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

<ol>
	<li>Cortvrindt, R., Smitz, J., VanSteirteghem, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+maturation,+fertilization+and+embryo+development+of+immature+oocytes+from+early+preantral+follicles+from+prepuberal+mice+in+a+simplified+culture+system.">In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system.</a> Human Reproduction. 11, 2656-2666 (1996).</li><li>Smitz, J., Cortvrindt, R., Hu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+combined+with+recombinant+human+chorionic+gonadotrophin+improves+meiotic+progression+in+mouse+follicle-enclosed+oocyte+culture.">Epidermal growth factor combined with recombinant human chorionic gonadotrophin improves meiotic progression in mouse follicle-enclosed oocyte culture.</a> Human Reproduction. 13, 664-669 (1998).</li><li>Xu, M., Banc, A., 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=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?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fetuin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>F3385</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen, Gibco</td>
<td>10082-139</td>
<td>&nbsp;</td>
<tr>
<td>Aprotinin</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10236624001</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>039-00475</td>
<td>40 mM</td>
</tr>
<tr>
<td>EGF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A412</td>
<td>&nbsp;</td>
<tr>
<td>rFSH</td>
<td>&nbsp;</td>
<td>A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CG-5</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1603</td>
<td>&nbsp;</td>
<tr>
<td>ITS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I1884-1VL</td>
<td>&nbsp;</td>
<tr>
<td>L-15</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;MEM+Gluta MAX</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>32561</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>TBS</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28379</td>
<td>&nbsp;</td>
<tr>
<td>Tisseel Fibrin kit</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>921030</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Alginate</td>
<td>&nbsp;</td>
<td>FMC BioPolymers</td>
<td>LF200DL</td>
<td>Mw 418kDa</td>
</tr>		</tbody>
		</table>
		</div>

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

Research

JoVE Journal

Bioengineering

24.2K 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

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

在三维自组装多肽水凝胶的人类神经祖细胞的培养

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

自我报告的支架3维细胞培养

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

新方法本地化记者荧光珠近细胞培养表面的牵引力显微镜

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

三维全层皮肤的生成等效和自动伤人

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

多井板膜插入系统中3D 组织模型渗透扩散的测定与模拟方法

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

3D 染色细胞的包覆和超弹性蛋白样凝胶的制备

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

透明质酸基水凝胶对3维患者源性胶质瘤细胞培养的研究

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

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

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

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

3D 动态培养系统构建多层间充质干细胞片材

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