我们感谢维罗妮卡 Schreiter 为她提供了出色的技术帮助和对已建立的冷冻保存技术和细胞培养模型的有益修改。此外, 我们还要感谢麻仁和 Swanhild Rodewald 在随后的分析中提供了出色的技术援助。

注意: 牛卵巢是从商业屠宰场获得的。屠宰副产品的收集不需要根据德国法律的道德批准。
1. 工作条件和准备
<ol><li>为了保证不孕, 在专门的细胞培养实验室中使用层流工作台, 进行所有培养基和组织准备以及所有的文化工作。</li>
	<li>准备1x 磷酸盐缓冲盐水 (PBS, pH 值 7.4) 补充100毫克青霉素, 0.1 镁/毫升链霉素和0.5 µg/µL 两性霉素为卵巢的运输。 注意: 在接受卵巢和隔离 GC 之间的最大持续时间是 2 h 在这个设置。</li>
</ol>2. 牛颗粒细胞的分离
<ol><li>
在 1x PBS 中多次冲洗卵巢 (含100微克青霉素, 0.1 毫克/毫升链霉素, 0.5 µg/µL 两性霉素), 在开始隔离手术前从表面清除任何血液。使用烧杯, 将卵巢放在里面, 用 1x pbs 填充, 然后再次丢弃 pbs。
	<ol>
<li>重复这个洗涤步骤 3–4x, 直到卵巢被清除任何剩余的血液。</li>
	</ol>
</li>
	<li>用70% 酒精浸泡过的实验室擦拭一个子房, 以尽量减少可能的污染物。</li>
	<li>用3毫升注射器和18克针, 通过穿刺小到中等大小的卵泡 (< 6 毫米, 用尺子测量) 吸入 GC, 并将滤泡液在一个50毫升离心管中池中。 注: 要滋润注射器, 请服用少量 1x PBS (含100毫克青霉素、0.1 毫升链霉素和0.5 µg/µL 两性霉素)。这有助于收集少量的滤泡液, 防止细胞黏附在注射器内。</li>
	<li>穿刺几个卵泡后, 用 1x PBS 冲洗注射器和针头, 进行中间清洗。</li>
</ol>3. 细胞的超低温保存
<ol><li>
使用台盼蓝染色, 数 hemocytometer 下的细胞。
	<ol>
<li>要计算活细胞的数量, 请准备一个1.5 µL 的0.25% 台盼蓝溶液的管。 注意: 活细胞将保持无瑕, 因为台盼蓝只能进入死细胞的细胞屏障, 然后出现蓝色。</li>
		<li>将颗粒细胞悬浮液中的15µL 加入到台盼蓝溶液中, 并轻轻搅拌。</li>
		<li>把混合物放在 hemocytometer 的两个房间里。计算每室一个大正方形的生活 (例如, 无瑕) 细胞数。 注意: 虽然可以看到一些血细胞, 但它们可以用非常小的尺寸来区分。</li>
		<li>根据一般准则计算两个腔室平方均值的活细胞数;活细胞的比例可以在卵巢之间变化, 但应超过 60%13。</li>
	</ol>
</li>
	<li>
从新鲜分离的颗粒细胞池中抽取样本作为进一步分析的参考。
	<ol>
<li>根据分离的 GC 的细胞计数, 预留适当数量的细胞作为参考样品为 RNA、脱氧核糖核酸或者蛋白质准备。 注意: 对于 RNA 的分离和随后对基因表达的评价, 1 x 105细胞是足够的, 而随后的其他分析可能需要不同的, 通常更高的细胞数。</li>
		<li>将一个或几个整除数的参考细胞在一个新鲜的管和离心机在室温和最大速度 (1.2万 x g), 以获得细胞颗粒。放弃上清。</li>
		<li>立即对液氮中的样品进行冲击冻结。将样品存放在摄氏-80 摄氏度。</li>
	</ol>
</li>
	<li>
按如下方式执行超低温保存。
	<ol>
<li>用80% 胎小牛血清 (FCS) 和20% 二甲基亚砜 (亚砜) 制备冷冻培养基。</li>
		<li>为了将 gc 轻轻地颗粒, 在室温和 500 x g上离心气相色谱为3分钟。</li>
		<li>仔细取出上清液, 轻轻地将100% 细胞 (如1 毫升) 并用重悬, 直到再也看不到细胞团簇。</li>
		<li>将所制备的冷冻介质的1体积 (例如1 毫升) 添加到细胞悬浮液中, 并将混合物转移到低温瓶中。 注: 冷冻保护介质的最终浓度将为 90% FCS 和10% 亚砜。</li>
		<li>将低温瓶放在专门的 (商用) 冷冻容器中, 防止快速结冰。样品的冷却速率不应超过-1 摄氏度/分钟. 将冷冻容器转移到-80 °c 冰箱中, 至少4小时。</li>
		<li>为了更长的贮存, 将低温瓶放在超低温容器中 (如液相氮气容器)。 注意: 该协议可以在这里停止, 因为冷冻保存允许更长的存储。</li>
	</ol>
</li>
</ol>4. 细胞培养用培养基和培养板的制备
<ol><li>
0.02% 胶原蛋白 R 的涂层细胞培养板。 注意: 胶原蛋白 R 被称为在培养中更好地附着 GC 的必要条件。<ol>
<li>制备0.02% 胶原 R 溶液, 纯化水适合细胞培养。</li>
		<li>在24井板 (= 2 厘米2/井) 中添加150µL, 并确保培养的整个表面都覆盖了胶原 R 溶液。</li>
		<li>让溶液干燥的盖子打开几个小时或一夜之间的层流罩。</li>
		<li>当培养皿完全干燥时, 用 UV 光照射它们, 以10–15最小的污染。</li>
		<li>如有必要, 将板材贮存在摄氏4摄氏度, 长达2月。</li>
	</ol>
</li>
	<li>在层流罩下为文化工作准备培养基和补充剂。为了成功回收 GC, 必须在解冻后立即处理。</li>
	<li>
准备以下媒体。
	<ol>
<li>补充α极小的基本培养基 (α-内存) 与2毫米 l-谷氨酰胺, 0.084% 碳酸氢钠, 0.1% 牛血清白蛋白 (BSA), 20 毫米 HEPES, 4 ng/毫升亚硒酸钠, 5 µg/毫升转铁蛋白, 10 ng/毫升胰岛素和1毫米非必需氨基酸。</li>
		<li>另外, 在培养基上加入100毫克青霉素和0.1 毫升链霉素。 注: 抗生素, 一旦加热, 是稳定的大约48小时。因此, 整除文化设置和计划媒体交换所需的媒体量。预热仅媒介整除数到37°c 在水浴。准备好的介质可以储存在4摄氏度, 不超过3月。</li>
		<li>在培养的牛气相色谱中诱导甾体活性, 另外辅以 20 ng/毫升卵泡刺激激素 (FSH)、50 IGF-1/ml R3 、2µM androstenedione。 注: 在文化或媒体交流开始前不久, 总是对这些组件进行补充。避免重复冻融循环的 FSH 和 IGF-1 的库存解决方案, 因为这可能会损害类固醇活动。</li>
	</ol>
</li>
</ol>5. 细胞培养工作
<ol><li>快速解冻细胞在水浴在37°c 为 3–4 min 并且转移细胞悬浮入37°c 预先预热的α-记忆 (没有激素补充)。对于离心, 建议最后的体积为10毫升。</li>
	<li>在室温和 500 x g处离心气相色谱3分钟。放弃上清。添加预热和补充的α记忆, 并并用重悬细胞仔细。</li>
	<li>将 GC 的种子密度为 1 x 105或 1 x 106个单元格, 并在最终体积为500µL. 包括每种条件下至少三个养殖井的技术复制。</li>
	<li>孵化细胞培养在一个孵化器在37°c 与 5% CO2为 8 d。</li>
	<li>每隔一天进行一次媒体交流。仅更换2/3 的培养基以减少压力, 并减轻细胞对新鲜介质的适应性。 注: 在培养几天后, GC 形成典型的成纤维细胞样表型, 并趋于聚集在一起。在高细胞密度的培养中, 比正常密度 GC 培养更频繁地发现更大的簇 (图 1, 左对右面板)。</li>
</ol>6. 培养颗粒细胞的后续分析
<ol><li>进行类固醇激素分析, 收集媒体和冻结在-20 摄氏度。使用适当的方法分析乏培养基中雌二醇和孕酮浓度 (如放射免疫 (RIA))14。</li>
	<li>对于不同靶分子 (RNA、DNA、蛋白质等) 的后续分析, 溶解细胞直接在培养基中与供应商推荐的合适的株溶藻剂结合。 注意: 对于大多数隔离过程, 有可能在这里停止协议, 因为裂解单元可以存储在-20 摄氏度, 长达1周。为了更长的贮存, 细胞应保持在摄氏-80 摄氏度。</li>
	<li>通过定量的实时聚合酶链反应 (qPCR) 技术, 确定转录丰度, 合成 cDNA 并测量特定转录量15。统计评价应包括至少三种不同细胞培养的复制, 其起源于不同的细胞制剂 (生物复制)。</li>
</ol>

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作者没有什么可透露的。

所提出的细胞培养模型为分析颗粒细胞的体外分化提供了工具。一些研究表明, 无血清培养是维持激素活性的先决条件, 在养殖牛 gc 或 gc 其他物种8,9。另外, 将培养皿涂上细胞外基质 (如胶原 R)13, 显著改善了细胞的附着。另一个重要特征是长时间的文化周期。最近, 已经证明, 一个长期的文化是必要的, 以获得足够的类固醇合成活动和平衡表达的颗粒细胞标识标记17。在隔离过程中, GC 似乎需要时间从物理压力中恢复。
培养基补充 FSH, IGF-1, 和 androstenedione 被称为诱导芳香化酶活性的培养气相色谱。特别是, 补充与 androstenedione 是绝对必要的, 因为 GC 需要一个前体雌二醇合成。这是以前发表的11,18 , 因此, 在本研究期间没有进一步调查。然而, 对于其他实验性的 IGF-1, 可能需要对 FSH、androstenedione 浓度的适应。
在这里描述的冷冻保存技术可以帮助改善组织培养实验, 使其更独立于不同的供应与卵巢。根据以往的测试, 冷冻保存不影响的 GC 表型或类固醇生产的文化。此外, 培养细胞中标记转录的丰度并没有显示出比较新鲜分离细胞与先前被冷冻保存16的样品相比有显著差异。
目前 GC 培养模型的关键参数是细胞电镀密度。如代表结果所示, 增加镀层密度引起了生理和分子特征的显著变化。一些基因是以特定的方式调节的, 类似于在体内4,19的 LH 刺激诱发的变化。在培养的牛气相色谱中, 细胞密度的增加可以驱动分化样的过程, 这一事实必须经过精心考虑, 以避免复制之间的冲突结果。因此, 与其他研究的矛盾结果可能归因于不同的细胞密度, 应该更密切地研究。
这里描述的文化模型显示对 LH 没有反应, 因为受体LHCGR的记录接近检测极限。因此, 模拟 LH 的浪涌在体内的情况下未能诱导分化13。然而, 该模型为研究雌二醇活性气相色谱在原代培养中的作用提供了有益的工具, 尤其是目前尚无功能性牛 gc 线。
在目前的 GC 培养模型中可以测试不同的治疗方案, 这有助于解开类固醇生产或 GC 分化的调控机制。此外, 可以单独分析参与发展过程的单个因素。因此, 这种区域性模型为许多不同的应用程序提供了基础。

成功的排卵和 luteinization 取决于细微调谐和精心编排的分子改变, 在不同的体细胞卵泡细胞类型。由于尚未充分了解这些发展进程的细节, 因此需要进一步澄清。在体内的方法是精心设计和昂贵的, 特别是未能解决在卵泡期间发生的特定分子机制。因此, 还原论的体外模型是需要的, 此外, 以提供洞察细胞和分子的细节。不同的研究描述在体外受精技术1,2,3的背景下, 整个卵泡的培养。由于研究人员对分化机制感兴趣, 许多研究侧重于滤泡 GC。这些细胞与卵子直接相关, 是雌激素产生的主要来源, 因此在整个卵泡和 luteinization4中起着至关重要的作用。
从不同的物种中开发出永生化的 GC 细胞系。然而, 他们中的大多数并没有显示出足够的类固醇激素生产5。到目前为止, 只有一条细胞线的牛 GC 已经建立了6, 但这条线失去了它的类固醇合成活动后, 几个通道7。因此, 由于类固醇激素分泌, 特别是雌二醇的生产是 GC 功能的一个基本特征, 所以最好在原细胞培养模型中研究这些方面。在以前的研究中, 它表明, 只有在无血清培养条件下, 才可以观察到相当多的雌二醇的产量8,9。此外, 补充雌二醇合成的前体是另一个先决条件, 因为 GC 未能表达出必要的酶, 可以将孕酮转化为 androstenedione10。此外, FSH 和 IGF-1在体外补充的协同作用揭示了芳香化酶的优化活性, 雌二醇合成的关键酵素11。在本议定书中, 还介绍了对 GC 文化模型产生重大影响的其他重要因素。特别是, 细胞的电镀密度对实验结果有巨大的影响12。此外, 还可以建立一种不显著干扰生物气相色谱生理的牛 gc 冷冻保存技术。该技术有助于改善细胞培养工作组织, 优化优选电镀密度。

<table><tbody><tr><td>PBS Instamed w/o Ca&lt;sup&gt;2+&lt;/sup&gt;, Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td>Merck-Millipore</td><td>L 182-05</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>penicillin / streptomycin (10,000 IU / 10,000 &amp;micro;g/mL)</td><td>Merck-Millipore</td><td>A 2213</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>amphotericin (250 &amp;micro;g/mL)</td><td>Merck-Millipore</td><td>A 2612</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>3 mL syringe (Omnifix Luer)</td><td>Braun</td><td>4616025V</td><td></td></tr><tr><td>18 G needle</td><td>Roth</td><td>C724.1</td><td></td></tr><tr><td>fetal calf serum</td><td>Merck-Millipore</td><td>S 0115</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>cryo-preservation vials (2 mL)</td><td>TPP Faust</td><td>TPP 89020</td><td></td></tr><tr><td>CoolCell LX cell freezing container</td><td>Corning</td><td>432004</td><td></td></tr><tr><td>culture dish, 24-well, flat bottom</td><td>TPP Faust</td><td>TPP 92424</td><td></td></tr><tr><td>collagen R (0.2 %)</td><td>Serva</td><td>47254</td><td></td></tr><tr><td>&amp;alpha;-MEM</td><td>Merck-Millipore</td><td>F 0915</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>L-Glutamin (200 mM)</td><td>Merck-Millipore</td><td>K 0282</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>sodium bicarbonate</td><td>Merck-Millipore</td><td>L 1713</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>BSA</td><td>Sigma</td><td>A3311</td><td></td></tr><tr><td>HEPES</td><td>Merck-Millipore</td><td>L 1603</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>sodium selenite</td><td>Sigma</td><td>S9133</td><td>dissolved in sterile water</td></tr><tr><td>transferrin</td><td>Sigma</td><td>T1283</td><td>dissolved in sterile water</td></tr><tr><td>insulin (10 mg/mL)</td><td>Sigma</td><td>I0516</td><td>dissolved in 1x PBS</td></tr><tr><td>NEA</td><td>Merck-Millipore</td><td>K 0293</td><td>originally purchased at Biochrom AG that is now part of the Merck-Millipore Company; the Cat.-No of Biochrom still exists</td></tr><tr><td>FSH</td><td>Sigma</td><td>F4021</td><td>we prefer to use a stock solution of 2 &amp;micro;g/mL, diluted in 0.9 % NaCl</td></tr><tr><td>LR3-IGF-1</td><td>Sigma</td><td>I1146</td><td>we use a stock solution of 2 &amp;micro;g/mL, dissolved in 10 mM HCl and 1x PBS with 1 mg/mL BSA</td></tr><tr><td>androstenedione</td><td>Sigma</td><td>46033</td><td>we use a stock solution of 10 mM, diluted in 100 % ethanol</td></tr></tbody></table>

a tissue culture model of estrogen-producing primary bovine granulosa cells

卵巢颗粒细胞 (GC) 是雌二醇合成的主要来源。由小鼠促黄体激素 (LH) 激增, 膜细胞, 特别是颗粒细胞层的诱导, 深刻改变其形态, 生理, 分子特征, 形成孕酮产生语料库负责维持妊娠的黄体。细胞培养模型是研究滤泡-黄体变换所涉及的基本调控机制的基本工具。本协议的重点是从中小卵泡 (< 6 毫米) 中提取牛气相色谱的分离过程和冷冻保存。利用这一技术, 可以获得几乎纯净的 GC 种群。冷冻保存程序极大地促进了细胞培养工作的时间管理, 独立于直接的初级组织 (卵巢) 供应。本协议描述一种无血清细胞培养模型, 模拟牛气相色谱的雌二醇活性状态。在整个《议定书》中讨论对成功的类固醇活性细胞培养必不可少的重要条件。实验表明, 增加细胞的镀层密度会诱发特定的反应, 如改变的基因表达谱和荷尔蒙产生。此外, 该模型为进一步研究 GC 分化和其它应用提供了依据。

GC 镀在 1 x 105细胞/以及显示一个典型的成纤维细胞样的外观, 因为它们形成一个细胞扩展可比成纤维细胞, 并倾向于建立集群 (图 1a和1c)。将电镀密度提高10倍至 1 x 106细胞/井没有改变形态学, 但可以观察到更多的细胞簇 (图 1b和1d, 箭头)。正如以前的出版物所显示的那样, 培养皿的胶原蛋白涂层在很大程度上改善了细胞13的附着。
与新鲜分离样品直接培养的细胞相比, 最初的超低温保存并没有显著改变其生理特性。如图 2所示, 当比较来自冷冻或新鲜隔离池的培养细胞时, 几个标记基因的转录丰度 (由 qPCR 测量) 没有差别。
图 3显示了在正常和高密度培养的牛 GC 中类固醇激素分析 (由 RIA 测量) 的代表性结果。与正常密度培养相比, 高密度培养中雌二醇浓度明显降低, 而孕酮产量趋于较高。
比较分析的几种基因 (由 qPCR 测量) 在高与正常密度的文化显示了一个显著的效果 (图 4)。CYP19A1, 编码的关键酶的雌二醇生物合成芳香化, 以及促性腺激素受体FSHR, 有明显下调。相反, 基因RGS2和VNN2显示了一个显著的上调节。这些结果清楚地表明, 通过增加细胞的电镀密度, 诱导细胞分化的具体过程。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58208/58208fig1.jpg"> 图 1: 在正常 (左面板) 和高细胞密度 (右面板) 培养的牛颗粒细胞.(a和c) 培养的细胞密度为 1 x 105细胞/以及显示典型的成纤维细胞样表型。(b和d)在高电镀密度 (1 x 106细胞/井) 培养的 GC 比正常密度下的细胞更频繁地形成大细胞簇 (箭头) (左面板)。刻度条 = 100 µm.<a href="https://www.jove.com/files/ftp_upload/58208/58208fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58208/58208fig2.jpg"> 图 2: 分离后和冷冻后直接培养的细胞的比较.细胞的培养密度为 1 x 105细胞每井。对几种标记转录物的基因表达进行了评价, 并揭示了未初始保存的培养细胞之间的差异。箱线图显示n = 3 的中值。两尾学生的t检验, 包括p值。这个数字是从 Baufeld 和 Vanselow16复制的。<a href="https://www.jove.com/files/ftp_upload/58208/58208fig2large.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/58208/58208fig3.jpg"> 图 3: 不同镀层密度培养的颗粒细胞甾体激素浓度.在高细胞密度下培养气相色谱时, 雌二醇 (E2) 浓度显著降低, 而孕酮 (P4) 浓度只趋于较高。对不同细胞数的 DNA 含量进行了校正, 使其激素浓度得以正常化。显示了n = 3 的平均值和 SEM。p > 0.05, 两尾学生的t检验。这个数字是从 Baufeld等复制的。15.<a href="https://www.jove.com/files/ftp_upload/58208/58208fig3large.jpg" target="_blank">请点击这里查看这个数字的更大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58208/58208fig4.jpg"> 图 4: 在正常与高镀层密度下培养的颗粒细胞中功能性关键转录的丰度.在无血清条件下培养的牛 GC 8 d 揭示了几种选定标记基因的特定调控。箱线图显示n = 3 个体复制的中值。p < 0.05, 两尾学生的t检验。<a href="https://www.jove.com/files/ftp_upload/58208/58208fig4large.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 Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells at JoVE.com

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

本文介绍了牛颗粒细胞在无血清条件下的长期培养模型。该模型允许研究者研究不同电镀密度对雌激素产生的牛颗粒细胞特性的影响。

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

a-tissue-culture-model-estrogen-producing-primary-bovine-granulosa

Research

JoVE Journal

Biology

9.1K 次观看.  Leibniz Institute for Farm Animal Biology (FBN). 本文介绍了牛颗粒细胞在无血清条件下的长期培养模型。该模型允许研究者研究不同电镀密度对雌激素产生的牛颗粒细胞特性的影响。

视频: A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

异型三维<em在体外</em&gt;卵巢癌的发生和发展过程中的间质 - 上皮细胞相互作用模型

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

科研

医学

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, regulation occurs at the hypothalamic-pituitary-gonadal axis level and on developing follicles in the ovary. Despite active studies, follicle development mechanisms are not clear because of the several steps involved from the dormant primordial follicle activation to ovulation, and because of the regulation complexity that differs at each follicular stage. To investigate the mechanisms of follicle development, and the dynamics of follicles throughout the estrus cycle, we developed a mouse ovarian tissue culture model that can be used to observe follicle development using a microscope. Systematic follicle development, periodical ovulation, and follicle atresia can all be reproduced in the cultured ovary model, and the culture conditions can be experimentally modulated. Here, we demonstrate the usefulness of this method in the study of the regulatory mechanisms of follicle development and other ovarian phenomena.

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

卵巢组织培养对小鼠卵巢现象的可视化

Mammalian females periodically ovulate an almost constant number of oocytes during each estrus cycle. To sustain such regularity and periodicity, ...

发育生物学

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

人类乳腺上皮细胞的激素反应3D文化模式

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

生物工程

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

工程三维上皮组织细胞外基质中嵌入

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

多细胞人类原发性子宫内膜器官的生成

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

In Vitro Culture Strategy for Oocytes from Early Antral Follicle in Cattle

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that during the reproductive life span only about 1% of the oocytes in an ovary mature and ovulate, several techniques have been developed to increase the exploitation of the ovarian reserve to the growing population of non-ovulatory follicles. Such technologies have allowed interventions of fertility preservation, selection programs in livestock, and conservation of endangered species. However, the vast potential of the ovarian reserve is still largely unexploited. In cows, for instance, some attempts have been made to support in vitro culture of oocytes at specific developmental stages, but efficient and reliable protocols have not yet been developed. Here we describe a culture system that reproduce the physiological conditions of the corresponding follicular stage, defined to develop in vitro growing oocytes collected from bovine early antral follicles to the fully-grown stage, corresponding to the medium antral follicle in vivo. A combination of hormones and a phosphodiesterase 3 inhibitor was used to prevent untimely meiotic resumption and to guide oocyte&#39;s differentiation.

We describe the procedures for isolation of growing oocytes from ovarian follicles at early stages of development, as well as the setup of an in vitro culture system which can support the growth and differentiation up to the fully-grown stage. &#160;

牛早期蚁卵泡卵母细胞的体外培养策略

The limited reserve of mature, fertilizable oocytes represents a major barrier for the success of assisted reproduction in mammals. Considering that ...

从 3D 牛回肠类结肠培养物中建立 2D 单层

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

作者聚焦：用于研究宿主-病原体相互作用的高级类肠模型 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

雌激素原代牛颗粒细胞的组织培养模型

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异型三维

卵巢组织培养对小鼠卵巢现象的可视化

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

人类乳腺上皮细胞的激素反应3D文化模式

工程三维上皮组织细胞外基质中嵌入

多细胞人类原发性子宫内膜器官的生成

牛早期蚁卵泡卵母细胞的体外培养策略

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

生成用于转化生物医学研究的牛原代肠源二维单层培养系统

生成用于转化生物医学研究的牛原代肠源二维单层培养系统