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瘤, 它涉及注射癌细胞悬浮液。然而, 这种简单方便的方法有其自身的缺点。为了产生细胞悬浮, 酶消化步骤将用于收获癌细胞。这导致了一些粘附蛋白的丢失, 这将降低移植细胞的植入效率。因此, 不能保证移植癌细胞会形成癌组织, 更不用说控制肿瘤大小的困难。此外, 这种方法还有另一个严重的缺点, 即移植癌细胞迅速过渡到血液循环或细胞植入成非靶器官13。
为了克服这些缺点, 研制了新型的细胞板技术。铃木等。14已成功地创建了结肠, 肝, 胰腺肿瘤动物使用癌细胞片移植方法。与通过肿瘤细胞移植方法产生的肿瘤相比, 这些肿瘤更大、更稳定。将小肿瘤组织植入动物体内诱发肿瘤, 高度依赖于使用不需要酶消化步骤的移植方法。蛋白水解酶会对细胞外基质造成损伤, 这会影响胞间相互作用。细胞板技术克服了这一限制使用侵入性采伐方法, 允许收集完整的单层细胞表, 连同其相关的细胞外基质和生长因子15。细胞板技术也允许长期植入16,17。这在活体研究中有很大的价值, 细胞必须能够在组织的整个生命周期中嫁接到宿主的组织中17。此外, 这项技术使开发一种新型的组织工程方法, 没有使用生物降解支架3,15。然而, 这种方法仍然有一些局限性。在使用细胞片技术时, 如果移植物太大, 移植组织的中心部分可能会坏死。反之, 移植的成功率如果太小, 可能会减少。此外, 为了控制肿瘤的后续生长, 移植组织的大小必须在所有实验中都是一致的。由于氧环境差18, 可移植组织的大小仅限于2毫米3 。
在本研究中, 用细胞片无支架移植成功地建立了肝癌动物模型。将三种细胞片移植到大鼠肝脏中: 肝癌细胞系细胞、肝癌细胞系细胞和骨髓基质细胞, 以及肝癌细胞系细胞和 UCMSCs 细胞片。所有三细胞片的移植足以诱发受体大鼠肿瘤。然而, 有与会者指出, 在床单上添加 MSCs 可以减少肿瘤的大小。这表明使用的 MSCs, 特别是 UCMSCs, 对肿瘤的发展有不利的影响。结论: 肿瘤细胞片移植是建立实体肿瘤动物模型的有效方法。有这样的体内模型可能会导致重要的临床信息与大量的应用。此外, 由于肿瘤大小的减少, UCMSCs 限制肿瘤的发展和传播。这表明一些 MSC 亚型可以用于一种基于细胞的治疗癌症的方法。

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。

<table><tbody><tr><td>&lt;strong&gt;Reagents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Gibco/Invitrogen</td><td>10270106</td><td></td></tr><tr><td>DMEM high glucose&amp;nbsp;</td><td>Sigma</td><td>D5671-500ML</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technology</td><td>15070063</td><td></td></tr><tr><td>Sterile physiologic saline</td><td>Sigma&amp;nbsp;</td><td>S0817-1GA</td><td></td></tr><tr><td>Human HepG2 cell line</td><td>ATCC, USA</td><td>HB-8065</td><td></td></tr><tr><td>Human bone marrow MSCs cell line</td><td>PromoCell, USA</td><td>C-12974</td><td></td></tr><tr><td>human umbilical cord tissue MSCs</td><td>PromoCell, USA&amp;nbsp;</td><td>C-12971</td><td></td></tr><tr><td>Ketamine 50%</td><td>Rompun, Bayer</td><td></td><td></td></tr><tr><td>Xylazine 2%</td><td>Rompun</td><td>23076-35-9</td><td></td></tr><tr><td>Alphadine&amp;reg;&amp;nbsp;solution.</td><td>Riyadh Pharma</td><td>LBL0816</td><td></td></tr><tr><td>&lt;strong&gt;Disposables:&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15mL Polypropylene High Clarity PP Centrifuge Tube</td><td>Falcon&amp;nbsp;</td><td>352097</td><td></td></tr><tr><td>3.5 cm sterile UpCell culture dishes with the filter paper (membrane)</td><td>Sigma</td><td>174904-1CS</td><td></td></tr><tr><td>100-1000 &amp;micro;l &amp;nbsp;Pipette Tips&amp;nbsp;</td><td>Sigma</td><td>CLS4868-1000EA&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Basic Procedure Drape</td><td>Thermofisher</td><td>PMD5293.0</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Plus pipette, variable volume</td><td>Eppendorf&amp;reg;&amp;nbsp;Research&amp;reg;</td><td>Z683779-1EA</td><td></td></tr><tr><td>Tissue culture incubator 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Biological safety cabinet</td><td>Any brand</td><td></td><td></td></tr><tr><td>Tissue culture incubator 20 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>Any brand</td><td></td><td></td></tr><tr><td>Sterile surgical tools and nude rats:&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>scalpel&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Nylon Suture&amp;nbsp; 5-0</td><td>Accutome</td><td>AB-3854S</td><td>Monofilament, Lancet</td></tr><tr><td>1 ml Tuberculin Syringes</td><td>Fisher Scientific</td><td>14-826-88</td><td></td></tr><tr><td>Nude rats&amp;nbsp;</td><td>Charles river</td><td></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:
非移植大鼠的苏木精和伊红 (H & E) 染色显示肝细胞排列在彼此吻合的板块中, 细胞核呈圆形, 有一或两个突出的核仁。相比之下, 所收集的肿瘤切片显示了可存活的发炎和坏死的皮下肝细胞的边缘, 核轮廓不规则和明显的变性 (图 4)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57805/57805fig1.jpg"> 图1: 用3.5 厘米温度响应培养皿制作细胞片.(a) 本小组显示受损的细胞表不适合移植。(B) 本小组显示完整的细胞表, 准备移植。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57805/57805fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57805/57805fig2.jpg"> 图 2: 裸鼠细胞片移植术.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57805/57805fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57805/57805fig3.jpg"> 图 3: 在一个月的细胞片移植后, 肿瘤在大鼠肝脏上发育.肿瘤是由 (a) HepG2 细胞片 (大小: 4.5 厘米), (B) HepG2 和 BMMSC 细胞表 (大小: 4 厘米) 和 (C) HepG2 和 UCMSC 细胞表 (大小: 2.5 厘米) 产生。蓝色圆圈显示肿瘤区域。此数字已从以前发布的工作10中进行了修改。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57805/57805fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57805/57805fig4.jpg"> 图 4: 大鼠肝脏的组织学分析.四周后细胞移植术后, 大鼠肝脏的 H & E 染色。(A) 本小组显示正常的大鼠肝细胞。(B) 本小组显示肿瘤细胞移植后的肝脏形态学。黑色箭头表示肝癌, 绿色箭头表示正常肝细胞, 蓝色箭头表示发炎的神经细胞, 橙色箭头表示坏死的肝癌细胞。刻度条表示20X 放大倍数。此数字已从以前发布的工作10中进行了修改。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57805/57805fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Universidad San Sebastian

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

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

在肝炎症和纤维化环境中产生的肝细胞癌的致癌肝细胞诱导正交小鼠模型

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

科研

癌症研究

A Biomimetic Model for Liver Cancer to Study Tumor-Stroma Interactions in a 3D Environment with Tunable Bio-Physical Properties

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a fibrotic environment actively supporting and driving hepatocarcinogenesis. Insight into hepatocarcinogenesis in terms of the interplay between the tumor stroma micro-environment and tumor cells is thus of considerable importance. Three-dimensional (3D) cell culture models are proposed as the missing link between current in vitro 2D cell culture models and in vivo animal models. Our aim was to design a novel 3D biomimetic HCC model with accompanying fibrotic stromal compartment and vasculature. Physiologically relevant hydrogels such as collagen and fibrinogen were incorporated to mimic the bio-physical properties of the tumor ECM. In this model LX2 and HepG2 cells embedded in a hydrogel matrix were seeded onto the inverted transmembrane insert. HUVEC cells were then seeded onto the opposite side of the membrane. Three formulations consisting of ECM-hydrogels embedded with cells were prepared and the bio-physical properties were determined by rheology. Cell viability was determined by a cell viability assay over 21 days. The effect of the chemotherapeutic drug doxorubicin was evaluated in both 2D co-culture and our 3D model for a period of 72h. Rheology results show that bio-physical properties of a fibrotic, cirrhotic and HCC liver can be successfully mimicked. Overall, results indicate that this 3D model is more representative of the&#160;in vivo&#160;situation compared to traditional 2D cultures.&#160;Our 3D tumor model showed a decreased response to chemotherapeutics, mimicking drug resistance typically seen in HCC patients.&#160;

This protocol presents a 3D biomimetic model with accompanying fibrotic stromal compartment. Prepared with physiologically relevant hydrogels in ratios mimicking the bio-physical properties of the stromal extracellular matrix, an active mediator of cellular interactions, tumor growth and metastasis.

肝癌的仿生模型，用于研究具有可调生物物理特性的3D环境中的肿瘤-斯特罗马相互作用

Hepatocellular carcinoma (HCC) is a primary liver tumor developing in the wake of chronic liver disease. Chronic liver disease and inflammation leads to a ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

皮下及肝内人肝癌裸鼠移植瘤的免疫缺陷小鼠的产生

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

医学

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

该<em&gt;卵内</em&gt;鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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 ...

生物工程

Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.

Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.

分离的CD133 +肝干细胞的克隆扩增

Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In ...

生物学

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

The preparation and transplantation of adipose tissue derived stem cell (ASC) sheets onto insufficiently sutured colorectal anastomoses in a rat model is presented. This novel application shows that ASC sheets can reduce colorectal anastomosis leakage.

脂肪组织干细胞移植对大鼠部分结肠切除术后渗漏的减少

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

从人类胚胎干细胞中引导肝细胞样细胞诱导的有效方法

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

免疫与感染

Assembloid Technology: Pioneering Liver Development Research

In Vitro Cultivation Techniques for Modeling Liver Organogenesis, Building Assembloids, and Designing Synthetic Tissues using Human Cell Lines

Chronic liver disease has reached epidemic proportions, affecting over 800 million people globally. The current treatment, orthotopic liver transplantation, has several limitations. Promising solutions have emerged in the field of liver regenerative medicine, with liver organogenesis holding significant potential. Early liver organogenesis, occurring between E8.5 and 11.5, involves the formation of epithelial-mesenchymal interactions leading to morphogenesis, hepatic cord formation, and collective migration. However, there is a lack of methods for in vitro modeling of this process. In this study, a detailed series of methods is presented, enabling the modeling of various stages and aspects of liver organogenesis with human cell lines. In one method series, assembloid technology with hepatic (HEP) and mesenchymal (MES) spheroids are utilized, replicating early structures found in liver organogenesis, modeling early morphogenesis, and demonstrating interstitial cell migration as seen in vivo. These innovative assembloid systems help identify factors influencing assembloid formation and migration. HEP spheroid cultivation systems were also employed to model collective migration and branching morphogenesis. Mesenchymal-conditioned media (M-CM) plays a significant role in initiating dose-dependent branching migration. All presented methods were shown to be highly reproducible with a high success rate. By linking these events together, our work will enable recreating sequential, morphogenetic events to reverse engineer organogenesis in vitro and in vivo.

Organoids revolutionize personalized tissue modeling for organ development, drug discovery, and disease research. Organoid engineering advances into creating intricate synthetic tissues. The aim is to integrate morphogenesis, assembloid technology, and biomatrices to advance tissue engineering. This protocol aids in modeling liver organogenesis and establishing guidelines for synthetic tissue construction.