作者感谢汉娜 Voersmann 建立了3D 脊髓黑色素瘤-球形皮肤模型, 并提供了优良的协议。作者还感谢 Silke 布希提供了宝贵的技术支持。这项工作得到了 BMBF e: 医学项目 "黑色素瘤敏感性" 031A423A 的支持。

所有人类组织工作都是使用批准的机构协议进行的。
1. 真皮和表皮与人皮肤的分离 (包皮环切术)
<ol><li>将包皮 (通常为 1-2 厘米2) 放入非粘附无菌细胞培养皿 (Ø10厘米), 并用 10-12 毫升磷酸盐缓冲盐水+ (PBS, 0.5 毫米氯化镁2, 0.9 毫米 CaCl2, pH 值 7.2 = PBS+)。使用无菌手术刀和镊子去除所有多余 (脂肪) 组织。</li>
	<li>将皮肤切成3毫米 x 5 毫米片, 用 PBS 冲洗, 并将其转移到无粘菌细胞培养皿 (Ø6厘米)。用 5-7 毫升的酶溶液覆盖组织片 (PBS 中2个 U/毫升)。用石蜡膜封住细胞培养皿, 在4摄氏度孵育16小时。</li>
	<li>用两个消毒钳从真皮部收回表皮。将解剖过的组织片转移到单独的非粘附细胞培养皿 (Ø6厘米), 并用 PBS+将其覆盖。</li>
</ol>2. 从表皮分离原代角质细胞
<ol><li>使用巴斯德吸管从细胞培养皿中小心移除 pbs+ , 并用 pbs 清洗表皮组织片 (1.3 节)。将表皮切成小块 (1 毫米2), 并将其收集在50毫升圆锥管中。添加10毫升1x 胰蛋白酶-EDTA (预热到37°c) 和孵化5分钟在一个水浴37°c。每2分钟涡旋组织悬浮。</li>
	<li>通过添加1毫升胎儿小牛血清 (FCS) 停止酶反应。并用重悬细胞通过吹打上下10毫升的塑料吸管, 以4分钟. 尽量避免气泡和泡沫形成。</li>
	<li>吸管细胞悬浮到细胞过滤器 (孔径100µm), 放置在一个新鲜的50毫升圆锥管顶部, 并冲洗3x 与5毫升 PBS。离心细胞在 200 x g 5 分钟并用重悬细胞颗粒在 2-6 毫升培养基中含有 1% (v/v) 庆大霉素。通过将 hemocytometer 和种子 6 x 105单元格中的单元格计数为 T75 细胞培养瓶来手动确定单元格号。</li>
</ol>3. 原代角质细胞的培养
<ol><li>在没有 FCS 的角质细胞培养基中, 在37°c 和 5% CO2中, 在2.3 步内孵化出第一个角质细胞, 到 50-70% 融合。</li>
	<li>要通过角质细胞仔细去除培养基, 添加5毫升 PBS, 并孵化细胞10分钟37摄氏度。检查细胞是否已经开始从培养瓶分离光显微镜下 (4X 放大: 细胞会出现圆形, 但仍然附加!)。如果没有, 更换旧的 pbs-edta 与新鲜 pbs-edta 和重新孵化10分钟, 在37摄氏度。</li>
	<li>添加5毫升1x 胰蛋白酶-edta 到圆细胞仍覆盖10毫升 PBS-edta 和重新孵化他们 1-3 分钟在37摄氏度。在50毫升圆锥管中加入1毫升的 FCS, 并收集分离的细胞, 停止酶反应。离心细胞在 200 x g 5 分钟, 并用重悬颗粒在角质形成培养基, 种子 6 x10 5 细胞进入新鲜的 T75 细胞培养瓶。 注: 要生成脊髓皮肤重建, 应使用的主要角质细胞不迟于 3-4。</li>
</ol>4. 从真皮中分离原发成纤维细胞
<ol><li>将真皮组织 (1.3 节) 切成小块 (1 毫米2) 并将其转移到50毫升圆锥管中。添加10毫升胶原酶-溶液 (PBS+中的 5 U/毫升), 并在37摄氏度的水浴中孵育45分钟。将组织件和细胞的混合物以 200 x g 为5分钟。</li>
	<li>用10毫升 DMEM (含4.5 克/升葡萄糖和 l-谷氨酰胺) 清洗细胞颗粒两次, 但无 l-丙酮酸。最后并用重悬了2毫升 DMEM 中含有 1% (v/v) 庆大霉素和10% 个 FCS 的组织片。将它们转移到一个 T25 细胞培养瓶中, 并在37摄氏度和 5% CO2过夜的细胞孵化器中孵化。 注意: 小体积允许成纤维细胞从组织碎片中移出, 迫使它们附着在培养瓶上。</li>
	<li>第二天早上, 添加另外6毫升 DMEM/庆大霉素/FCS 和孵化 2-3 天在37摄氏度, 直到细胞已经达到 80-90% 融合。</li>
</ol>5. 原代成纤维细胞的培养
<ol><li>通过 PBS 移除培养基, 并将黏附细胞洗净。添加5毫升1x 胰蛋白酶-EDTA 和孵育3分钟37摄氏度。</li>
	<li>通过添加5毫升 DMEM/FCS, 并收集50毫升圆锥管中的分离细胞, 停止酶反应。离心细胞在 200 x g 5 分钟, 并用重悬颗粒在 DMEM 培养基, 种子 6 x10 5 细胞成一个新鲜的 T75 细胞培养瓶。 注: 要生成脊髓皮肤重建, 应使用的主要成纤维细胞不迟于通道 4-6。</li>
</ol>6. 3D 黑色素瘤的产生球体通过悬挂滴法
<ol><li>根据一般协议, 使用含有 10% FCS15的 RPMI, 将黑色素瘤细胞 (例如, 451 路, 或任何黑色素瘤细胞系) 按常规。<ol>
<li>为了生成类似大小和质量的黑色素瘤球体, 在 pbs 中冲洗黑色素瘤细胞, 在 pbs 中加入5毫升1x 胰蛋白酶-EDTA, 在 T175 细胞培养瓶中的细胞中, 在室温下孵育 3-5 分钟。加入5毫升 RPMI/10%fcs 中和胰蛋白酶。以 200 x g 为5分钟, 以离心的方法收割细胞, 在最后浓度为1万细胞/毫升的 RPMI 中重新悬浮细胞颗粒, 通过计数在 hemocytometer 中确定。</li>
	</ol>
</li>
	<li>40 x 25 µL (= 250 细胞) 的细胞悬浮在一个无菌的非粘细胞培养皿 (Ø10厘米) 的内表面上使用电子多吸管。快速但平滑的运动, 打开盖子周围, 并把它放在各自的细胞培养皿包含5毫升 PBS。<ol>
<li>文化 "垂悬的菜" 在细胞孵化器在37°c 和 5% CO2为 10-14 天, 取决于使用的细胞类型。 注意: 与 RGP 或 VGP 的黑色素瘤细胞相比, MM 生长阶段的细胞生长速度较快, 并且在悬滴中形成更坚实的球体。对于个体评价, 观察在双筒望远镜或光显微镜下的球状生长 (4X 放大)。通常情况下, 球体在双筒望远镜或光镜下 (4X 放大倍数) 下, 可探测到48小时。10-14 天后, 它们是可见的, 没有任何放大装置。</li>
	</ol>
</li>
	<li>在最初的下落发现5天之后, 增加10µL 新鲜 RPMI/10%fcs 培养基到每个下落。随后, 每隔一天交换10µL。在这个步骤中使用电子分配器非常有用。</li>
	<li>根据细胞类型, 收获球体 (参见部分10为细节) 在 10-15 天以后通过轻轻地漂洗他们从细胞培养皿盖子与 PBS。收集他们在一个新鲜的不粘细胞培养皿。 注: 培养期取决于黑色素瘤细胞最初获得时的肿瘤生长阶段,例如, 从451路细胞中提取的球体500µm 在12天内在悬挂下降15.</li>
</ol>7. 脊髓全皮肤重建真皮隔层的生成
<ol><li>使用24井细胞微孔膜插入 (孔尺寸8µm), 放置在24井板上, 生成皮肤模型。 注: 确保不要使用悬挂的, 但站立的插入物, 因为它们将被放置在一个6井板的空气液体种植在稍后阶段。</li>
	<li>准备凝胶中和溶液 (GNL)15, 以及称为 MM 和内皮生长介质 (EGM)16,17的区域性媒体。为防止凝血, 将胶原蛋白 i 型 (通常从鼠尾, 3.5-4 毫克/毫升在 0.02 N 乙酸) 在冰上, 直到使用, 因为它开始凝胶在 RT。</li>
	<li>在 GNL 中重新挂起 1 x 105的成纤维细胞, 并快速将细胞悬浮与胶原蛋白混合, 其比例为 1:3, 最终体积为500µL/插入。混合通过轻轻吹打, 以避免气泡形成气泡可能会损害皮肤重建的质量。</li>
	<li>允许单独的真皮凝胶解决, 使他们没有培养基在 RT 30 分钟的无菌罩。随后覆盖每一个凝胶与 DMEM 含有4.5 克/升葡萄糖, 1% l-谷氨酰胺, 10% FCS, 没有 l-丙酮酸, 和孵化隔夜在37摄氏度。</li>
</ol>8. 脊髓全皮肤重建表皮细胞室的生成
<ol><li>第二天从真皮凝胶中取出培养基, 并将其与 EGM 培养基 (10% FCS, 1% PenStrep, 10 毫克/毫升庆大霉素) 平衡, 2 小时37摄氏度。提取中和小心种子 1 x 105角质细胞悬浮在100µL EGM 在真皮凝胶的顶部。</li>
	<li>孵育1.5 小时的重建, 在37°c, 使角质形成细胞坚持真皮室。 注: 在此潜伏期, 凝胶将开始收缩, 由于成纤维细胞引起的收缩, 因此, 至少部分分离从插入壁。</li>
	<li>用大约800µL EGM 覆盖皮肤当量, 并小心地从插入壁上取出残留凝胶, 用一个小 (白色) 吸管尖。在37摄氏度、5% CO2的细胞孵化器中, 在 EGM 中浸泡7天的皮肤当量, 每隔一天更换一次培养基。 注意: 在这段时间内, 皮肤等价物将显著萎缩。</li>
</ol>9. 脊髓全皮肤重构的气液培养
<ol><li>在8天, 把每个插入到一个6井板的个人井。仅增加 1.2-1.4 毫升毫米中等到每个井, 因此皮肤重建从井的底部提供中等, 但不被媒介覆盖。 注: 在空气液界面的培养允许表皮部分的分层和建立一个完整的 cornified 层 (角质层)。</li>
	<li>在接下来的 10-17 天内, 更改每隔一天9.1 节中所述的介质。在此期间, 添加药物或其他刺激, 如需要的培养基。仔细去除从微孔膜插入的完整皮肤重建使用弯镊子进一步分析,例如, 免疫组化分析 (图 1)。</li>
</ol>10. 脊髓黑色素瘤球形皮肤模型的生成
<ol><li>为了将黑色素瘤球体到脊髓全皮肤重建的真皮室中, 小心地将球体 (步骤6.4 后) 从吊滴细胞培养皿的盖子上冲洗出来。收集 10-20 球体/插入无菌的非粘附细胞培养皿。用巴斯德吸管小心地去除过多的 PBS。</li>
	<li>在 EGM 的最小体积内吸球体。在这一点上观察和计数的球体与肉眼, 没有任何进一步的放大装置。采取 10-20 球体每皮肤模型/凝胶, 如步骤10.1 所述。</li>
	<li>将它们转移到含有成纤维细胞的 GNL (步骤7.3 中), 并与 i 型胶原混合。从这一步骤开始, 如 7.3-9.1 节所述。 注: 肿瘤球体在真皮凝胶中可见为白色斑点 (图 2)</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+three-dimensional+multicellular+spheroid+culture+for+biomedical+research.">Recent advances in three-dimensional multicellular spheroid culture for biomedical research.</a> Biotechnol. J. 3 (9-10), 1172-1184 (2008).</li></ol>

作者声明他们没有竞争的财政利益。

本文介绍的脊髓黑色素瘤球形皮肤模型, 为深入了解肿瘤内和肿瘤宿主相互作用提供了新的见解, 并可为研究肿瘤发展的分子机制和未来的治疗耐药性。
为了保证最佳的生理和体内模拟皮肤重建的条件, 主要细胞的质量是至关重要的。如上所述, 幼年或产前皮肤细胞表现出最低的分化等级, 因此最适合生成全厚度皮肤当量。第一个可能的质量控制是皮肤萎缩的程度在2天后, 播种角质细胞和平衡凝胶到 EGM (协议节8.2 和 8.3)。真皮凝胶将最有效地收缩成纤维细胞的质量。此外, 太少或太多的成纤维细胞的整合可能会损害皮肤收缩, 从而将表皮附着到真皮间。这反过来会损害表皮分化, 因为这个过程需要在真皮和表皮细胞之间进行广泛的交叉交谈。
主要细胞的质量也取决于细胞融合以及细胞的通过。如果角质形成细胞生长到≥80% 融合他们立即停止增殖和开始分化。因此, 在传代期间将它们培养到 40-70% 融合是很重要的, 并且不迟于通过 3-4 的时间使用它们。成纤维细胞较不敏感, 但不应使用较晚于 4-6。还请注意, 用于生成脊髓黑色素瘤球形皮肤模型的所有主要细胞和细胞系均无抗生素培养, 因此污染风险很高。然而, 抗生素的添加改变了个体细胞的生理, 从而降低了皮肤当量的质量。
一般来说, 肿瘤球体的形成是可能的几乎所有类型的肿瘤细胞系, 也从肿瘤细胞新鲜分离的病人材料。获得最佳球体尺寸的初始细胞数和/或培养时间可能因所使用的细胞类型而异。高达 150-200 µm 的大小, 所有在球体中的细胞仍然可以通过简单的扩散充分提供养分。只有球体大小≥500µm 显示高度的细胞分化, 代表非血管瘤组织的典型特征22。
在这里开发的脊髓黑色素瘤-球形皮肤模型特别适合研究肿瘤宿主相互作用和黑色素瘤药物测试在体内样情况下15。然而, 黑色素瘤的环境,在体内更复杂, 窝藏各种肿瘤相关的细胞类型, 包括免疫细胞和内皮细胞。由于适当的初级免疫细胞的添加面临着组织相容性的问题, 这些模型尚不能充分监测免疫治疗方法。
然而, 黑色素瘤球体可以由新鲜隔离的病人材料产生, 一旦纳入完整的皮肤重建, 可以作为个别的药物筛选平台。即使将黑色素瘤转移到皮肤重建中, 也可以用一种量身定做的治疗方法来测试药物组合。包括脊髓3D 皮肤黑色素瘤模型在临床前试验中可能有助于确保只有最有前途的新的治疗概念, 以进行的医疗测试, 从而降低了潜在的新疗法的磨损率疾病和提高治疗成功率的临床试验。

人体皮肤由两个不同的隔间组成, 在保护身体免受不良环境影响的情况下1。下真皮室由纤维弹性结缔组织组成。它由松质连接的胶原蛋白和纤维蛋白合成的弹力纤维素组成, 为机械屏障功能服务。真皮与上表皮分离, 基底叶片是由于两个皮肤间的恒定通信而产生的细胞外基质。与真皮相比, 表皮是一种鳞状上皮, 主要由角质形成, 可分化为四层。地层 basale包括未分化的基底角质形成细胞, 它不断地从皮肤祖细胞分层通过地层棘和层 granulosum进入角质层的阶段. 保护身体不受脱水和感染2。黑色素细胞在基底膜上排列, 通过树突状延伸与多个角质形成沟通。他们生产色素黑色素, 以保护皮肤组织免受紫外线辐射的不利影响, 如皮肤老化, 免疫抑制, 炎症, 并诱导非黑色素瘤皮肤癌。然而, 紫外线辐射对黑色素细胞转化为恶性黑色素瘤的贡献仍在辩论中3。
黑色素瘤的发展被区分成不同的肿瘤进展阶段, 并以一定的遗传、形态学和组织学变化为特征4。它们起源于从头或从先天或获得的痣, 由于局部增加的黑色素扩散造成良性的肿瘤。这种前体病变可转化为结构改良的肝硬化组织, 含有 atypic 细胞, 可能持续到第一个恶性阶段, 即径向生长阶段 (RGP)。这个早期的肿瘤进展阶段的特点是细胞在表皮内呈放射状增殖, 在乳突真皮内几乎没有局部侵入细胞。在随后的垂直生长阶段 (VGP) 黑色素瘤细胞已经显示了转移和侵入性表型通过打破基底层, 渗透到真皮的更深部分以及皮下组织5。最后, 转移性黑色素瘤 (MM) 代表最积极的进展阶段, 转移细胞系统地扩散到整个血液和淋巴体系侵入远端器官, 如肝, 肺和大脑的6。
迄今为止, 早期诊断和手术仍然是恶性黑色素瘤最有效的治疗方法。然而, 远距离转移患者的预后仍然特别差7, 因为经典化疗方案只授予很少的生存好处8,9。然而, 经过数十年的停滞, 最近在靶向治疗的进展大大改善了恶性黑色素瘤的预后。
失调两个主要的丝裂原活化通路, RAS-皇家空军-PI3K-AKT-PTEN 和信号通路, 是黑色素瘤进展的关键驱动因素, 特别是当组成性激活点突变的原始癌基因 BRAFV600 和NRAS 当前10。因此, 靶向激酶抑制剂的发明承诺治疗转移性黑色素瘤患者的疗效。在这一点上进行的许多临床试验对转移性黑色素瘤患者没有取得显著的好处。几乎所有的反应都是局部的, 有一个亚群的患者显示主要的抵抗。此外, 大多数患者在11、12中观察到了导致复发的二次耐药性的获得。
很明显, 单纯的变异状态分析不足以形成最有效的治疗策略。为了系统记录和分析单个癌细胞的反应能力, 需要新的快速、可靠的诊断工具。目前可获得的关于人类黑色素瘤的绝大多数数据都是从二维 (2D) 黑色素瘤细胞培养得到的。然而, 肿瘤细胞在3D 生长, 允许分化癌细胞亚群之间的细胞间串扰以及癌细胞与非转化的周围宿主组织之间的相互干扰。因此, 最好重构黑色素瘤开发的3D 环境, 用作临床前筛选模型13,14。

<table><tbody><tr><td>fetal serum albumin (FCS)</td><td>Thermo Fisher Scientific</td><td>10270106</td><td>add 10 % to cell culture medium</td></tr><tr><td>Trypsin-EDTA</td><td>Thermo Fisher Scientific</td><td>25300054</td><td>1X dilute in PBS</td></tr><tr><td>RPMI</td><td>Thermo Fisher Scientific</td><td>61870010</td><td>for cultivation of melanoma cell lines</td></tr><tr><td>DMEM&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>41965062</td><td>for cultivation of primary fibroblasts</td></tr><tr><td>Dispase</td><td>Gibco life technologies&amp;nbsp;</td><td>17105041</td><td>2U/ml, dissolve in PBS</td></tr><tr><td>Collagen type I</td><td>BD Biosciences</td><td>354249</td><td>usually from rat tail, concentration should be 3.5-4 mg/ml</td></tr><tr><td>24-well inserts Nunclon&amp;nbsp;</td><td>Nunc</td><td>140629</td><td>8 &amp;micro;m pore size; use standing, not hanging inserts!</td></tr><tr><td>cell strainer</td><td>BD Falcon</td><td>352360</td><td>yellow</td></tr><tr><td>Gentamycine</td><td>Thermo Fisher Scientific</td><td>15750060</td><td>dissolve in PBS</td></tr><tr><td>Keralife keratincyte medium</td><td>Cell Systems</td><td></td><td>do not add FCS or antibiotics</td></tr><tr><td>Collagenase</td><td>SERVA</td><td>17454</td><td>NB4 standard grade</td></tr><tr><td>juvenile human keratinocytes</td><td>Cell Systems</td><td>FC-0007</td><td>alternative to own preparation from juvenile foreskin</td></tr><tr><td>juvenile human fibroblasts</td><td>Cell Systems</td><td>FC-0001</td><td>alternative to own preparation from juvenile foreskin</td></tr><tr><td>DMEM&lt;br/&gt; [+] 4,5 g/l Glucose,&lt;br/&gt; [+] L- Glutamine,&lt;br/&gt; [-] L- Pyruvate</td><td>Gibco life technologies</td><td>41965</td><td>mix 1:1 with Ham&amp;acute;s F-12 for MM medium;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mix 3:1 with Ham&amp;acute;s F-12 for EGM medium</td></tr><tr><td>Ham&amp;acute;s F-12 [+] L-Glutamine</td><td>Gibco life technologies</td><td>&amp;nbsp;21765-029</td><td>add to DMEM for the generation of EGM and MM medium (see lane 15)</td></tr><tr><td>EGF&amp;nbsp;</td><td>Gibco life technologies</td><td>&amp;nbsp;13247-051</td><td>add 10 ng/ml only for the generation of EGM medium</td></tr><tr><td>Calcium chloride</td><td>Merck Chemicals</td><td>2381</td><td>add 1.9 mM for the generation of EGM and 3.8 mM for MM medium</td></tr><tr><td>Selenic acid</td><td>Alfa Aesar</td><td>18851</td><td>add 53 nM for the generation of EGM and MM medium</td></tr><tr><td>Insulin</td><td>Lilly</td><td>HI0219 Hum Pen 3ml 100 E.I./ml</td><td>add 5 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>E-0135</td><td>add 0.1 mM for the generation of EGM and MM medium</td></tr><tr><td>P-Ethanolamine</td><td>Sigma-Aldrich</td><td>P-0503</td><td>add 0.1 mM for the generation of EGM and MM medium</td></tr><tr><td>Holo-Transferrine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>T-0665</td><td>add 5 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Triiodthyronine</td><td>Sigma-Aldrich</td><td>T-6397</td><td>add 20 pM for the generation of EGM and MM medium</td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H-088816</td><td>add 0.4 &amp;micro;g/ml for the generation of EGM and MM medium</td></tr><tr><td>Progesterone</td><td>Sigma-Aldrich</td><td>P-8783</td><td>add 10 nM only for the generation of EGM medium</td></tr><tr><td>Hepes</td><td>Sigma-Aldrich</td><td>H-3784</td><td>add 15 mM for the generation of EGM and MM medium</td></tr><tr><td>Serine</td><td>Sigma-Aldrich</td><td>S-4311</td><td>add 1 mM for the generation of EGM and MM medium</td></tr><tr><td>Cholinchloride</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C-7017</td><td>add 0.64 mM for the generation of EGM and MM medium</td></tr><tr><td>dFCS</td><td>Sigma-Aldrich</td><td>F-0392 Lot 086K0361</td><td>add 2 % for the generation of EGM and MM medium</td></tr><tr><td>Adenine</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A-9795</td><td>add 0.18 mM for the generation of EGM and MM medium</td></tr><tr><td>L-Glutamine</td><td>PromoCell</td><td>C-42209</td><td>add 7.25 mM for the generation of EGM and MM medium</td></tr><tr><td>Strontiumchloride</td><td>Sigma-Aldrich</td><td>255521</td><td>add 1 mM for the generation of EGM and MM medium</td></tr><tr><td>anti cytokeratin 10 antibody</td><td>Dako</td><td>M7002</td><td>1:50</td></tr><tr><td>anti cytokeratin 14 antibody</td><td>Santa Cruz</td><td>sc-53253</td><td>1:200</td></tr><tr><td>&amp;nbsp;anti laminin 5 antibody</td><td>Santa Cruz</td><td>sc-32794</td><td>1:50</td></tr><tr><td>anti filaggrin antibody</td><td>Thermo Fisher Scientific</td><td>MA5-13440</td><td>1:50</td></tr><tr><td>anti involucrin antibody</td><td>Acris Antibodies</td><td>AM33368PU</td><td>1:50</td></tr><tr><td>secondary polyclonal goat anti-mouse IgG FITC labeled</td><td>LifeSpan Biosciences</td><td>LS-C153907</td><td>1:50</td></tr><tr><td>secondary polyclonal goat anti-mouse IgG Cy3 labeled</td><td>Jackson Immuno Research</td><td>115-165-003</td><td>1:1000</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>10236276001</td><td>1:100</td></tr></tbody></table>

a 3d organotypic melanoma spheroid skin model

黑色素细胞的恶性转化, 人类皮肤的色素干细胞, 导致黑素瘤的形成, 一种高度侵袭性的癌症, 增加转移潜能。最近, 单化疗继续改善由黑色素瘤特定的组合疗法与靶向激酶抑制剂。然而, 转移性黑色素瘤仍然是一种危及生命的疾病, 因为肿瘤表现出主要的抵抗力或发展抗新疗法, 从而恢复癌变能力。为了提高恶性黑色素瘤的治疗成功率, 有必要确定分子机制, 以抵抗常规治疗方法;但是, 它需要创新的蜂窝体外模型。在这里, 我们介绍一个体外三维 (3D) 脊髓黑色素瘤球形模型, 它可以刻画恶性黑色素瘤的体内体系结构, 并可能对肿瘤内和肿瘤宿主相互作用有新的洞察力。该模型纳入了定义的数量成熟和分化黑色素瘤球体在一个3D 人全皮肤重建模型, 由主要皮肤细胞组成。嵌体黑色素瘤球体的细胞组成和分化状况与皮肤黑色素瘤转移在体内相似。利用这种脊髓黑色素瘤球形模型作为药物筛选平台, 可以支持对选择的组合疗法的应答者的识别, 同时又能为非应答者节省不必要的治疗负担, 从而提高治疗干预。

成功治疗黑色素瘤转移可受肿瘤细胞之间的交叉交谈以及肿瘤和非转化宿主细胞的影响。开发肿瘤的脊髓模型的目的体外是提供合适的临床前测试系统, 概括3D 组织和复杂性的人黑色素瘤在体内.这允许研究脊髓环境中对肿瘤的治疗作用以及对周围的主要组织的不良影响。
为了开发最好的脊髓皮肤模型, 主要细胞的质量是至关重要的。它是有利的使用幼原成纤维细胞和角质形成细胞, 因为它们通常较少区分相比, 成人主要皮肤细胞。青少年皮肤细胞可以从幼年包皮分离, 如《议定书》 1-5 节所述, 但也可以从公司购买产前主要成纤维细胞和角质形成细胞。如果购买, 需要从不同的捐赠者的细胞, 以避免捐助者特定的结果,例如, 为药物敏感性。整个协议显示为图 3中的方案。
3D 全皮肤当量的质量控制需要免疫组化分析。用苏木精-伊红 (H & E) 染色的石蜡嵌段 (3 µm) 可以获得第一印象。详细分析表皮分化的质量和真皮与表皮之间基底层的形成需要用特异抗体对表皮分层标记进行免疫组化分析。这允许区分未分化的, 高度增生的细胞位于靠近基底膜和高度分化和角质细胞在角质层通过形成不同的表皮层之间的.如免疫组织学染色 ( 图 4) 所示, 表皮细胞的分化可以与正常皮肤相似: 主要是下部表皮层的未分化细胞 (地层basale和地层棘) 对角蛋白14着色阳性, 而从超基底层 (地层 granulosum和层角质层) 中分化的细胞则为角蛋白10和 involucrin 着色阳性。因此, filaggrin 染色只能观察到高度分化的细胞的角质层。最重要的是, 层粘连蛋白5染色显示, 产生了一个基底叶片, 以生理连接表皮与真皮室的人工皮肤重建。这证明了一个通信脊髓微环境已经产生的宿主黑色素瘤细胞或球体的生理和病理生理学分析。
为黑色素瘤药物筛选的目的, 单黑色素瘤细胞也可以集成到真皮的全皮肤等值, 以允许从头黑色素瘤巢形成18,19。因此, 黑色素瘤细胞与原发性成纤维细胞相结合, 比例为 5:1, 离心在 200 x g 为5分钟和悬浮在 GNL 之前与胶原蛋白混合。因此, 黑色素瘤细胞巢将自发地形成在真皮室。根据我们的经验, 只有转移生长阶段的细胞形成适当的巢, 与 RGP 或 VGP 的黑色素瘤细胞相比,15。这类模型的一个主要缺点是, 不能预测黑色素瘤巢的数量和大小, 并且在个体皮肤重建之间可能会有所不同, 独立于任何治疗。例如, 1000 个被植入真皮隔间的细胞可以获得10个巢, 由100个细胞或100个巢组成, 每个单元包括10个细胞 (图 5)。这些生物变量存在三缺陷: 第一, 黑色素瘤巢的数量和大小是不可预知的;其次,体内的转移通常大于黑色素瘤巢, 表现出更复杂的肿瘤内多样性;第三, 由于肿瘤巢模型的寿命有限, 及早开始治疗, 从而抑制肿瘤的生长, 而不是导致现有的肿瘤巢的回归。
为了克服这些局限性, 脊髓黑色素瘤可以产生球形皮肤模型。通过培养250个转移性黑色素瘤细胞14天20, 球体是由可行的黑色素瘤细胞构成的重现性, 呈现一个紧凑的结构, 最终直径约500µm 模仿非血管化肿瘤淋巴结, 微转移, 或毛细血管间微观区域的实体肿瘤21,22。一般情况下, 任何黑色素瘤细胞系均适用于通过悬吊滴法生成球体;然而, 从较先进的转移瘤阶段产生的细胞比从早期进展阶段的细胞系更坚实的球体,例如, RGP。
对于某些细胞来说, 适当的球体形成有利于提高悬滴培养基的粘度。这可以通过增加 10-50% 甲基纤维素到培养基中来实现。对于甲基纤维素溶液, 蒸压釜1.2 克甲基纤维素和100毫升玻璃瓶中的磁力搅拌棒。添加100毫升预热 (60 °c) 培养基和搅拌20分钟在室温下, 另 1-2 h 在4°c。离心机的库存解决方案为2小时在 5000 x g 和存储粘性上清到4°c, 直到使用。
充分的皮肤黑色素瘤球形模型的正确验证提供的事实是, 一个定义的数量的黑色素瘤球体可以-至少统计-被纳入真皮成纤维细胞/胶原 i 脚手架1天的皮肤模型建设, 使他们在表皮分化期间共同开发 25-27 天。球体的产量可以直接分析在播种后, 因为球体出现在透明真皮凝胶内的白色斑点, 可以看到没有任何放大设备 (图 2)。因此, 产生了一个3D 皮肤模型, 球体成熟的黑色素瘤, 它已经培养了体外总共约42天, 显示最高水平的肿瘤细胞分化15。
皮肤黑色素瘤球形模型的 H & E 染色揭示了黑色素瘤球体的组织学表现和细胞分布, 与非血管性人黑色素瘤皮肤转移瘤 (体内15 ) 非常相似 (图6).在这些条件下, 黑色素瘤细胞的两个亚群明显地是区分的: 一个外围增殖群和一个主要由萎缩、凋亡或坏死细胞组成的中心群, 形成所谓的 "坏死"中心。免疫组化生存和增殖亚群可以检测使用抗体对增殖标记 KI-67, 而坏死中心的细胞可以可视化的15。肿瘤细胞亚群的这种特殊分布是由球体大小 (≥500µm) 所保证的, 这是由于代谢废物积累的中心部分缺乏养分和氧气造成的。根据这里提供的协议将允许生成一个可靠的和可重现的脊髓人的全厚度皮肤模型与嵌入人类黑色素瘤球体, 模仿人类黑色素瘤皮肤转移。该模型的应用包括药物检测、毒素筛选、化妆品化合物或激光治疗对黑色素瘤的影响以及治疗。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57500/57500fig1.jpg"> 图 1: 3D 脊髓皮肤的培养, 在空气液界面中浸泡.在0天, 主角质形成细胞被播种在真皮室的顶部, 其中主要成纤维细胞嵌入到胶原 i 型基质中。3D. 用 EGM 浸泡7天的皮肤重建, 从插入壁分离, 开始收缩。在8天, 插入物被转移到6井和耕种在空气-液体接口允许表皮分层。<a href="/files/ftp_upload/57500/57500fig1large.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/57500/57500fig2.jpg"> 图 2: 将黑色素瘤球体嵌入真皮隔间会出现白色斑点.在准备3D 皮肤重建的真皮隔间时, 有一个定义的黑色素瘤球体可以添加到成纤维细胞胶原 i 型混合。一旦真皮凝胶已经解决, 黑色素瘤球体成为可见的白色斑点。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57500/57500fig3.jpg"> 图 3: 3D 脊髓皮肤模型构建方案.从皮肤标本中取出脂肪组织, 切成小块。孵化与酶溶液隔夜在4°c 促进表皮的分离从真皮。分离的原发成纤维细胞和角质细胞应单独培养, 并在 4-6 和 3-4 之间分别使用。随后, 3D 皮肤模型的生成可以按照协议中的描述进行。<a href="/files/ftp_upload/57500/57500fig3large.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/57500/57500fig4.jpg"> 图 4: 3D 脊髓皮肤重建显示出与正常人皮肤相似的分化水平。与正常人体皮肤 (B) 相比, 皮肤的石蜡切片 (A) 被染色为角质14的表达 (红色: λex 554 毫微米; λem 568 nm) 和 10, involucrin (绿色: λ前490 nm; λem 525 nm), filaggrin (绿色: λex 490 毫微米; λem 525 毫微米) 和层粘连蛋白 5 (绿色: λ前490 nm; λem 525 nm), 并用共聚焦荧光显微镜进行分析。细胞细胞核通过 DAPI 染色 (蓝色: λ前340 nm; λem 488 nm) 进行可视化。免疫组化检查的3D 全厚度皮肤等值显示适当的表皮分层形成明显的表皮层, 在正常的人皮肤上看到。当细胞从下部表皮层染色阳性的角蛋白 14, 更分化的细胞从上部基底层显示角蛋白10和 involucrin 染色。高度分化的细胞接近角质层表达 filaggrin。层粘连蛋白5染色显示, 基底叶片产生的生理连接表皮与真皮间隔 (最低的面板)。此数字已取自 Voersmann et 。15具有权限。<a href="/files/ftp_upload/57500/57500fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57500/57500fig5.jpg"> 图 5: 无法预测自发形成的黑色素瘤巢的数量和大小.从头黑色素瘤巢形成在真皮室的全皮肤当量可以通过混合一个定义的黑色素瘤细胞与原发成纤维蛋白, 以嵌入两种细胞类型的胶原 i 型基质。自发形成的黑色素瘤巢的数量和大小只能从成熟的3D 皮肤重建21天后进行分析。如两个样本 (A) 和 (B) 所描述的, 黑色素瘤巢的数量和大小可能因个体皮肤重建而异。因此, 很难验证这些模型并预测治疗效果。<a href="/files/ftp_upload/57500/57500fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57500/57500fig6.jpg"> 图 6: 黑色素瘤球体纳入皮肤等值概述人皮肤黑色素瘤转移的关键特征.H & E 染色石蜡切片的肿瘤球体嵌入到皮肤等值显示球体分享关键特征与非血管性人皮肤黑色素瘤转移在体内。细胞的两个亚群明显可见: 外周生物群和中心群主要由萎缩、凋亡或坏死细胞组成, 形成 "坏死" 中心。肿瘤细胞亚群的分布由球体大小保证。此数字已从 Voersmann et . 中进行了修改。15具有权限。<a href="/files/ftp_upload/57500/57500fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A 3D Organotypic Melanoma Spheroid Skin Model at JoVE.com

A 3D Organotypic Melanoma Spheroid Skin Model

在这里, 我们提出一个协议来生成一个3D 脊髓黑色素瘤球形皮肤模型, 概括的结构和多细胞复杂性的器官/肿瘤在体内, 但同时容纳系统实验干预。

3D 脊髓黑色素瘤球形皮肤模型

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

a-3d-organotypic-melanoma-spheroid-skin-model

Nanyang Technological University

Research

JoVE Journal

Cancer Research

15.6K 次观看.  TU-Dresden. 在这里, 我们提出一个协议来生成一个3D 脊髓黑色素瘤球形皮肤模型, 概括的结构和多细胞复杂性的器官/肿瘤在体内, 但同时容纳系统实验干预。

视频: A 3D Organotypic Melanoma Spheroid Skin Model

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

科研

生物工程

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

黑色素瘤患者衍生异种移植模型

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

癌症研究

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

<meta charset="utf8"></head><body>患者来源的黑色素瘤类器官：癌症免疫治疗的前沿

Unveiling Therapeutic Opportunities with Melanoma Patient-derived Organoid Models

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While traditional two- and three-dimensional models can offer insights into cancer development and progression, these lack crucial aspects that hinder a faithful mimic of native tumors. An alternative model that has gained a lot of attention is the patient-derived organoid. The development of these organoids recapitulates the complex intercellular communication, tumor microenvironment, and histoarchitecture of tumors. This paper describes the protocol for establishing melanoma patient-derived organoid (MPDO) models. To validate these models, we assessed the immune cell composition, including the expression levels of T-cell activation markers, to confirm the cellular heterogeneity of the organoids. Additionally, to describe the potential utility of MPDOs in cellular therapies, we evaluated the cytotoxic capabilities of treating the organoids with &#947;&#948; T-cells. In conclusion, the MPDO models offer promising avenues for understanding tumor complexity, validating therapeutic strategies, and potentially advancing personalized treatment.

<meta charset="utf8"></head><body>作者聚焦：使用患者来源的类器官重建黑色素瘤复杂性以进行免疫治疗评估

Here, we present a protocol to generate melanoma patient-derived organoids by culturing disassociated cell suspensions from fresh melanoma tissues. These organoids faithfully recapitulate patient-specific tumors in vitro, offering an innovative approach to exploring tumor immunosuppressive mechanisms, drug screening, drug resistance mechanisms, and cancer surveillance approaches.

揭示黑色素瘤患者来源的类器官模型的治疗机会

With the development of immunotherapy, there is an ongoing need to develop models that can recapitulate the tumor microenvironment of native tumors. While ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

医学

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

<meta charset="utf8"></head><body>高效的 3D 皮肤模型，用于灵活的研究应用

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

<meta charset="utf8"></head><body>作者聚焦：使用经济高效的 3D 细胞模型增强皮肤模型的多样性

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

3D 脊髓黑色素瘤球形皮肤模型

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用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

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揭示黑色素瘤患者来源的类器官模型的治疗机会

揭示黑色素瘤患者来源的类器官模型的治疗机会

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

大规模培育三维重建人类表皮

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

3D 脊髓黑色素瘤球形皮肤模型

摘要

探索更多视频

三维人体皮肤重建模型：一种工具来研究正常皮肤恶性黑色素瘤进展

黑色素瘤患者衍生异种移植模型

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

揭示黑色素瘤患者来源的类器官模型的治疗机会

揭示黑色素瘤患者来源的类器官模型的治疗机会

揭示黑色素瘤患者来源的类器官模型的治疗机会

单元位置的Imaging-和流式细胞仪为基础的分析和3D球体黑色素瘤细胞周期

大规模培育三维重建人类表皮

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养