Name: co-transplantation of human ovarian tissue with engineered endothelial cells: a cell-based strategy combining accelerated perfusion with direct paracrine delivery
Uploaded: 2018-05-16T14:00:00.000+00:00
Duration: 7 min 40 s
Description: 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

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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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&amp;rsquo;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&amp;trade; 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

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

科研

生物工程

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

癌症研究

Orthotopic Ovarian Transplantation Procedures to Investigate the Life- and Health-span Influence of Ovarian Senescence in Female Mice

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is being used to investigate the health-protective effects of young ovaries in young females. The current procedures employ an orthotopic transplantation method, where allogenic ovaries are transplanted back to their original position in the ovarian bursa. This is in contrast to the more commonly used heterotopic transplantation of ovaries/ovarian tissue subcutaneously or under the kidney capsule. All three locations provide efficient revascularization of the transplanted tissues. However, orthotopic transplantation provides the ovary with the most natural signaling environment and is the only procedure that provides the opportunity for the animal to reproduce naturally post-operatively. One must take care to remove all endogenous ovarian tissue during the ovariectomy procedure. If any endogenous tissue remains or if only one ovary is removed, the transplanted tissue will remain dormant until the existing tissue becomes senescent. While revascularization of the transplanted ovaries occurs very quickly, the transplant recipient can take a considerable amount of time to adapt to a new hypothalamic/pituitary/gonadal/adrenal (HPG/A) axis signaling regime associated with the transplanted tissue. This normally takes about 100 days in the mouse. Therefore, transplantation experiments should be designed to accommodate this adaptation period. Typical results with ovarian transplantation will include changes in the health of the recipient that reflect the age of the transplanted ovary, rather than the chronological age of the recipient.

Described here are orthotopic ovarian transplantation protocols for modifying ovarian influence on the physiology of the recipient mouse. This surgical procedure is used to expose female mice to various types of ovarian influence and may include heterochronic transplantations and transplantation of ovaries that have undergone various ex vivo treatments.

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

Ovarian transplantation was first conducted at Utah State University in 1963. In more recent work, heterochronic transplantation of mammalian ovaries is ...

发育生物学

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

组织工程血管在小鼠模型的生成和嫁接

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

人卵巢上皮类器官：组织修复和药物检测模型

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. 
Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

作者聚焦：使用表面上皮类器官推进人类卵巢修复和癌症研究

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

人卵巢表面上皮类器官作为研究组织再生的平台

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

移植到小鼠卵巢脂肪垫

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

医学

Characterization of Blood Outgrowth Endothelial Cells (BOEC) from Porcine Peripheral Blood

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, inflammation, and homeostasis. The endothelium also plays an important role in pathophysiologies such as atherosclerosis, hypertension, and diabetes. Endothelial cells form the inner lining of blood and lymphatic vessels and display heterogeneity in structure and function. Various groups have evaluated the functionality of endothelial cells derived from human peripheral blood with a focus on endothelial progenitor cells derived from hematopoietic stem cells or mature blood outgrowth endothelial cells (or endothelial colony-forming cells). These cells provide an autologous resource for therapeutics and disease modeling. Xenogeneic cells may provide an alternative source of therapeutics due to their availability and homogeneity achieved by using genetically similar animals raised in similar conditions. Hence, a robust protocol for the isolation and expansion of highly proliferative blood outgrowth endothelial cells from porcine peripheral blood has been presented. These cells can be used for numerous applications such as cardiovascular tissue engineering, cell therapy, disease modeling, drug screening, studying endothelial cell biology, and in vitro co-cultures to investigate inflammatory and coagulation responses in xenotransplantation.

Characterization of Blood Outgrowth Endothelial Cells BOEC from Porcine Peripheral Blood

猪外周血血液生长内皮细胞（BOEC）的表征

The endothelium is a dynamic integrated structure that plays an important role in many physiological functions such as angiogenesis, hemostasis, ...

Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress. 
The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

使用脱细胞小鼠肺和原代人内皮细胞移植生物工程肺

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor ...

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

摘要

探索更多视频

此视频中的章节

自体内皮祖细胞播种技术和生物相容性在大动物模型的心血管设备的测试

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

原位卵巢移植术对雌性小鼠卵巢衰老的影响及寿命及健康范围研究

组织工程血管在小鼠模型的生成和嫁接

人卵巢表面上皮类器官作为研究组织再生的平台

人卵巢表面上皮类器官作为研究组织再生的平台

人卵巢表面上皮类器官作为研究组织再生的平台

移植到小鼠卵巢脂肪垫

猪外周血血液生长内皮细胞（BOEC）的表征

使用脱细胞小鼠肺和原代人内皮细胞移植生物工程肺