奥马尔. 亚历山大的人为例证。 L.M. 获得了康奈尔临床和翻译科学中心的试验奖和 ASRM 研究补助金的支持。 作者感谢詹姆斯实验室成员对手稿的批判性阅读。

所有涉及动物的程序都已由威尔康奈尔医学院的机构动物护理和使用委员会 (IACUC) 批准。所有使用卵巢组织的异种移植实验均按照相关的指导原则和规定进行。人卵巢组织收集的病人计划化疗或放疗的癌症治疗或前骨髓移植。威尔康奈尔医学院的机构审查委员会批准了为潜在自体使用的组织的收集, 并在患者知情同意下, 捐赠多达10% 的卵巢组织供研究使用。
1. 人类卵巢组织的收集
注意: 当卵巢组织从远程设施中转运输时, 时间不应超过 5 h18,19。
<ol><li>收集卵巢组织和冲洗与无菌盐水溶液。</li>
	<li>将卵巢置于无菌容器中。</li>
	<li>倒入莱博维茨的 L-15 培养基, 直到卵巢完全浸入培养基中。</li>
	<li>关闭并密封容器。</li>
	<li>把容器运到冰上。</li>
</ol>2. 加工所采购的卵巢组织, 改编自施密特et 。18
<ol><li>
卵巢加工和冷冻的准备工作
	<ol>
<li>准备100毫升的培养基进行加工: 莱博维茨的 L-15 中99毫升 + 1 毫升抗生素防霉溶液。使用0.2 µm 过滤器过滤介质。保持中间冷藏。</li>
		<li>用亚砜作为冷冻防护, 制备100毫升冷冻溶液。添加69.64 毫升莱博维茨的 L-15 培养基, 17.66 毫升胎牛血清 (血清), 3.42 克蔗糖 (创建最终浓度0.1 摩尔/升), 10.65 毫升亚砜 (以创建最终浓度的1.5 摩尔/升), 和1毫升抗生素防霉解决方案。使用150毫升过滤器单元过滤介质, 0.2 µm. 将培养基存放在4摄氏度。</li>
		<li>使用用于包装固体灭菌循环的高压釜消毒手术工具。</li>
	</ol>
</li>
	<li>
组织到达后
	<ol>
<li>在生物安全柜中设置无菌手术工具: 手术刀与刀片 21, 锋利的细弯剪刀和钳在不同的大小: 长 (大约150毫米长度) 和2中等大小 (大约110毫米长度)。</li>
		<li>戴上无菌手套, 在安全柜内打开容器。将卵巢放在150毫米培养皿中, 然后在卵巢的2.1.1 上倒入冷培养基, 以防止卵巢组织脱水。</li>
		<li>将卵巢从任何残留的组织中分离出来, 用冷媒冲洗, 直到没有组织和血液。</li>
		<li>将卵巢放在一个干净的150毫米培养皿中, 加入冷媒, 在台阶2.1.1 中准备, 直到卵巢半淹没在它里面。</li>
	</ol>
</li>
	<li>
卵巢组织解剖
	<ol>
<li>对开卵巢, 先用弯细剪刀取出髓质。然后用一把21刀片的无菌手术刀刮掉髓质。之前, 直到皮质组织是 1-1. 5 毫米的厚度。</li>
		<li>将皮质组织切成2-3 毫米宽的薄片, 该条的长度将是所处理的卵巢片的整个长度。</li>
	</ol>
</li>
</ol>3. 卵巢组织缓慢冰冻, 适应牛顿et al。和 Oktay et 。6,20
<ol><li>
冷冻准备
	<ol>
<li>标签 cryovials (1.8 毫升)。</li>
		<li>在步骤2.1.2 中添加1.5 毫升的冷冻溶液至每瓶。</li>
		<li>每一个含冷冻溶液的低温小瓶转移一皮质带。</li>
		<li>在旋转板上平衡皮质带20分钟, 在4摄氏度处应用温和搅拌。</li>
	</ol>
</li>
	<li>
卵巢组织缓慢冰冻
	<ol>
<li>将 cryovials 装入可编程的刨床冷冻机, 从0摄氏度开始。</li>
		<li>冷却在2°c/分钟到-7 °c。</li>
		<li>保持组织在这个恒定的温度为10分钟。</li>
		<li>执行人工播种的冰晶成核诱导, 通过触摸每个 cryovial 与棉花尖端浸泡在液氮 (在2)。</li>
		<li>继续冷却在0.3 °c/分钟, 直到样品温度达到-40 摄氏度。</li>
		<li>冷却速度快10°c/分钟到-140 摄氏度。</li>
		<li>将 cryovials 转移到 LN2 (-196 °c) 中用于存储的杜瓦瓶中。</li>
	</ol>
</li>
</ol>4. 手术准备 (双边卵巢切除术和联合移植)
<ol><li>
板材的制备
	<ol>
<li>解冻卵巢组织移植前的一天<ol>
<li>将一块塑料石蜡膜放在50毫米培养皿的底部, 用70% 乙醇喷雾, 直到完全覆盖。</li>
			<li>把培养皿放在安全柜里过夜。每只老鼠准备2培养皿。</li>
		</ol>
</li>
		<li>在手术当天<ol>
<li>在生物安全柜内含有塑料石蜡膜的培养皿中吸入乙醇。</li>
			<li>离开培养皿铅半开, 直到乙醇完全蒸发。</li>
			<li>当塑料石蜡膜完全干燥时, 关闭培养皿的盖子。把它放在安全柜里。</li>
			<li>6井板标签井相应, 0.1 摩尔/升蔗糖 + 1 摩尔/升亚砜, 0.1 摩尔/升蔗糖 + 0.5 摩尔/升亚砜, 0.1 摩尔/升蔗糖, 基本解冻溶液 (基站), 培养基。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
解决方案的准备工作
	<ol>
<li>准备100毫升的基站: 莱博维茨的 L-15 中等79毫升 + 血清20毫升 + 抗生素防霉溶液1毫升。过滤器与0.22 µm 过滤系统。</li>
		<li>用0.1 摩尔/升蔗糖制备10毫升的基站。0.342 克的蔗糖和添加10毫升的4.2.1 在步骤中制备, 轻轻搅动, 直到蔗糖完全溶解。使用注射器过滤器0.22 µm 过滤解决方案。</li>
		<li>根据表 1准备解决方案。带铝箔的含亚砜的盖管。保持冷藏, 直到使用。</li>
	</ol>
</li>
</ol>5. 卵巢组织快速解冻
<ol><li>取出2杜瓦瓶中含有冰冻卵巢组织的瓶子, 并将其保持在室温下三十年代。</li>
	<li>用纸巾擦干净瓶子。</li>
	<li>将瓶子浸泡在30摄氏度的水浴中1-2 分钟, 直到它的内容解冻。</li>
	<li>打开瓶子, 并将皮质带放在第一个井中, 其中第一个溶液中含有 3ml (0.1 摩尔/升蔗糖 + 1 摩尔/升亚砜, 在步骤4.2.3 中制备)。 注: 所有涉及打开板盖的步骤将在层流下完成。</li>
	<li>在4摄氏度的旋转板上孵化6井板, 5 分钟. 用铝箔盖住这一步。</li>
	<li>将皮质带转入第二个含3毫升第二溶液的井中 (0.1 摩尔/升蔗糖 + 0.5 摩尔/升二甲基亚砜, 在步骤4.2.3 中制备)。</li>
	<li>在旋转板上孵育6井板, 使其在4摄氏度处进行温和搅拌, 5 分钟. 用铝箔盖住这一步。</li>
	<li>将皮质带转入第三井, 含4毫升溶液 (基站与0.1 摩尔/L 蔗糖, 在步骤4.2.2 中制备)。</li>
	<li>在旋转板上孵育, 以在4°c 5 分钟的温和搅拌。</li>
	<li>将皮质带移植到含有4毫升溶液的第四井中 (4.2.1)。</li>
	<li>在旋转板上孵育, 以在4°c 5 分钟的温和搅拌。</li>
	<li>将皮质带移植到最后一个含4毫升冷介质的井中 (在步骤2.1.1 中制备)。保持解冻的卵巢组织在冰上, 直到执行移植。</li>
</ol>6. 卵巢组织的封装
<ol><li>
准备以下解决方案
	<ol>
<li>在0.9% 盐水中准备25毫升20毫摩尔 HEPES 缓冲液。过滤和存储在4摄氏度。</li>
		<li>准备1毫升的 CaCl2在浓度为1摩尔/l 过滤器和存储在4°c。</li>
	</ol>
</li>
	<li>
制备纤维蛋白原50毫克/毫升库存溶液
	<ol>
<li>将1克纤维蛋白原加入20毫升20毫摩尔/升 HEPES 缓冲液中, 在0.9% 盐水中慢慢将纤维蛋白原混合到 HEPES 缓冲的盐水中, 在37摄氏度以上数小时。</li>
		<li>标签1.7 毫升微离心管。</li>
		<li>过滤解决方案通过0.45 µm 注射器过滤器, 然后通过0.2 µm 注射器过滤器。</li>
		<li>整除将溶液分为200µL 微离心管, 贮存在-20 摄氏度。</li>
	</ol>
</li>
	<li>
制备凝血酶 100 U/毫升库存溶液 注: 凝血酶溶液吸附在玻璃上, 整除溶液中的塑料管/瓶。<ol>
<li>添加2.5 毫升0.9% 不育生理盐水到 250 U 的凝血酶。</li>
		<li>轻轻摇动, 直到溶液完全溶解。</li>
		<li>标签1.7 毫升微离心管。</li>
		<li>过滤通过0.2 µm 注射器过滤器, 整除50µL 入微离心管并且存放在-80 °c。</li>
	</ol>
</li>
	<li>
使纤维蛋白凝块70µL, 获得最终浓度10毫克/毫升纤维蛋白原和 5 U/毫升凝血酶 注意: 确保快速工作, 解决方案在几秒钟内凝结。此外, 避免添加气泡的混合物。<ol>
<li>在1.7 毫升微离心管中制备纤维蛋白原溶液。<ol>
<li>在0.9% 盐水溶液中添加360µL 20 毫摩尔/升 HEPES 缓冲液, 用于纤维蛋白原库存的 aliquoted 200 µL, 在步骤6.2.4 中制备。</li>
			<li>由温和的吹打混合。</li>
			<li>把它放在冰上。</li>
		</ol>
</li>
		<li>在第二微离心管中制备凝血酶溶液。<ol>
<li>在步骤6.3.4 中加入148.7 µL 的 DMEM, aliquoted 50 µL 的凝血酶。</li>
			<li>由温和的吹打混合。</li>
			<li>添加1.3 µL 1 摩尔/升 CaCl2。</li>
			<li>由温和的吹打混合。</li>
			<li>把它放在冰上。</li>
		</ol>
</li>
		<li>在第三个微离心管中制备单个细胞悬浮液。<ol>
<li>用酶细胞脱离培养基从板中分离出工程化的内皮细胞。</li>
			<li>用 hemocytometer 和盖玻璃旋转并计数单元格。</li>
			<li>每1毫米2的卵巢组织使用2万个细胞。在 mm2中计算卵巢组织的20,000x 面积。</li>
			<li>旋转的工程内皮细胞, 并重新悬浮在基本培养基 (DMEM), 以达到16.8 µL 的总容量。</li>
			<li>把它放在冰上。</li>
		</ol>
</li>
		<li>将一块卵巢组织放在无菌纱布海绵上晾干几秒钟。</li>
		<li>将卵巢组织放在50毫米培养皿内的塑料石蜡膜上。</li>
		<li>添加39.2 µL 的纤维蛋白原溶液, 准备在步骤 6.4.1.2, 进入细胞悬浮在步骤6.4.3.4 准备, 混合由温和的吹打。把它放在冰上。</li>
		<li>添加14µL 在步骤6.4.2.4 中制备的凝血酶溶液。轻轻地混合吹打一次。工作快点。</li>
		<li>将混合物放在卵巢组织的顶端, 吸管它以拉长的水滴的形式。</li>
		<li>在37摄氏度的孵化器中放置培养皿与血栓。</li>
	</ol>
</li>
</ol>7. 人卵巢组织与工程化内皮细胞对核型核型小鼠的双边卵巢切除术和联合移植
注: 十至十四周前的雌性核供应国小鼠使用21 (杰克逊实验室)。
<ol><li>准备所有的外科工具, 如步骤2.1.3 所阐述的, 确保你有所有的缝合, 垫, 和外科磁带方便。</li>
	<li>
用异氟醚的麻醉系统麻醉老鼠。
	<ol>
<li>将鼠标放在感应腔内, 关闭盖子, 打开适当的旋塞阀。</li>
		<li>打开流量计到1000毫升/分. 打开蒸发器并设置它提供 2-3. 5%。</li>
		<li>观察麻醉效果;失去知觉, 缓慢和定期呼吸。</li>
		<li>转到鼻锥保持麻醉。根据生命体征, 将蒸发器送出1-3%。</li>
		<li>验证没有踏板或尾部反射。如果反射存在, 增加异氟醚, 直到它缺席。</li>
	</ol>
</li>
	<li>修剪鼠标的背部头发, 从尾部到锁骨线, 使用一个电动理发。</li>
	<li>把鼠标放在手术台上的俯卧位置上。</li>
	<li>用手术胶带将鼻锥固定在手术平台上。</li>
	<li>用1.25 厘米宽的外科胶带将鼠标的四肢轻轻地贴到手术平台上。</li>
	<li>使用无菌润滑剂果冻和插入温度计 PR. 用1.25 厘米宽的穿孔塑料手术胶带将温度计固定在手术平台上。</li>
	<li>用2.5 厘米宽的外科纸带将尾部胶带贴到手术平台上。</li>
	<li>加热, 使用红外线或热水加热垫, 并监测整个过程中鼠标的体温。保持体温在35-37 摄氏度范围内。</li>
	<li>用无菌聚维酮碘溶液拭子清洁修剪区域, 用无菌酒精进行擦拭。</li>
	<li>重复步骤6.9 两次。</li>
	<li>在鼠标的眼睛上放置一滴不育的眼部润滑剂兽医软膏, 以防止脱水和角膜损伤。</li>
	<li>用不育的外科悬垂盖住老鼠。</li>
	<li>注射丁丙诺啡 (1 毫克/千克) 亚皮肤 (S/C)。</li>
	<li>使用手术刀 (刀片 #21) 进行纵向内侧背切口。</li>
	<li>
执行双侧卵巢切除术, 使用背侧方法。
	<ol>
<li>用剪刀把皮下结缔组织用钝夹层释放出来。</li>
		<li>将筋膜侧向中线, 用锋利的剪刀切开并到达腹膜腔。</li>
		<li>用镊子轻轻抓住卵巢脂肪垫和卵巢, 将其拉出腹腔。</li>
		<li>用血管钳抓住卵巢和脂肪垫。</li>
		<li>结扎卵巢和脂肪垫使用4/0 单丝可吸收缝合在卵巢的基础上, 在输卵管远端。</li>
		<li>将子房夹在领带的远端。确保从树桩底部没有出血。</li>
		<li>将纸巾放回腹腔, 用6/0 编织可吸收缝线缝合筋膜。</li>
		<li>重复阶段 7.15.1-7.15.7 在对侧。</li>
	</ol>
</li>
	<li>
联合移植卵巢组织。
	<ol>
<li>在臀高肌上方的筋膜上进行水平切口, 长度与凝块尺寸相符。通过打开筋膜下面的剪刀, 在这个虚拟空间内创建一个口袋。</li>
		<li>拿起一个包裹的皮质组织, 准备在步骤 6.4.8, 并把它放在这个口袋里。</li>
		<li>用6/0 编织可吸收缝线缝合筋膜。</li>
	</ol>
</li>
	<li>重复阶段 7.16.1-7.16.3 在对侧, 以及。</li>
	<li>用4/0 单丝可吸收缝线闭合背部壁, 使用简单的间断缝线。</li>
	<li>在切口部位注射利多卡因 0.5% (1.2 毫升/千克) 的 S/C。</li>
	<li>使用无菌酒精的准备清洁皮肤。</li>
	<li>在监视鼠标的温度时关闭异氟醚。</li>
	<li>提供1-2 分钟 O2, 不带异氟醚。继续变暖鼠标, 直到它开始移动和恢复知觉。</li>
	<li>把鼠标放在一个干净的恢复笼子里, 然后把老鼠放在一个单独的笼子里, 直到手术伤口完全愈合。</li>
	<li>每8-12 小时注射丁丙诺啡 (1 毫克/千克), 术后24小时。</li>
	<li>当所有的食物、水、被褥和笼子都是蒸压的时候, 把老鼠放在单独的笼子里, 直到实验的终点。把笼子放在有压力的通风室里。在处理小鼠时使用个人防护设备。</li>
</ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2017.">Cancer Statistics, 2017.</a> CA Cancer J Clin. 67 (1), 7-30 (2017).</li><li>Magelssen, H., Melve, K. K., Skjaerven, R., Fossa, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parenthood+probability+and+pregnancy+outcome+in+patients+with+a+cancer+diagnosis+during+adolescence+and+young+adulthood.">Parenthood probability and pregnancy outcome in patients with a cancer diagnosis during adolescence and young adulthood.</a> Hum Reprod. 23 (1), 178-186 (2008).</li><li>Donnez, J., Dolmans, M. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+paramagnetic+resonance+as+a+tool+to+evaluate+human+ovarian+tissue+reoxygenation+after+xenografting.">Electron paramagnetic resonance as a tool to evaluate human ovarian tissue reoxygenation after xenografting.</a> Fertil Steril. 92 (1), 374-381 (2009).</li><li>Nugent, D., Newton, H., Gallivan, L., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effect+of+vitamin+E+on+ischaemia-reperfusion+injury+in+ovarian+grafts.">Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.</a> J Reprod Fertil. 114 (2), 341-346 (1998).</li><li>Kim, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+ischemic+tissue+damage+in+ovarian+cortical+tissue+with+or+without+antioxidant+(ascorbic+acid)+treatment.">Quantitative assessment of ischemic tissue damage in ovarian cortical tissue with or without antioxidant (ascorbic acid) treatment.</a> Fertil Steril. 82 (3), 679-685 (2004).</li><li>Abir, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+posttransplantation+survival+of+human+ovarian+tissue+by+treating+the+host+and+graft.">Improving posttransplantation survival of human ovarian tissue by treating the host and graft.</a> Fertil Steril. 95 (4), 1205-1210 (2011).</li><li>Friedman, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possible+improvements+in+human+ovarian+grafting+by+various+host+and+graft+treatments.">Possible improvements in human ovarian grafting by various host and graft treatments.</a> Hum Reprod. 27 (2), 474-482 (2012).</li><li>Shikanov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+encapsulation+and+vascular+endothelial+growth+factor+delivery+promotes+ovarian+graft+survival+in+mice.">Fibrin encapsulation and vascular endothelial growth factor delivery promotes ovarian graft survival in mice.</a> Tissue Eng Part A. 17 (23-24), 3095-3104 (2011).</li><li>Soleimani, R., Heytens, E., Oktay, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+neoangiogenesis+and+follicle+survival+by+sphingosine-1-phosphate+in+human+ovarian+tissue+xenotransplants.">Enhancement of neoangiogenesis and follicle survival by sphingosine-1-phosphate in human ovarian tissue xenotransplants.</a> PLoS One. 6 (4), e19475 (2011).</li><li>Israely, T., Dafni, H., Nevo, N., Tsafriri, A., Neeman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+ectopic+ovarian+xenotransplantation:+multiparameter+characterization+of+the+neovasculature+by+dynamic+contrast-enhanced+MRI.">Angiogenesis in ectopic ovarian xenotransplantation: multiparameter characterization of the neovasculature by dynamic contrast-enhanced MRI.</a> Magn Reson Med. 52 (4), 741-750 (2004).</li><li>Buratini, J., Price, C. 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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcomes+of+transplantations+of+cryopreserved+ovarian+tissue+to+41+women+in+Denmark.">Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark.</a> Hum Reprod. 30 (12), 2838-2845 (2015).</li><li>Oktay, K., Newton, H., Aubard, Y., Salha, O., Gosden, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+immature+human+oocytes+and+ovarian+tissue:+an+emerging+technology?.">Cryopreservation of immature human oocytes and ovarian tissue: an emerging technology?.</a> Fertil Steril. 69 (1), 1-7 (1998).</li><li>Shultz, L. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E1(-)E4(+)+adenoviral+gene+transfer+vectors+function+as+a+&amp;#34;pro-life&amp;#34;+signal+to+promote+survival+of+primary+human+endothelial+cells.">E1(-)E4(+) adenoviral gene transfer vectors function as a &amp;#34;pro-life&amp;#34; signal to promote survival of primary human endothelial cells.</a> Blood. 93 (9), 2936-2944 (1999).</li><li>Seandel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+functional+and+durable+vascular+niche+by+the+adenoviral+E4ORF1+gene.">Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.</a> Proc Natl Acad Sci U S A. 105 (49), 19288-19293 (2008).</li><li>Meirow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+fibrosis+and+blood-vessels+damage+in+human+ovaries+exposed+to+chemotherapy.+Potential+mechanisms+of+ovarian+injury.">Cortical fibrosis and blood-vessels damage in human ovaries exposed to chemotherapy. 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K., Clinton, M., Webb, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+anti-M&#252;llerian+hormone+(AMH)+during+follicle+development+in+a+monovulatory+species+(sheep).">The role of anti-M&#252;llerian hormone (AMH) during follicle development in a monovulatory species (sheep).</a> Endocrinology. 153 (9), 4533-4543 (2012).</li><li>Donnez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+ovarian+activity+and+pregnancy+after+transplantation+of+cryopreserved+ovarian+tissue:+a+review+of+60+cases+of+reimplantation.">Restoration of ovarian activity and pregnancy after transplantation of cryopreserved ovarian tissue: a review of 60 cases of reimplantation.</a> Fertil Steril. 99 (6), 1503-1513 (2013).</li><li>Ferreira, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+sample+size+on+the+outcome+of+ovarian+tissue+cryopreservation.">The effects of sample size on the outcome of ovarian tissue cryopreservation.</a> Reprod Domest Anim. 45 (1), 99-102 (2010).</li><li>Gavish, Z., Peer, G., Roness, H., Cohen, Y., Meirow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Follicle+activation+and+'burn-out'+contribute+to+post-transplantation+follicle+loss+in+ovarian+tissue+grafts:+the+effect+of+graft+thickness.">Follicle activation and 'burn-out' contribute to post-transplantation follicle loss in ovarian tissue grafts: the effect of graft thickness.</a> Hum Reprod. 30 (4), 1003 (2015).</li><li>Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+Preservation+in+Women.">Fertility Preservation in Women.</a> N Engl J Med. 377 (17), 1657-1665 (2017).</li><li>Salama, M., 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=New+advances+in+ovarian+autotransplantation+to+restore+fertility+in+cancer+patients.">New advances in ovarian autotransplantation to restore fertility in cancer patients.</a> Cancer Metastasis Rev. 34 (4), 807-822 (2015).</li><li>Donnez, J., Dolmans, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cortex+transplantation:+60+reported+live+births+brings+the+success+and+worldwide+expansion+of+the+technique+towards+routine+clinical+practice.">Ovarian cortex transplantation: 60 reported live births brings the success and worldwide expansion of the technique towards routine clinical practice.</a> J Assist Reprod Genet. 32 (8), 1167-1170 (2015).</li><li>Meirow, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantations+of+frozen-thawed+ovarian+tissue+demonstrate+high+reproductive+performance+and+the+need+to+revise+restrictive+criteria.">Transplantations of frozen-thawed ovarian tissue demonstrate high reproductive performance and the need to revise restrictive criteria.</a> Fertil Steril. 106 (2), 467-474 (2016).</li><li>Kalich-Philosoph, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophosphamide+triggers+follicle+activation+and+"burnout";+AS101+prevents+follicle+loss+and+preserves+fertility.">Cyclophosphamide triggers follicle activation and "burnout"; AS101 prevents follicle loss and preserves fertility.</a> Sci Transl Med. 5 (185), 185ra162 (2013).</li><li>Kano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMH/MIS+as+a+contraceptive+that+protects+the+ovarian+reserve+during+chemotherapy.">AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy.</a> Proc Natl Acad Sci U S A. 114 (9), E1688-E1697 (2017).</li></ol>

迈克尔金斯伯格是 Angiocrine 生物科学公司的雇员, 圣地亚哥, 加利福尼亚, 92130, 美国, 那隔绝, 转染与 E4-ORF-1 并且标记了我们使用的内皮细胞。

在这里我们证明, 联合移植的执行官提供了一个重要的好处, 卵巢组织的生存能力和功能后, 移植的小鼠。未设置卵巢组织自动移植的临床应用标准和最佳参数 (大小, 移植部位, 移植时间,等)。32,33,34对于滤泡池的增强恢复仍未定义。当进行自动移植时, 解冻皮层卵巢组织的缺血性移植主要表现在骨盆部位, 如其余的卵巢、?...

癌症仍然是发达国家死亡的主要原因之一, 但数十年的研究已经取得了重大进展, 在大多数类型的癌症, 并在某些情况下, 几乎加倍的生存率1。不幸的是, 化疗药物经常 gonadotoxic, 耗尽卵巢原始卵泡的储备, 并减少生育率2。这种日益增长的人口可以受益于多种生育方法, 包括卵母细胞和/或胚胎超低温保存, 但是, 需要迅速启动癌症治疗和青春期前患者的患者不符合这些选择。另外, 一些患者选择在进行治疗方案之前 cryopreserve 卵巢组织, 并在恢复和缓解后, 自动移植组织恢复生育率3。然而, 到目前为止, 自动移植后的移植生存和卵泡输出仍然相对较低4, 主要是由于组织缺血和缺氧5,6,7。尽管许多努力改善卵巢皮质移植的生存能力使用抗氧化剂8,9, 支持血管生成素10,11,12,1 3或机械操作14, 5 至7天窗口移植后的移植物缺血损害了移植7的生存和生存。为了解决这一问题, 我们制定了一个基于细胞的策略, 以促进宿主和移植血管的吻合, 从而加速卵巢组织的再灌注。
除了在移植后的窗口中对接枝卵巢组织的缺血性侮辱外, 卵泡间信号的中断可能导致池的耗尽15,16。由于外源性内皮细胞 (执行官) 有助于在移植周围的稳定和功能的血管, 他们提供了一个独特的机会, 向移植组织传达定义的分子输入。作为一项原则的证明, 执行官们被设计来表达超生理水平的抗苗勒激素 (AMH), 一个成员的转化生长因子 beta (TGFβ) 超家族, 已被证明限制卵泡生长17。用控制和 AMH 表达细胞联合移植移植滤泡分布的比较验证了工程主管的生物活性和效力。
总之, 通过提高移植的生存能力和抑制卵泡池的过早动员, 这种方法可以提高在生育保护患者中自动移植卵巢组织的生产率。此外, 基于 ExEC 的平台还能对与卵泡发育有关的分子调控器进行实验性审讯。

<table>
	<tbody>
		<tr>
			<td>Leibovitz&#8217;s L-15 medium</td>
			<td>Gibco</td>
			<td>11415064</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td>Anti-Anti X100</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S 1888</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma</td>
			<td>F 8630</td>
			<td>from bovine plasma</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma</td>
			<td>T 1063</td>
			<td>from human plasma</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D 2650</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>12491015</td>
			<td></td>
		</tr>
		<tr>
			<td>Enzyme Cell Detachment Medium</td>
			<td>Invitrogen</td>
			<td>00-4555-56</td>
			<td>Accutase</td>
		</tr>
		<tr>
			<td>Plastic paraffin film</td>
			<td>Bemis NA</td>
			<td></td>
			<td>Parafilm M</td>
		</tr>
		<tr>
			<td>Surgical paper tape 2.5 cm</td>
			<td>3M</td>
			<td>1530-1</td>
			<td>Micropore</td>
		</tr>
		<tr>
			<td>Surgical Paper tape 1.25 cm</td>
			<td>3M</td>
			<td>1530-0</td>
			<td>Micropore</td>
		</tr>
		<tr>
			<td>Perforated plastic Surgical tape 1.25 cm</td>
			<td>3M</td>
			<td>1527-0</td>
			<td>Transpore</td>
		</tr>
		<tr>
			<td>Monofilament Absorbable Suture</td>
			<td>Covidien</td>
			<td>UM-203</td>
			<td>Biosyn</td>
		</tr>
		<tr>
			<td>Braided Absorbable Suture</td>
			<td>Covidien</td>
			<td>GL-889</td>
			<td>Polysorb</td>
		</tr>
		<tr>
			<td>Povidone-iodine Solution USP 10%</td>
			<td>Purdue Products</td>
			<td>67618-153-01</td>
			<td>Betadine Solution Swab Stick</td>
		</tr>
		<tr>
			<td>Cryoviales</td>
			<td>Nunc</td>
			<td>377267</td>
			<td>CryoTube</td>
		</tr>
		<tr>
			<td>sterile ocular lubricant</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td>Puralube</td>
		</tr>
		<tr>
			<td>1.7 ml micro-centrifuge tube</td>
			<td>Denville</td>
			<td>C-2172</td>
			<td>Eppendorf</td>
		</tr>
		<tr>
			<td>Anasthesia system</td>
			<td>VetEquip</td>
			<td>V-1 table top system with scavenging</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cells</td>
			<td>Angiocrine Biosciences, Inc., San Diego, CA, USA</td>
			<td></td>
			<td>Isolated, transfected with E4-ORF- 1 and labeled endothelial cells</td>
		</tr>
		<tr>
			<td>Trichrome stain</td>
			<td>Sigma</td>
			<td>HT15-1kt</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Isolectin</td>
			<td>Invitrogen</td>
			<td>I32450</td>
			<td>isolectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor&#8482; 647 Conjugate</td>
		</tr>
	</tbody>
</table>

co-transplantation of human ovarian tissue with engineered endothelial cells: a cell-based strategy combining accelerated perfusion with direct paracrine delivery

不孕是化疗和/或放疗的经常性副作用, 对某些患者来说, 冷冻的卵母细胞或胚胎不是一种选择。作为替代, 越来越多的这些患者选择 cryopreserve 卵巢组织的移植后恢复和缓解。尽管保存的卵巢组织自动移植患者的预后有所改善, 但移植组织的有效血管重建仍然是一个主要障碍。为了减轻缺血, 从而改善自动移植患者的预后, 我们开发了一种以血管细胞为基础的加速卵巢组织灌注的策略。本文描述了一种在小鼠同种异体模型中联合移植保存卵巢组织的外源性内皮细胞 (执行官) 的方法。我们扩大这一做法, 雇用已设计为组成性表达抗苗勒激素 (AMH) 的执行官, 从而使持续分泌信号输入到卵巢移植。联合移植与执行官增加卵泡体积和改善窦卵泡发育, 和 AMH 表达的执行官促进保留静态原始卵泡。这一联合战略可能是一个有用的工具, 以减轻缺血和调节卵泡激活的背景下的生育率保存和/或不育的大。

为了确定联合移植的执行官是否为患者的组织提供了好处, 解冻后的卵巢皮质带被分成同等大小的部分, 并嫁接为免疫性损害, 点头, 伽玛 (核供应), 小鼠。在单独嵌入纤维蛋白血块 (无 ECs) 和其他包含执行官 (图 1a) 的一侧, 每只鼠标都充当自己的控制项。执行官是通过从人脐带分离的原发性内皮和随后的治疗与腺病毒基因片段 E4-ORF1, 如前所述<sup class="...

Watch this Scientific Journal Video about Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Deli

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

对某些患者来说, 保留生育能力的唯一选择是保存卵巢组织。不幸的是, 延迟血管重建破坏卵泡活力。在这里, 我们提出一个协议, 共同移植人卵巢组织与内皮细胞作为一种基于细胞的战略结合加速灌注与直接分泌交付的生物活性分子。

人卵巢组织与工程化内皮细胞联合移植: 一种以细胞为基础的快速灌注与直接分泌的融合策略

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

co-transplantation-human-ovarian-tissue-with-engineered-endothelial

Research

JoVE Journal

Bioengineering

7.4K 次观看.  Weill Cornell Medical College. 对某些患者来说, 保留生育能力的唯一选择是保存卵巢组织。不幸的是, 延迟血管重建破坏卵泡活力。在这里, 我们提出一个协议, 共同移植人卵巢组织与内皮细胞作为一种基于细胞的战略结合加速灌注与直接分泌交付的生物活性分子。

视频: Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

阻隔组织破坏与有机电化学晶体管传感

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

科研

生物工程

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

肿瘤微环境基质与应用癌细胞侵袭组织工程

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

交货蛋白质，多肽或细胞不可渗透的小分子进入活细胞被孵化与核内体溶解试剂dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

人工程心脏组织的收缩自动分析心脏药物安全筛选

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

微流控装置中组织模型的 Perfusable 血管网

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

一种新型的腱绦缝合技术，采用组织工程胶原蛋白移植物修复大肌腱缺损

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced antibiotic resistance, which poses potential health dangers, but can also be beneficial for environmental applications such as purification of drinking water. The further development of anti-bacterial therapeutics and biofilm-inspired applications will require the development of reproducible, engineerable methods for biofilm creation. Recently, a novel method of biofilm preparation using a modified three-dimensional (3D) printer with a bacterial ink has been developed. This article describes the steps necessary to build this efficient, low-cost 3D bioprinter that offers multiple applications in bacterially-induced materials processing. The protocol begins with an adapted commercial 3D printer in which the extruder has been replaced with a bio-ink dispenser connected to a syringe pump system enabling a controllable, continuous flow of bio-ink. To develop a bio-ink suitable for biofilm printing, engineered Escherichia coli bacteria were suspended in a solution of alginate, so that they solidify in contact with a surface containing calcium. The inclusion of an inducer chemical within the printing substrate drives expression of biofilm proteins within the printed bio-ink. This method enables 3D printing of various spatial patterns composed of discrete layers of printed biofilms. Such spatially-controlled biofilms can serve as model systems and can find applications in multiple fields that have a wide-ranging impact on society, including antibiotic resistance prevention or drinking water purification, among others.

This article describes a method of transforming a low-cost commercial 3D printer into a bacterial 3D printer that can facilitate printing of patterned biofilms. All necessary aspects of preparing the bioprinter and bio-ink are described, as well as verification methods to assess the formation of biofilms.

工程生物膜的三维造型

Biofilms are aggregates of bacteria embedded in a self-produced spatially-patterned extracellular matrix. Bacteria within a biofilm develop enhanced ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

使用微流体芯片以确定性方式建立基于单细胞的共培养物

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...