蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装
Summary
此处介绍的两个协议用于在三生培养条件下封装猪卵母细胞。在第一种，积卵-卵母细胞复合物（COCs）封装在纤维蛋白-藻酸珠中。第二，它们被用氟化乙烯丙烯粉末颗粒（微生物反应器）封闭。两个系统都确保以最佳条件维护其 3D 组织。
Abstract
在生殖生物学方面，从人工授精和胚胎移植技术开始的生物技术革命导致辅助生殖技术的发展，如卵母细胞体外成熟（IVM）、体外受精（IVF）和通过从体细胞核转移克隆家畜。IVM 是特别重要的方法。它是为商业重要或濒危物种的生成间隔缩短、体外人类生殖研究以及细胞疗法的转基因动物生产等应用提供成熟、优质卵母细胞的平台技术。卵母细胞质量一词包括其完成成熟、受精的能力，从而产生健康的后代。这意味着优质卵母细胞对于成功受精至关重要，包括试管婴儿手术。这给开发一种可靠的培养方法带来了许多困难，这种方法不仅支持人类卵母细胞的生长，而且支持其他大型哺乳动物物种的生长。IVM 的第一步是卵母细胞的体外培养。本文描述了猪卵母细胞的3D培养的两个协议。在第一个，3D模型累积-卵母细胞复合物（COCs）封装在一个纤维蛋白-藻酸盐珠渗透网络中，其中纤维蛋白和藻酸盐的混合物同时凝胶化。在第二种材料中，COC悬浮在一滴介质中，用氟化乙烯丙烯（FEP;六氟丙烯和四氟乙烯的共聚物）粉末颗粒封装，形成定义为液体大理石（LM）的微生物反应器。两个 3D 系统都维护体外气态培养环境。他们还通过防止其扁平化和由此破坏间隙结来维护 COC 3D 组织，从而保持卵母细胞与周围卵泡细胞之间的功能关系。
Introduction
各种培养系统的发展，包括三维（3D）的培养系统，旨在为从卵泡分离的卵母细胞的生长和成熟提供最佳条件，即使在发育初期。这对辅助生殖技术（ART）非常重要，特别是鉴于癌症治疗后与不孕症作斗争的妇女越来越多。体外条件（IVM）卵母细胞的成熟已经是一种成熟的技术，主要用于体外胚胎生成，用于牲畜繁殖2。然而，在大多数哺乳动物物种中，即使能达到高成熟率的积卵-卵母细胞复合物（COCs）（范围60至90%），它们的发展能力仍然不足以满足需求。这是因为以这样的方式获得的受精卵的发展，甚至到囊肿阶段是低的，在转移到代孕动物后，其期限的生存能力降低。因此，有必要提高从接受IVM程序4的卵母细胞获得的胚胎的发育能力。因此，新的成熟介质5正在设计，并测试不同时期的体外培养6，77以及6补充各种生长因子和分子的培养媒体,8，9。
任何完整的 IVM 系统的第一步是为卵母细胞在体外培养期间的可持续生长创造最佳条件。卵母细胞生长是卵母细胞恢复卵母细胞能力的具体指标之,一。此外，适当的体外培养系统必须能够支持其核成熟和细胞质分化12。积卵-卵母细胞复合物的形态是 ART 诊所使用的另一个重要指标，用于选择最佳卵母细胞，用于人类和牲畜12、13的体外受精 （IVF） 程序的后续步骤。考虑的COC的形态特征包括：卵母细胞直径、细胞质造粒和第一极体完整性14、15。14,此外，卵母细胞的发育潜力与积细胞的外观和压实及其围绕卵母细胞的层数相关。对于适当的卵母细胞体外培养系统来说，非常重要的也是维持卵母细胞-累积细胞的适当相互作用和细胞骨架稳定性16、17、18、19。16,17,18,19到目前为止，在人体体外卵母细胞生长的人类COC中已经证明20。使用牛科动物也导致活产。这些从不成熟的卵巢卵泡分离，然后培养14天，直到卵母细胞足够大，接受IVF程序21。类似地，从大猩猩蚁卵泡中分离出的COCs，在体外培养后受IVM影响，产生能够重新形成卵母细胞的卵母细胞，以正常出现的主轴结构22。元相II阶段。然而，在这项研究中，作者并没有试图给他们施肥。然而，这些结果表明，类似的程序不仅可应用于这些特定的哺乳动物物种，而且也适用于从卵泡中获得的人类积云-卵母细胞复合物，这些复合物应能获得适合成功的IVF技术的优质卵母细胞。
上述结果通过应用传统的IVM协议获得，在二维（2D）系统中培养卵母细胞。2D培养系统中的常规程序是覆盖卵母细胞，浸入一滴适当的培养介质中，与矿物油23，24。23,24假定体外卵母细胞培养期间的油覆盖有助于防止液体蒸发，从而确保培养物中适当的pH值和渗透压力的维持。虽然这种2D培养系统允许获得，甚至高达87%的成熟猪卵母细胞25，已经证明，矿物油覆盖导致脂质可溶性材料的实质性扩散，这是适当的卵母细胞发展所必需的26。此外，由于类固醇（黄体酮和雌激素）在卵母细胞培养期间扩散到矿物油中，观察到核成熟延迟和猪卵母细胞发育能力成就下降。这可能导致获得少量的酶，此外，其特点是发育能力低到囊肿阶段，并转移到接受动物27后生存能力差。因此，正在尝试通过创造最佳条件，实现与CC一起培养的卵母细胞作为复合物，特别是使用三维（3D）系统，提高从IVM程序后接收的卵母细胞衍生的胚胎的发育能力。各种创新的3D体外培养系统已经发展，近二十年来，28，29。,29这些旨在保持细胞的自然空间组织，并避免它们在培养皿中扁平化，这是传统2D培养中无法实现的。养殖COC的结构和功能活动可以通过维护其适当的架构和不受干扰的沟通，通过各种隔间之间的间隙结30，可以确保其结构和功能活动。生物支架对于3D细胞复合物体外培养的适宜性已经使用天然生物材料进行了评估，如细胞外基质（ECM;胶原蛋白和透明质酸）的各种成分（ECM;胶原蛋白和透明质酸）31或惰性聚合物（藻酸盐）32。这些尝试在几个物种的测试带来了有希望的结果，在卵母细胞梅病恢复和实现他们的完全能力33，34，35。33,34,35然而，到目前为止，还没有开发适合从大型家畜（包括猪）分离出的COC成熟3D系统。
本文描述了可用于猪 COC 的 3D 培养的两种协议。第一个协议描述了纤维蛋白-藻酸盐珠（FAB）中的封装。FAB 可以通过同时混合藻酸盐和纤维蛋白溶液而形成，这种溶液经过同步凝胶过程。这种组合提供了一个动态的机械环境，因为两个组件都有助于矩阵刚度。类似的解决方案以前也用于小鼠卵巢卵泡培养和成熟36。在所提出的协议中，为了避免藻酸纤维蛋白网络过早降解，使用适当浓度更高的氯化钙溶液，确保快速稳定的凝胶过程。动态机械环境在 COC 驻留和尺寸增加的自然卵泡环境中创造了类似这样的条件。此外，该作品还展示了COC 3D培养系统的代表性结果，其中这些培养系统悬浮在一滴介质中，并用氟化乙烯丙烯（FEP;六氟丙烯和四氟乙烯的共聚物）粉末颗粒封装，形成微生物反应器（液体大理石、LM）。LM是一种3D生物反应器，以前已经证明支持活微生物37，肿瘤球类38和 胚胎干细胞39的生长。LMs也已成功用于羊卵母细胞培养40。在大多数使用LMs的实验中，生物反应器是使用粒径为1μm41的聚四氟乙烯（PTFE）粉床制备的。提出的协议使用FEP，其成分和性质与氟聚合物PTFE非常相似。但FEP比PTFE更容易形成和柔软，尤其重要的是，它是高度透明的。
两个 3D 系统都维护体外气态培养环境。他们还通过防止其扁平化和由此破坏间隙结，保持卵母细胞和周围卵泡细胞之间的功能关系，来维持COC 3D组织。
Protocol
Representative Results
Discussion
能够保持体外生长的卵母细胞，但也在其周围的积细胞，同时支持其成熟是极其必要的成功的辅助生殖技术，并促进对体细胞/卵母细胞相互作用的理解，特别是在物种，如人类或猪。不断改进的IVM技术正在成为在多囊卵巢综合征（PCOS）、卵巢过早衰竭或最终不育（肿瘤治疗）的情况下保留生殖选择的重要工具。此外，由于某些卵巢功能障碍可能是由调节性卵泡生长不良引起的，了解控制卵母细?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者非常感谢：Waclaw Twozydlo博士（贾吉洛尼亚大学动物学和生物医学研究所发育生物学和无脊椎动物形态学系）在TEM的技术设施;Beata Snakowska女士（贾吉洛尼亚大学动物学和生物医学研究所内分泌学系）提供技术援助;到日本济隆大学动物学和生物医学研究所细胞生物学和成像系，JEOL JEM 2100HT（日本东京，日本 JEOL）。波兰国家科学中心2018/29/N/NZ9/00983赠款支持这项工作。
Materials
| General | |||
| Antibiotic Antimycotic (100x) 100ml | Thermo Fisher | 15240062 | 2.5% final concentration for Handling Medium. 1% in PBS (step 1.2) |
| DMEM/F12 (500ml) | Sigma-Aldrich | D8062 | Handling and Maturation Medium |
| DPBS (w/o Ca, Mg), 1x, 500ml | Thermo Fisher | 14190144 | |
| FCS (100 ml) | Thermo Fisher | 16140063 | 10% final concentration for both Handling Medium and Maturation Medium. (steps: 1.5. 2.6.) |
| PBS (1x, pH 7.4) 500ml | Thermo Fisher | 10010023 | |
| TBS Stock Solution (10x, pH 7.4) 500 ml | Cayman Chemicals | 600232 | 1x final concentration. Other brand can be use |
| Maturation Medium | |||
| hCG (1 VIAL of 10 000 U) | Sigma-Aldrich | CG10 | |
| PMSG | BioVendor | RP1782721000 | |
| Fibrin-alginate beads | |||
| Alginate Lyase | Sigma-Aldrich | A1603 | (Step 2.11.1) |
| Thrombin | Sigma-Aldrich | T9326-150UN | (Step 2.1) |
| Calcium Chloride | Sigma-Aldrich | C5670 | (Step 2.1) |
| Fibrinogen (250mg) | Sigma-Aldrich | F3879 | (Step 2.2) |
| Sodium Alginate | Sigma-Aldrich | W201502 | (Step 2.3) use for alginate solution |
| Liquid Marble | |||
| FEP | Dyneon GmbH 3M AdMD | A-66670 | |
| Morphological examination | |||
| LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells | Thermo Fisher | L3224 | (Step 4.1.) Emitted fluorescence: 494 nm for calcein, 528 nm for EthD-1; measure: 517 nm for calcein, 617 nm – EthD-1 |
| VECTASHIELD Antifade Mounting Medium | Vector Laboratories | H-1000 | mounting medium |
| Ultrastructure examination | |||
| Glutaraldehyde solution | Sigma-Aldrich | G5882 | 2.5% final concentraion (Step 4.2.1.) |
| LR White resin | Sigma-Aldrich | L9774 | (Step 4.2.4.) |
| Methylene blue | Sigma-Aldrich | M9140 | (Step 4.2.5.) |
| Osmium Tetroxide | Sigma-Aldrich | O5500 | (Step 4.2.3.) |
| Sodium cacodylate trihydrate | Sigma-Aldrich | C0250 | Use for preparing 0.1M sodium cacodylate buffer (pH 7.2) |
| Uranyl Acetate | POCH | 868540111 | (Step 4.2.4.) |
| Specific instruments, tools | |||
| 30 mm Pteri dish | TPP | 93040 | |
| 60 mm IVF Petri dish | Falcon | 353653 | |
| Ez-Grid Premium Cell Handling Pipettor | RI Life Sciences | 8-72-288 | |
| Ez-Tip | RI Life Sciences | 8-72-4155/20 | |
| Heating Table | SEMIC | Other brands can be used | |
| Incubator | Panasonic | MCO-170AIC-PE | Other brands can be used |
| Sterile petri dish (10 cm) | NEST Biotechnology | 704002 | |
| Sterile syringe filters with 0.2 µm | GOOGLAB SCIENTIFIC | GB-30-022PES | |
| Thermos | Quechua | 5602589 | Other brands can be used |
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