这项工作是由美国国立卫生研究院（U54HD41857和PL1EB008542，P30内Oncofertility联盟路线图资助的生物材料的核心）。

1。卵泡的分离动物实验是由美国国立卫生指南研究院提出的指导方针和规定，对实验动物的照顾和使用和建立的机构使用动物，并在西北大学保健协议的规定执行。 为了达到最佳效果，所有解剖进行了pH值控制在L15的媒体在37 ° C加热温度控制的阶段，并在一个洁净工作台，空气中的CO 2水平，以减少细菌污染。剥离媒体（DM）的准备与L15媒体50 IU / mL青霉素和50μg/ mL链霉素和1％FBS的补充。维护媒体（MM）是准备αMEM媒体50 IU / mL青霉素和50μg/ mL链霉素和1％FBS的补充。 <ol><li>从16日龄小鼠转移到一个新的35毫米的Petri盘1-2毫升含0.1％胶原酶和0.1％的DNA酶MM新鲜解剖卵巢。在37 ° C和5％的CO 2孵育15分钟内的孵化器。 </li><li>孵化后，在DM执行几个洗涤步骤来删除胶原酶，然后转移到一个35毫米的Petri菜用新鲜DM。隔离轻轻弹（或切割）卵泡，通过使用两个28 5 / 8号一次性注射器针头从整个卵巢的卵泡从卵巢。删除尽可能多的基质，而不损害卵泡的完整性。解剖出20-40％卵巢的次级卵泡（130 - 150mm的）。这些卵泡通常有2-3层的体细胞。 </li><li>加入1毫升的MM到60毫米的试管婴儿（体外受精）培养皿中，3毫升MM外圈中央。与体外受精菜，简单地冲洗外圈吸管传输完整的毛囊，然后有选择地转移到中央的井（见 1.4 ）。商店在孵化器这个试管婴儿的菜。 </li><li>收集所有的卵泡后，选择步骤与5 - 8X放大解剖显微镜下进行。健康的毛囊有以下形态特征: <ul><li> 2-3体细胞层</li><li> 130-150毫米</li><li>卵母细胞和体细胞之间没有分离</li><li>完整的和全面的卵母细胞 进行封装试管婴儿菜中心转移到健康的毛囊。 </li></ul></li></ol> 2。卵泡封装，方法1 - "滴法" <ol><li>添加到中间的试管婴儿菜40毫米氯化钙2 1毫升50 IU / mL的凝血酶的TBS。在冰上解冻，并保持纤维蛋白原的股票（50毫克/毫升的TBS）。使纤维蛋白原至室温前使用。 </li><li>准备的纤维蛋白原/海藻酸钠溶液混合1X PBS，PBS液和50毫克/毫升纤维蛋白原溶液1.7 mL无菌的离心管中的2:1:1的比例的0.5％海藻酸钠。避免引入到解决方案中的气泡。轻轻涡旋和自旋向下。该解决方案出现略微浑浊，应立即使用前的准备。 </li><li>纤维蛋白原/海藻酸钠解决方案在体外受精菜外圈放置两个水滴:90μL封装和10μL的洗涤液滴液滴。为了减少蒸发，关闭加热阶段，在解剖显微镜。转让10-15卵泡到10μL与文化传媒（&lt;5μL）使用200微米的微管尖端，并迅速混合少量液滴。 90μL的解决方案少量（&lt;5μL）从第一个下拉，和混合液滴转移到所有的卵泡。 </li><li>同时吸一个卵泡和5μL纤维蛋白原/海藻酸钠使用10μL枪头，并释放到凝血酶/ CA 2 +解决方案在体外受精培养皿的解决方案。重复此步骤，直到所有的毛囊都被封装。 </li><li>交联珠凝血酶/ CA 2 +溶液中5-7分钟。珠将成为纤维蛋白胶暗。珠转移到培养皿中含有的MM，开始与较深的珠。孵育15-30分钟inthe孵化器冲洗掉剩余的凝血酶的菜。 </li><li> 96孔培养板含有100μL卵泡的生长介质，图像立即传送到珠。生长介质（GM）是10 MIU / mL的重组FSH，3 mg / mL的牛血清白蛋白补充αMEM媒体的准备，1毫克/毫升牛胎球蛋白，5微克/毫升的胰岛素，5微克/毫升转，和5毫微克/毫升的硒。 </li></ol> 3。卵泡封装，方法2 - "封口膜法" <ol><li>准备在1.7 mL无菌的离心管中按1:1的比例混合0.5％的海藻酸钠溶液和50 mg / mL的纤维蛋白原溶液的纤维蛋白原/海藻酸钠溶液。 </li><li>移液器7.5μL滴的纤维蛋白原/海藻酸钠混合到一个3毫米的垫片的封口膜镀膜玻璃幻灯片。转移到一个极小值每下降1卵泡媒体的升量。 </li><li>凝血酶/ CA 2 +溶液中加入7.5μL，每下降。混合是不必要的，因为凝胶形式几乎在瞬间。 </li><li>第二封口膜镀膜玻璃幻灯片的覆盖胶，放幻灯片，倒挂在100毫米的培养皿中，并转移为5分钟的孵化器。 </li><li>转移到一个Petri菜的MM的珠子，然后进入以及前面所述的文化。如果珠片粘在一起，这是由于交联之间的珠子，他们可以用血管钳轻轻分离。 </li></ol> 4。卵泡成像和媒体变化<ol><li>每2天，卵泡培养成像，使用光学显微镜和卵泡直径测量软件程序ImageJ。 </li><li>每2天，生长介质（50μL）的一半是新鲜，预平衡生长介质取代。 </li></ol> 5。从3D矩阵和卵母细胞体外成熟（IVM）的卵泡恢复<ol><li>经过8天的文化，水凝胶出现明确的完成降解纤维蛋白的​​组成部分，和毛囊扩大到直径300-400微米。其余的海藻酸钠海藻酸钠 - 裂解酶，植物来源的酶，特别是降低海藻酸钠，并不会影响动物细胞的退化。 </li><li>生长介质，含珠的水井中删除，加入100毫升10 IU /毫升的褐藻胶裂解酶在αMEM。离开25-30分钟的孵化器板。 </li><li>取出用钝尖底溶解珠卵泡。传输第一个试管婴儿马克盘的外圈，然后到中心好，还含有DM洗。 </li><li>洗涤后，卵泡转移到外，然后内心环试管婴儿菜含有成熟的媒体在体外成熟媒体（IVM）的αMEM，10％FCS，1.5 IU /毫升的hCG组成，5毫微克/毫升表皮生长因子（EGF） </li><li>转移到CO 2培养箱为15-16小时。 检查卵泡卵丘细胞扩展。要删除从卵母细胞的卵丘细胞，添加透明质酸酶的解决方案，以终浓度为0.1 mg / mL和孵化器孵育2-3分钟。使用75毫米的微管尖端剪切从卵母细胞的卵丘细胞吹打向上和向下几次。确定一盏灯或解剖显微镜下的卵母细胞成熟阶段。可能的阶段，从最少到最成熟的，是: <ul><li>堕落。卵母细胞分裂成两个或更多件。 </li><li>生发泡（GV）的阶段。没有恢复的卵母细胞减数分裂绒毛膜促性腺激素的曝光，这是显而易见的，核的卵母细胞（GV）的继续存在。 </li><li>生发泡破裂（GVBD）。核膜是缺席的卵母细胞，但目前没有极体。因此，卵母细胞在减数分裂我仍然是。 </li><li>中期- II被捕卵母细胞（MII）。卵母细胞恢复减数分裂，并正处于中期- II被捕。极体，应该是一盏灯或解剖镜下可见。除非在以后的实验受精的卵母细胞会留在信息产业部。 </li></ul></li></ol> 6。代表性的成果: 我们描述了在FA - IPN的卵泡在体外培养（图1）一个新型的封装方法。卵巢滤泡组成一个由数层的体细胞包围的卵母细胞。多个蜂窝舱室之间的沟通是健康的卵泡发育和卵母细胞成熟必不可少的。卵泡封装在一个三维凝胶支持毛囊扩张，同时保持毛囊 9,11,12的架构。由于毛囊的发展，其体积膨胀指数和封装的生物材料应允许没有抑制压缩力的发展扩张。 FA - IPN是两个天然材料，其中纤维素是由纤维蛋白溶酶激活蛋白水解降解毛囊和海藻酸钠生物惰性13（图2）建立了一个网络。在卵泡的生长，纤维蛋白降解开始在当地附近的毛囊，并继续进行，直到纤维蛋白凝胶清除。非降解的海藻酸钠的组成部分，它在整个养殖期保持完整，支持三维结构的凝胶。 在中学阶段的发展（直径150-180微米）和卵泡分离扩大到400微米胃窦FA - IPN凝胶的发展阶段。 HCG刺激，可以接受培养的卵泡卵丘细胞扩展，卵母细胞能恢复减数分裂和受精进步中期第二。这些结果表明，封装方法和封装材料允许在体外 （图3）卵泡的文化和成功的成熟。 纤维蛋白降解周围encapsulated卵泡文化的第一天开始，6​​天完成。抑肽酶，可溶性纤维蛋白溶酶抑制剂，可用于改变纤维蛋白降解和封装材料（图4），以延长机械梯度。如果毛囊培养与0.01 TIU / mL的抑肽酶，纤维蛋白降解为第4天，慢。尽管如此，毛囊仍然可以发展到胃窦阶段的卵母细胞仍然主管恢复减数分裂中期二后抑肽酶removed.A抑肽酶高浓度（0.1 TIU /毫升），显着抑制纤维蛋白降解，矩阵刚度，防止毛囊扩张和体细胞观察到矩阵的入侵。 <img src="/files/ftp_upload/2695/2695fig1.jpg" alt="图1" /> 图1。包括由一个或多个图层支持卵母细胞发育的体细胞，包围了位于市中心的卵母细胞卵泡卵泡发育的流程图。卵泡从中学排卵胃窦阶段的发展，卵母细胞周围的体细胞增殖和分化，卵母细胞的大小增加。 体外成熟（IVM）的卵泡文化的最后一步，当相邻的体细胞卵母细胞，称为卵丘细胞，扩大HCG刺激后，卵母细胞恢复减数分裂和发展到一个中期II（MII）的阶段。 <img src="/files/ftp_upload/2695/2695fig2a.jpg" alt="图2a" /> 图2a。海藻酸钠和3D卵泡体外培养的纤维蛋白-海藻酸钠（一）海藻酸钠是一种天然的生物材料，是由于其温和的凝胶和生化特性，如网目尺寸，可控的刚性和生物惰性，在体外卵泡文化。海藻酸钠是一种α- L - guluronic酸（G）和β- D - mannuronic酸（M）的线性多糖聚合物。重复摹单体的地区，被称为"G块"，交联，形成水凝胶中的二价钙离子的存在。 <img src="/files/ftp_upload/2695/2695fig2b.jpg" alt="图2b" /> 图2b（BI）小型封装卵泡经验，在文化开始在海藻酸钠低压缩力。然而，由于毛囊扩大珠流离失所量不断增加，这在更大的压缩力相反的方向（B - II）在毛囊扩张的结果。 <img src="/files/ftp_upload/2695/2695fig2c.jpg" alt="图2c" /> 图2c。个别聚合物链中的纤维蛋白，海藻酸钠溶液是完全纠缠交联前。 FA - IPN的两个组成部分，开始交叉链接，因为它们是立即暴露凝血酶和钙的混合物。 <img src="/files/ftp_upload/2695/2695fig3a.jpg" alt="图3a" /> 图3a。卵泡的隔离和封装在一个FA - IPN的次级卵泡的流程图是孤立的，从16日龄小鼠（ AI）。生殖器官解剖（A - II），并分离出的毛囊被转移到一碟与维护媒体（A - III）。 <img src="/files/ftp_upload/2695/2695fig3b.jpg" alt="图3b" /> 图3b为卵泡封装中的纤维蛋白原海藻酸钠溶液滴法（B:I - IV） 。两滴纤维蛋白原海藻酸钠溶液，冲洗滴（10μL）和封装下降（90μL）吸管入菜（BI）。未来3-5卵泡转移到漂洗下降（B - II）。冲洗和媒体去除后，卵泡转移到封装下降（B - III）。每个毛囊吸入5μL纤维蛋白原 - 海藻酸钠10μL枪头的解决方案，然后成凝血酶/钙溶液（B - IV）驱逐出境。 <img src="/files/ftp_upload/2695/2695fig3c.jpg" alt="图3c" /> 图3c珠交联5分钟。封口膜封装毛囊中的纤维蛋白原的海藻酸钠溶液的方法（C:I - III）。纤维蛋白原的海藻酸钠溶液（7.5μL）吸管封口膜镀膜玻璃幻灯片和毛囊转入单独漂洗后每下降（CI，II）。凝血酶溶液（7.5μL）被添加到每下降（C - III）。 <img src="/files/ftp_upload/2695/2695fig3d_e.jpg" alt="图3D - E" /> 图3D - E.滴都覆盖着第二次封口膜镀膜玻璃幻灯片的FA - IPN是在孵化器的交联5分钟。 （四）转移到封装的卵泡生长介质，在96孔板。 （e）在一个封装中的FA - IPN（白色箭头）卵泡的形象。 <img src="/files/ftp_upload/2695/2695fig4.jpg" alt="图4" /> 图4。纤维蛋白降解日益增长的FOLLICLE。在文化时期，证明这是一个清晰的圆圈周围的卵泡，卵泡退化的FA - IPN的纤维蛋白组成。卵泡的3D架构，支持由剩余的海藻酸钠。在当天0（D0）的纤维蛋白仍然是完整的，和周围的毛囊基质是阴天文化术后第1天（D1），出现毛囊周围的明确环（白色箭头）和纤维蛋白降解方面继续呈放射状扩大天2（D2）和4天的文化（D4）。 <img src="/files/ftp_upload/2695/2695fig5.jpg" alt="图5" /> 图5。卵泡发育的图片 。代表图像ofsecondary卵泡在FA - IPN的增长0天（一），4（B）6（C）和8（四）文化。 8天之后， 在体外成熟的卵泡，并造成信息产业部期卵母细胞（E极体是用黑色箭头所示） 。 <img src="/files/ftp_upload/2695/2695fig6.jpg" alt="图6" /> 图6。纤维蛋白降解是由抑肽酶抑制毛囊生长在第2天，4，10和12中的第一行所示。抑肽酶浓度0.01 TIU /毫升（第二行）和0.1 TIU / mL的是添加到0，2，4天的培养基。只有卵泡0.01 TIU / mL的抑肽酶退化抑肽酶搬迁后的纤维蛋白的​​文化和达到胃窦阶段。培养卵泡在0.1 TIU / mL的抑肽酶没有在FA - IPN的增长。 <table border="1"><tbody><tr><td> 缩写 </td><td> 全名 </td></tr><tr><td> 二氧化碳培养箱</td><td> 37 °彗星孵化器与5％的CO 2 </td></tr><tr><td>中国奥委会</td><td>卵丘卵母细胞复合体</td></tr><tr><td> 3D </td><td> 三维 </td></tr><tr><td>马克</td><td> 夹层媒体 </td></tr><tr><td>表皮生长因子</td><td> 表皮生长因子 </td></tr><tr><td> FA - IPN </td><td> 海藻酸钠纤维蛋白互穿网络 </td></tr><tr><td>胎牛血清</td><td> 胎牛血清 </td></tr><tr><td>通用汽车公司</td><td>生长介质</td></tr><tr><td>货车</td><td>生发泡</td></tr><tr><td>绒毛膜促性腺激素</td><td> 人绒毛膜促性腺激素 </td></tr><tr><td>及其</td><td> 胰岛素转硒 </td></tr><tr><td>试管婴儿菜</td><td>中心以及60毫米体外受精盘</td></tr><tr><td> MM </td><td> 维护媒体 </td></tr><tr><td> MII阶段卵母细胞</td><td>中期第二阶段卵母细胞</td></tr><tr><td> RFSH </td><td>重组卵泡刺激素</td></tr><tr><td>日本TBS电视台</td><td> Tris缓冲生理盐水</td></tr><tr><td>调景</td><td>胰蛋白酶抑制单位</td></tr></tbody></table> 表1。缩写

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+Follicle+Growth+and+Oocyte+Maturation+by+Culture+in+Alginate+Hydrogel+Following+Cryopreservation+of+the+Ovary+or+Individual+Follicles.">Secondary Follicle Growth and Oocyte Maturation by Culture in Alginate Hydrogel Following Cryopreservation of the Ovary or Individual Follicles.</a> Biotechnology and Bioengineering. 103, 378-386 (2009).</li><li>Smitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+achievements+and+future+research+directions+in+ovarian+tissue+culture,+in+vitro+follicle+development+and+transplantation:+implications+for+fertility+preservation.">Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.</a> Human Reproduction Update. 16, 395-414 (2010).</li><li>Shikanov, A., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpenetrating+fibrin-alginate+matrices+for+in+vitro+ovarian+follicle+development.">Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development.</a> Biomaterials. 30, 5476-5485 (2009).</li><li>West, E. R., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+alginate+hydrogels+and+their+effects+on+in+vitro+follicle+development.">Physical properties of alginate hydrogels and their effects on in vitro follicle development.</a> Biomaterials. 28, 4439-4448 (2007).</li><li>Kreeger, P. K., Fernandes, N. N., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mouse+follicle+development+by+follicle-stimulating+hormone+in+a+three-dimensional+in+vitro+culture+system+is+dependent+on+follicle+stage+and+dose.">Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose.</a> Biology of Reproduction. 73, 942-950 (2005).</li><li>Pangas, S. A., Saudye, H., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approach+for+the+three-dimensional+culture+of+granulosa+cell-oocyte+complexes.">Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.</a> Tissue Engineering. 9, 1013-1021 (2003).</li><li>Xu, M., West, E., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+stage-specific+permissive+in+vitro+culture+environment+for+follicle+growth+and+oocyte+development.">Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development.</a> Biology of Reproduction. 75, 916-923 (2006).</li><li>Xu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+Three-Dimensional+Culture+Supports+Development+of+Nonhuman+Primate+Secondary+Follicles.">Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles.</a> Biology of Reproduction. 81, 587-594 (2009).</li><li>West, E. R., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+follicle+micro+environment.">Engineering the follicle micro environment.</a> Seminars in Reproductive Medicine. 25, 287-299 (2007).</li><li>Xu, M., Kreeger, P. K., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+follicles+produce+live,+fertile+offspring.">Tissue-engineered follicles produce live, fertile offspring.</a> Tissue Engineering. 12, 2739-2746 (2006).</li><li>Ebisch, I. M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+role+of+the+plasminogen+activator+system+and+vascular+endothelial+growth+factor+in+subfertility.">Review of the role of the plasminogen activator system and vascular endothelial growth factor in subfertility.</a> Fertility and Sterility. 90, 2340-2350 (2008).</li></ol>

所提出的卵泡在FA - IPN的封装方法允许在体外的3D环境中的卵泡文化。一个FA - IPN是一个动态的，细胞的反应矩阵最初的机械性能相结合的纤维蛋白和海藻酸钠决定。在文化，封装毛囊激活蛋白酶降解的IPN只有一个组成部分，纤维蛋白，它在一个逐渐下降的凝胶的刚性，完全是由文化的剩余海藻酸钠贡献的结果。与FA - IPN获得的动态力学性能与自然环境的发展毛囊相一致，并已提出改善率比较海藻酸钠单独的卵母细胞减数分裂成熟做出了贡献。 FA - IPN示范轻度和快速凝胶，与每个系统交叉连接，由一个独立的机制的组成部分。我们已经描述了其他地方5，可以通过纤维蛋白原和凝血酶浓度控制凝胶形成的速度。由抑肽酶抑制纤维蛋白水解，降解率可调节。小鼠次级卵泡通常为8-12天，需要较长的文化时期的其他物种的卵泡培养。因此，延迟纤维蛋白降解由抑肽酶可能提供一个较长的文化扩展的动态环境。 可以应用于其他系统，如封装和文化的微观组织或胚体细胞与细胞接触，其中还可以保留的总额，可以部分降低矩阵，并创建一个扩展的空间，描述的封装方法。的FA - IPN封装方法的结论，提出了一个充满活力，细胞反应的机械性能和可控降解率的无菌水凝胶文化系统。

<table><tbody><tr><td>Fetuin</td><td>Sigma-Aldrich</td><td>F3385</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>10082-139</td><td></td></tr><tr><td>Aprotinin</td><td>Roche Group</td><td>10236624001</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Wako Pure Chemical Industries, Ltd.</td><td>039-00475</td><td>40 mM</td></tr><tr><td>EGF</td><td>Sigma-Aldrich</td><td>A412</td><td></td></tr><tr><td>rFSH</td><td>National Institute of Diabetes and Digestive and Kidney Diseases</td><td></td><td></td></tr><tr><td>hCG</td><td>Sigma-Aldrich</td><td>CG-5</td><td></td></tr><tr><td>Hyaluronidase</td><td>Sigma-Aldrich</td><td>A1603</td><td></td></tr><tr><td>ITS</td><td>Sigma-Aldrich</td><td>I1884-1VL</td><td></td></tr><tr><td>L-15</td><td>GIBCO, by Life Technologies</td><td>11415</td><td></td></tr><tr><td>&amp;alpha;MEM+Gluta MAX</td><td>GIBCO, by Life Technologies</td><td>32561</td><td></td></tr><tr><td>Pen-Strep</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>TBS</td><td>Pierce, Thermo Scientific</td><td>28379</td><td></td></tr><tr><td>Tisseel Fibrin kit</td><td>Baxter Internationl Inc.</td><td>921030</td><td></td></tr><tr><td>Sodium Alginate</td><td>FMC BioPolymers</td><td>LF200DL</td><td>Mw 418kDa</td></tr></tbody></table>

a method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system

卵泡是卵巢分泌性激素的功能单元，并支持卵母细胞成熟 。体外卵泡技术提供了一个工具，以调查的基本生物学模型卵泡发育，并进一步被作为一种技术，保留生育开发诊所1-4。我们在体外培养系统，采用水凝胶以模仿卵巢原生环境，保持3D滤泡结构，细胞间的相互作用和旁分泌的信号，直接卵泡发育5。在此之前，卵泡培养成功的海藻酸钠，一种惰性藻类衍生的多糖，经过与钙离子凝胶6-8。在浓度为0.25％W / V形成的海藻酸钠水凝胶是最宽松的卵泡文化，并保留了最高的发展能力9。海藻酸钠水凝胶不降解，从而增加卵泡直径结果在压缩力的毛囊，会影响卵泡发育10。我们后来发展为基础的文化系统，纤维蛋白 - 海藻酸钠互穿网络（FA - IPN），其中纤维蛋白和褐藻胶的混合物凝胶同时。这种组合提供了一个动态的力学环境，因为这两个组件有助于矩阵刚性最初，然而，日益增长的卵泡分泌的蛋白酶降解，只留下海藻酸钠提供支持的矩阵中的纤维蛋白。使用IPN，海藻酸钠含量可减少0.25％以下，这是不符合海藻酸钠仅5。因此，随着卵泡的扩大，它会遇到由于减少固体含量减少压缩力。在这里，我们描述了一种封装方法，并在在FA - IPN的卵巢卵泡体外培养体系。动态力学环境模仿自然卵巢的环境中，小卵泡驻留在一个刚性的皮质，并移动到一个更宽松的髓质，因为它们增加在大小11。为了支持大于10的6倍量的增加，人类毛囊通常在体内进行降解成分，可能是特别为临床翻译的关键。

Watch this Scientific Journal Video about A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System at JoVE.com

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

描述一个卵巢卵泡封装在三维纤维蛋白 - 海藻酸钠的互穿网络的新方法。此系统结合蛋白水解降解结构的支持，以支持发展不成熟的卵泡产生成熟的卵母细胞。这种方法可应用于文化细胞聚集，保持不限制扩张的细胞 - 细胞接触。

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

a-method-for-ovarian-follicle-encapsulation-culture-proteolytically

Nanyang Technological University

Research

JoVE Journal

Bioengineering

24.3K 次观看.  Northwestern University. 描述一个卵巢卵泡封装在三维纤维蛋白 - 海藻酸钠的互穿网络的新方法。此系统结合蛋白水解降解结构的支持，以支持发展不成熟的卵泡产生成熟的卵母细胞。这种方法可应用于文化细胞聚集，保持不限制扩张的细胞 - 细胞接触。

视频: A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

文化和共同培养小鼠的卵巢和卵巢卵泡

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

科研

发育生物学

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

卵巢和输卵管的三维器官培养的海藻酸钙凝胶

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

生物工程

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 ...

医学

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

体外模拟微重力下小鼠腔卵泡生长的研究

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

组织工程细胞在三维凝胶封装

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

生物学

标准化卵巢癌类器官生物样本库

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in research and patient care, standardization remains a challenge due to the heterogeneity of this lethal malignancy, combined with the inherent complexity of organoid technology. This adaptable protocol provides a systematic framework to realize the full potential of ovarian cancer organoids considering a patient-specific variability of progenitors. By implementing a structured experimental workflow to select optimal culture conditions and seeding methods, with parallel testing of direct 3D seeding versus a 2D/3D route, we obtain, in most cases, robust long-term expanding lines suitable for a broad range of downstream applications. 
Notably, the protocol has been tested and proven efficient in a great number of cases (N = 120) of highly heterogeneous starting material, including high-grade and low-grade ovarian cancer and stages of the disease with primary debulking, recurrent disease, and post-neoadjuvant surgical specimens. Within a low Wnt, high BMP exogenous signaling environment, we observed progenitors being differently susceptible to the activation of the Heregulin 1 &#223; (HER&#223;-1)-pathway, with HER&#223;-1 promoting organoid formation in some while inhibiting it in others. For a subset of the patient's samples, optimal organoid formation and long-term growth necessitate the addition of fibroblast growth factor 10 and R-Spondin 1 to the medium. 
Further, we highlight the critical steps of tissue digestion and progenitor isolation and point to examples where brief cultivation in 2D on plastic is beneficial for subsequent organoid formation in the Basement Membrane Extract type 2 matrix. Overall, optimal biobanking requires systematic testing of all main conditions in parallel to identify an adequate growth environment for individual lines. The protocol also describes the handling procedure for efficient embedding, sectioning, and staining to obtain high-resolution images of organoids, which is required for comprehensive phenotyping.

作者聚焦：推进卵巢癌的个性化医疗 

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...

癌症研究

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

功能性与极化胆囊肿的生成与定量特征

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

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功能性与极化胆囊肿的生成与定量特征

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装