨砂、手套、口罩、头和鞋套。</li>
		<li>在手术开始时使用清洁和灭菌的外科工具 (通过热处理; 高压下饱和茎)。</li>
		<li>在老鼠之间换手套。</li>
	</ol>
</li>
	<li>
外科动物的制备
	<ol>
<li>使用8到12周大的裸鼠。</li>
		<li>为大鼠建立注射麻醉。<ol>
<li>为准备麻醉溶液, 氯胺酮-甲苯噻嗪, 混合2毫升氯胺酮, 1 毫升甲苯噻嗪 (浓度为20毫克/毫升), 1 毫升的无菌的生理生理盐水 (如0.9% 氯化钠) 在一个不育的10毫升血清收集瓶和震动好之前使用。</li>
			<li>应用麻醉, 注射0.2 毫升的氯胺酮-甲苯噻嗪溶液每100克的体重的大鼠腹腔。 注: 总剂量的溶液是100毫克/公斤氯胺酮和10毫克/公斤的甲苯噻嗪。</li>
		</ol>
</li>
		<li>清除手术部位的可见污垢/碎片。</li>
		<li>检查麻醉深度通过测试踏板撤退反射 (脚垫捏在两个后腿)。 注: 如果脚垫捏引起反应, 重复剂量为 0.05 mL/100 g (大约每30分钟), 然后复查麻醉深度。</li>
		<li>用聚维酮-碘/异丙醇对手术区进行二级擦洗消毒。</li>
		<li>用不育的褶皱盖住老鼠, 以避免切口受到污染。</li>
		<li>在切口前注射丁丙诺啡 (0.01-0.05 毫克/千克)8的大鼠。</li>
		<li>使用无菌手术刀, 在皮肤和底层肌肉之间进行7到10厘米的切割以暴露肝脏。<ol>
<li>用手术刀的平后边缘将腹部肌肉与皮肤分开。</li>
			<li>用手术刀仔细刺linea , 用锋利的剪刀伸展切口。</li>
		</ol>
</li>
	</ol>
</li>
</ol>4. 细胞片移植
注意: 细胞片移植是在肝脏上进行的。
<ol><li>从培养皿中收集细胞表。<ol>
<li>轻轻地点击在长凳上的文化碟, 从它的表面分离床单。</li>
		<li>使用不育的吸管尖, 用半圆圈刮去薄片的边缘, 将细胞片从盘子的表面分离出来。</li>
		<li>从培养皿中轻轻移除整个培养基。</li>
		<li>确保床单完好无损, 不损坏;否则, 无法使用它 (图 1)。 注: 所产生的床单可以直接检测到眼睛。</li>
	</ol>
</li>
	<li>准备一张无菌膜 (滤纸) 从盘子里收割细胞。<ol>
<li>首先, 用生理盐水浸泡膜。然后, 用无菌棉垫去除多余的盐水。</li>
		<li>将湿膜放在电池板上, 同时避免膜与细胞片之间的折痕和气泡。</li>
		<li>在膜和细胞片中加入少量的生理盐水来收集它们。 注意: 为了避免打碎细胞片, 不要使用冷盐水, 用镊子提起膜和细胞片。</li>
		<li>离开膜几秒钟, 以确保细胞片正确地附着在膜上, 然后收集。</li>
	</ol>
</li>
	<li>为细胞片移植准备肝脏。<ol>
<li>通过用无菌棉垫轻轻地敲击它, 确保肝脏表面干燥。</li>
		<li>使用无菌钳, 将细胞膜与细胞片放在大鼠肝脏的单叶上。</li>
		<li>添加几滴不育的盐水, 并对膜施加一个温和的压力, 以分离细胞表。</li>
		<li>先等5分钟, 再仔细取出膜。 注意: 这样做是为了确保细胞表被正确地附着在肝脏上。</li>
		<li>最后, 用5-0 尼龙缝线关闭底层肌肉和皮肤切口。</li>
		<li>用聚维酮碘对手术区进行消毒。 注:图 2显示了裸鼠肝脏的细胞表移植程序图。</li>
	</ol>
</li>
</ol>5. 术后护理
<ol><li>手术后, 立即将老鼠单独放在一起, 以避免食人或窒息, 并定期监测 (至少每15分钟), 直到它清醒并完全 ambulant。</li>
	<li>然后, 每天对老鼠进行 5-14 维的评估, 手术后至少 5 d, 以确保没有并发症。在日常评估中, 请确保以下内容。<ol>
<li>伤口闭合, 缝合完好, 不太紧。</li>
		<li>切口部位无感染迹象, 如发热、过度肿胀或化脓性放电。</li>
		<li>没有与疼痛相关的迹象, 如食物和水的消耗减少, 体重减轻, 脱水, 皮肤 tenting, 或快速和张开嘴呼吸。为了控制中度至严重术后疼痛, 如果有的话, 注射以丁丙诺啡 (0.01-0.05 毫克/千克) 皮下的大鼠每 6-8 小时9 , 术后至少48至72小时。</li>
	</ol>
</li>
</ol>6. 对移植区域的分析
<ol><li>移植后一个月, scarifice 大鼠, 收集移植区进行组织学和免疫组化分析。</li>
</ol>

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作者没有什么可透露的。

大量的研究致力于开发一种与人类癌症相似的活体前临床动物模型。目前, 用于创建癌症动物模型的主要方法涉及基因工程和细胞移植11。转基因动物模型是识别和验证目标基因的好工具, 也是了解药物诱导毒性的分子机制的良好手段。但是, 该模型与某些局限性相关;例如, 对某一特定基因的修饰并不总是导致预期的表型12。细胞移植法更常用于诱发动物肿...

肝癌是原发性肝癌, 与预后差有显著相关性。每年, 几乎有一半的新病人被诊断为肝癌, 占全世界1的肝癌患者的85%。肝癌不是一种单一形式的疾病;相反, 它是一个疾病的集合, 有不同的病理学特征和基因和基因组变异, 除了不同的预后结果1。因此, 对肝癌的有效治疗策略的发展面临的主要挑战是对肝癌生物学的认识有限, 缺乏合适的实验动物模型来帮助了解这种复杂的疾病。模拟人类癌症的体内动物模型对于选择候选基因和确定与癌症诱导相关的预后/预测指标以及调查可能影响癌症反应的不同因素都是必要的。治疗药物。
体外肿瘤的研究仍与主要的局限性有关。这是由于癌症细胞在培养时失去了许多体内的特性。细胞体外发生的变化是由于体内的组织生理没有形成。癌细胞对细胞的相互作用 (基质、免疫、血管、上皮等) 在肿瘤微环境中对癌细胞的特征有很大的反映2。除了 angiogenetic 和转移电位外, 肿瘤微环境可以改变癌细胞的基因/蛋白表达和表型特征。二维 (2 维)体外培养系统也缺乏合适的组织基质, 对肿瘤进展有一定的调节作用。因此, 由于这些限制,体内模型应始终用于支持的初步结果的体外模型。在本研究中, 我们利用细胞板技术开发了一种活体动物模型, 阐明了肝癌的完整生物学过程。
十年前, 冈野实验室建立了一种基于细胞片技术的组织工程新方法3。这种技术利用热响应的培养塑料, 以允许可逆的细胞粘附/脱离通过控制表面疏水性。这种方法允许在完整的三维 (3 维) 格式 (即细胞片) 中温和地收获培养细胞, 并保持良好的细胞外基质 (ECM) 和细胞对细胞的相互作用。细胞板技术需要预涂的文化菜肴与温度反应聚合物聚 (N-丙烯酰胺) (琵琶 Am), 这是商业上可用和准备使用。在摄氏20摄氏度以下的温度下, 琵琶的聚合物变得水合并溶解于水溶液中, 而在较高温度 (37 摄氏度) 时, 聚合物变得脱水, 转化成混浊沉淀。该聚合物含有亲水性酰胺链和疏水性侧链 (异丙基团)。在高温下, 水分子的布朗运动被强化, 而在低温下, 水合结构击穿的异丙基群周围的水分子和疏水性异丙基集群因为疏水性的相互作用。因此, 整个链的聚合物聚合和沉淀4。
在本文的研究中, 采用该技术开发了三种不同细胞片的肝癌动物模型。第一张工作表仅由肝癌细胞系细胞组成, 而另两片则由两种不同来源的肝癌细胞系细胞和 mscs 组成: 骨髓间充质干细胞 (BMMSCs) 和脐带 mscs (UCMSCs)。MSCs 是非造血基质细胞, 能够区分 intocell 衍生物的间充质谱系, 包括脂肪细胞, 骨, 软骨, 和肌细胞5。我们在创建癌细胞表时使用这些细胞的原因是关于 MSCs 对癌症的影响的报告不一致。有人建议, MSCs 可以有两个不同的表型: "MSC1", 促炎型, 和 "MSC2", 免疫抑制表6。MSCs 表达的类似的收费受体 (TLRs)。TLR4 启动的 MSCs 增加其分泌的促炎因子, 而 TLR3 启动增加其分泌免疫抑制因子6。对这两种表型的体外研究报告说, MSC1 与癌细胞株的共培养会削弱癌细胞的生长, 而 MSC2 共培养则有相反的作用7。这表明, MSCs 可以是亲癌或抗癌, 取决于他们的表型。因此, 除了利用细胞板技术开发肝癌动物模型外, 我们还要探讨 MSC 移植对肿瘤发展的影响, 以及使用这些细胞是否会增强或减少该模型的发展。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Reagents&#160;</td>
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			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">FBS&#160;</td>
			<td style="width:192px;">Gibco/Invitrogen</td>
			<td style="width:119px;">10270106</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">DMEM high glucose&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">D5671-500ML</td>
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			<td height="31" style="height:31px;width:309px;">Penicillin/streptomycin&#160;</td>
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		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Sterile physiologic saline</td>
			<td style="width:192px;">Sigma&#160;</td>
			<td style="width:119px;">S0817-1GA</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">Human HepG2 cell line</td>
			<td style="width:192px;">ATCC, USA</td>
			<td style="width:119px;">HB-8065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Human bone marrow MSCs cell line</td>
			<td style="width:192px;">PromoCell, USA</td>
			<td>C-12974</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:309px;">human umbilical cord tissue MSCs</td>
			<td style="width:192px;">PromoCell, USA&#160;</td>
			<td style="width:119px;">C-12971</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Ketamine 50%</td>
			<td style="width:192px;">Rompun, Bayer</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Xylazine 2%</td>
			<td style="width:192px;">Rompun</td>
			<td style="width:119px;">23076-35-9</td>
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			<td height="22" style="height:22px;">Alphadine&#174;&#160;solution.</td>
			<td style="width:192px;">Riyadh Pharma</td>
			<td style="width:119px;">LBL0816</td>
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			<td height="22" style="height:22px;width:501px;">Disposables:&#160;</td>
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			<td style="width:119px;">352097</td>
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		<tr height="48">
			<td height="48" style="height:48px;width:309px;">3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">174904-1CS</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:309px;">100-1000 &#181;l &#160;Pipette Tips&#160;</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">CLS4868-1000EA&#160;&#160;</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;">Basic Procedure Drape</td>
			<td style="width:192px;">Thermofisher</td>
			<td style="width:119px;">PMD5293.0</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:501px;">Equipment&#160;</td>
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			<td style="width:119px;"></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Plus pipette, variable volume</td>
			<td style="width:192px;">Eppendorf&#174;&#160;Research&#174;</td>
			<td style="width:119px;">Z683779-1EA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Tissue culture incubator 37 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:309px;">Biological safety cabinet</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:309px;">Tissue culture incubator 20 &#176;C, 5% CO2</td>
			<td style="width:192px;">Any brand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Sterile surgical tools and nude rats:&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="22">
			<td colspan="2" height="22" style="height:22px;width:501px;">Forceps</td>
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			<td colspan="2" height="22" style="height:22px;width:501px;">Scissors</td>
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			<td colspan="2" height="22" style="height:22px;width:501px;">scalpel&#160;</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:309px;">&#160;Nylon Suture&#160; 5-0</td>
			<td style="width:192px;">Accutome</td>
			<td style="width:119px;">AB-3854S</td>
			<td>Monofilament, Lancet</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">1 ml Tuberculin Syringes</td>
			<td style="width:192px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-88</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:309px;">Nude rats&#160;</td>
			<td style="width:192px;">Charles river</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

exploring the potential of mesenchymal stem cell sheet on the development of hepatocellular carcinoma in vivo

一种模拟人类癌症的体内动物模型可以有各种应用来提供重要的临床信息。目前用于体内肿瘤模型发展的技术有很大的局限性。因此, 本研究旨在实现细胞板技术在体内肿瘤模型的建立。肝细胞癌 (hcc) 是在裸鼠中成功开发的, 使用细胞片创建的癌细胞系细胞。癌细胞片是通过细胞内黏附和形成的分层结构, 由细胞外基质控制。这使得肝癌片移植到肝脏, 并在一个月内创建一个肿瘤动物模型。此外, 研究了骨髓间充质干细胞 (MSC) 在肿瘤模型发展中的作用。除了肝癌细胞线外, 还创建了另外两个细胞片: 一张肝癌细胞和骨髓间充质干细胞 (BMMSCs) 和一张肝癌细胞和脐带 MSCs (UCMSCs)。有肝癌细胞和骨髓间充质干细胞组合的床单也能产生一种含肿瘤的动物。然而, 骨髓间充质干细胞的增加减少了肿瘤的大小, 这对肿瘤发育的不利影响取决于所使用的 mscs 的来源。这表明用某些 MSC 亚型制成的细胞片可以用于肿瘤的管理和控制。

大鼠移植细胞致瘤性的研究:
移植后一个月, 在大鼠肝脏移植的所有细胞片都已发育成肿瘤 (图 3)。被开发的肿瘤的平均大小从 HepG2, HepG2/BMMSC 和 HepG2/UCMSC 细胞片是 4.5 cm, 4 cm 和 2.5 cm, 分别10。
肝组织组织学分析:
<p...

Watch this Scientific Journal Video about Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo at JoVE.com

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

在这里, 我们提出了一个使用细胞板技术开发体内肿瘤模型的协议。这种模型对肿瘤治疗的评价有很大的参考价值。

骨髓间充质干细胞片对肝癌体外发育的潜能探讨

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

exploring-potential-mesenchymal-stem-cell-sheet-on-development

Research

JoVE Journal

Bioengineering

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

5.8K 次观看.  Ministry of National Guard Health Affairs. 在这里, 我们提出了一个使用细胞板技术开发体内肿瘤模型的协议。这种模型对肿瘤治疗的评价有很大的参考价值。

视频: Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

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

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

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

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

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

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

科研

生物工程

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 ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

发展多深的圆形横截面微内皮化的一个芯片的程序

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

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 ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

在符合标准的基质上对人类胚胎干细胞菌落的几何结构进行模式化，以控制组织级力学

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

山羊窒息关节髌下脂肪垫间充质干细胞的分离、扩增和分化

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

骨髓间充质干细胞片对肝癌体外发育的潜能探讨

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