我们的研究得到了国家自然科学基金 （82171603）、上海市卫生健康委员会基金 （202140341）、上海市科学技术委员会 （23JC1403803） 以及国家卫健委和上海市重点实验室创新促进计划（RC2023-02）的支持。

小鼠处理和实验研究根据上海生物医学与制药技术研究所动物委员会批准的方案进行。确保安全程序: 处理化学品或生物材料时，请始终佩戴适当的个人防护设备 （PPE）。
1. 小鼠胚胎的获取
注意:本节介绍获取小鼠胚胎的过程。成年雌性小鼠 （6-8 周龄，杂交 C57BL/6 和 DBA/2） 在 20-25 °C 和 40-60% 湿度下维持 12 小时光照和 12 小时黑暗的标准环境条件下， 随意提供食物和水。
<ol><li>腹膜内注射雌性小鼠 10 IU 妊娠母马血清促性腺激素 （PMSG），然后以 42-48 小时的间隔注射 10 IU 人绒毛膜促性腺激素 （HCG）。</li>
	<li>将每只雌性小鼠与同等年龄的雄性小鼠交配，并在第二天早上检查阴道塞。 注意:阴道栓是精液在阴道口凝固后形成的浅黄色栓剂块。</li>
	<li>通过吸入 CO2 对带有阴道塞的雌性小鼠实施安乐死。</li>
	<li>打开腹部，找到女性生殖器;用镊子拉伸输卵管、卵巢和脂肪垫;用剪刀剪断输卵管和卵巢;然后，切开输卵管和子宫（图 1）。</li>
	<li>将获得的输卵管转移到预装有 M2 培养基的 35 mm 培养皿中。 注:M2 培养基应在培养箱（37 °C，5% CO2 ）中预热 30 分钟。</li>
	<li>用 25 G 针刺穿扩大的输卵管壶腹，以释放或捏住卵丘-受精卵复合物（图 2）。</li>
	<li>使用镊子将卵丘-合子复合物转移到含有 0.5 mg/mL 透明质酸酶的 M2 溶液中，并孵育几分钟直至卵丘细胞解离（图 3）。</li>
	<li>使用鸡蛋转移移液管收集受精卵，并在含有 4 mg/mL BSA 的 M2 培养基中洗涤，以去除残留的透明质酸酶、卵丘细胞和杂质。</li>
</ol>2. 小鼠胚胎的 体外 培养
注:本节详细介绍了从受精卵到囊胚的 小鼠 胚胎体外培养的过程。KSOM 培养微滴应提前 24 小时制备，以确保温度和气体平衡。
<ol><li>在 35 mm 培养皿的底部准备 12 个 KSOM 培养基微滴，每个微滴的体积为 20 μL。用 3-5 mL 矿物油覆盖微滴（图 4），并将培养皿放入 37 °C 和 5% CO2 的培养箱中以平衡。</li>
	<li>使用移液管将小鼠胚胎从 M2 培养基转移到 KSOM 培养基中，以进行彻底洗涤。 注意:此步骤需要具有 37 °C 加热台的立体显微镜。尽量减少在培养箱外处理胚胎的时间。</li>
	<li>将洗涤过的胚胎放入准备好的 KSOM 微滴中，并在 37 °C 和 5% CO2 下孵育 4 天。 注:为确保营养充足，每 20 μL 微滴培养 5-10 个胚胎。</li>
	<li>根据它们的大小 （100-130 μm）、内细胞质量（内细胞团由许多全部压实的细胞组成）、滋养层细胞的数量（滋养外胚层由许多紧密结合并堆积在一起的细胞组成）和扩张程度（囊胚腔占据胚胎的 80-100%）选择有利的囊胚。</li>
</ol>3. 子宫内膜细胞的制备
<ol><li>在生物安全柜内，使用移液管吸出 1 mL 2% 明胶，并将其分配到 12 孔培养板中。轻轻旋转板以确保明胶完全覆盖底部。将板置于 37 °C 培养箱中 30 分钟。然后，吸出多余的明胶，让板底完全干燥。</li>
	<li>包被过程完成后，用 DMEM/F12（详见 材料表 ）、1 mM 丙酮酸钠、2 mM L-谷氨酰胺、100 μg/mL 链霉素和 100 IU/mL 青霉素培养人子宫内膜腺癌细胞（Ishikawa），将它们接种到明胶包被的 12 孔板（2 × 105个细胞/孔）中，并孵育 24 小时。</li>
</ol>4. 胚胎附着
<ol><li>轻轻地将 5-15 个新鲜囊胚放入含有上一步制备用于共培养的 Ishikawa 细胞的 12 孔板的每个孔中（图 5）。</li>
	<li>孵育 48 小时后，在显微镜下观察胚胎以评估其附着状态。要观察胚胎附着，请快速移动板 3 次。未附着的胚胎将在 Ishikawa 细胞表面表现出漂浮或滚动运动。</li>
	<li>使用等式 （1） 计算胚胎着床率;使用胚胎粘附率表示植入效果。 胚胎着床率 = （附着的胚胎数/植入的胚胎总数） × 100% （1）</li>
</ol>

在与 Ishikawa 细胞的体外共培养模型中评估胚胎粘附

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</ol>

作者无需声明任何财务或其他利益冲突。

所提出的胚胎植入体外模型的简单性和快速性为早期药物筛选和其他研究应用提供了显着的优势。简单的方案，加上 Ishikawa 细胞系的高通量特性，使其成为大规模筛选的理想选择，尤其是在药物发现的早期阶段。Liang13 发现 TAGLN2 敲低降低了滋养层细胞的侵袭能力。Green14 表明胰岛素样生长因子提高了基于小鼠胚胎-Ishikawa 细胞系统的小鼠囊胚的粘附能力?...

胚胎植入是成功怀孕的第一步，对于了解其生物学基础和应对不孕症的挑战至关重要。然而，伦理限制对收集人类临床胚胎样本构成了重大限制，阻碍了对怀孕早期人类胚胎与子宫内膜之间复杂相互作用的研究1。对这些复杂机制的深刻理解对于推进基础生物学研究、药物发现和生殖健康至关重要。
以前的体外模型采用粘附模型，其中单层人子宫内膜上皮细胞 （RL95-2）2 与胚胎替代品（如 BeWo、JAr 或 Jeg-33 绒毛膜癌细胞）共培养。然而，球体大小的选择（范围为 70-100 μm）至关重要，因为更大或更小的细胞聚集体可能会影响实验的准确性。值得注意的是，每种制剂中只有 25% 的球体符合合适大小4 的标准，并且与囊胚的生理条件相比存在显着差异。
此外，子宫内膜和囊胚的三维 （3D） 培养系统提供了更具生理相关性的环境5，但它们需要高技术专长、细胞生长缓慢、资源和维护成本高、标准化困难和可重复性低6。
在该协议中，我们提出了一种经济高效且快速的胚胎植入体外模型，该模型可以在大多数实验室中开发和使用。该方法的总体目标是为在受控环境中研究胚胎和子宫内膜之间的相互作用提供一个可靠且可重复的平台。通过模拟 体外胚胎植入的关键事件，该模型旨在弥合动物模型和临床环境之间的差距，从而实现更精确和有针对性的研究。
与胚胎替代品相比，小鼠囊胚的使用提供了体内胚胎植入过程的更生理相关性的表示7。此外，源自人子宫内膜腺癌的 Ishikawa 细胞系具有腺上皮和子宫腔上皮1 的混合特性，使其能够表达类似于正常子宫内膜中的酶、结构蛋白和功能性类固醇受体，从而为胚胎植入提供生理相关的底物。共培养系统的简单性和快速性可实现高通量筛选和药物检测 8,9,10。通过提供一个强大且可重复的平台，这种方法为促进我们对胚胎植入及其对人类健康影响的理解提供了机会。

<table><tbody><tr><td>17-&amp;beta;-estradiol</td><td>SIGMA</td><td>3301</td><td>Most potent mammalian estrogenic hormone</td></tr><tr><td>BD Falcon</td><td>BD</td><td>353001</td><td>Bacteriological Petri Dishes 35 x 10 mm style w/tight lid, crystal-grade virgin polystyrene, sterile</td></tr><tr><td>Biosafety Cabinet</td><td>ESCO</td><td>class &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66873/66873eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;BSC</td><td>Aseptic operations, making culture dishes, aliquoting reagents, etc</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Thermo</td><td>8000DH</td><td>Embryo culture</td></tr><tr><td>Culture plate</td><td>Corning</td><td>3506</td><td>Cell culture</td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>1133032</td><td>DMEM/F12 (1x), liquid 1:1,Contains L-glutamine and 15 mM HEPES buffer</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10099141</td><td>Fetal Bovine Serum, Qualified, Australia Origin</td></tr><tr><td>Gelatin</td><td>SIGMA</td><td>G9391</td><td>Type B, powder, BioReagent, suitable for cell culture</td></tr><tr><td>HCG</td><td>Nanjing Aibei</td><td>M2520</td><td>Sterilization reagent, intraperitoneal injection, 50 IU/mL</td></tr><tr><td>Hyaluronidase&amp;nbsp;</td><td>SIGMA</td><td>V900833</td><td>Reagent grade, powder&amp;nbsp;</td></tr><tr><td>KSOM</td><td>Merck</td><td>MR-020P-D</td><td>(1x), Powder, w/o Phenol Red, 5 x 10 mL</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030081</td><td>L-glutamine-200 mM (100x), liquid</td></tr><tr><td>M2</td><td>Merck</td><td>MR-015-D</td><td>EmbryoMax M2 Medium (1x), Liquid, with Phenol Red</td></tr><tr><td>Mineral oil&amp;nbsp;</td><td>SIGMA</td><td>M8410</td><td>Mineral oil is suitable for use as a cover layer to control evaporation and cross-contamination in various molecular biology applications</td></tr><tr><td>Penicillin-Streptomycin, liquid</td><td>Gibco</td><td>15140122</td><td>10,000 Units penicillin (base) and 10,000 units streptomycin (base), utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline</td></tr><tr><td>PMSG</td><td>Nanjing Aibei</td><td>M2620</td><td>Sterilization reagent ,intraperitoneal injection, 50 IU/mL</td></tr><tr><td>Progesterone</td><td>SIGMA</td><td>5341</td><td>Steroid hormone secreted by the corpus luteum during the latter half of the menstrual cycle</td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360070</td><td>Sodium pyruvate is commonly added to cell culture media as a carbon source in addition to glucose</td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX7</td><td>Embryo retrieval and observation of embryo development</td></tr></tbody></table>

establishment of an embryo implantation model in vitro

胚胎植入是成功怀孕的第一步。胚胎植入的 体外 模型对于基础生物学研究、药物开发和筛选至关重要。本文提出了一种简单、快速、高效的 胚胎 植入体外模型。在该方案中，我们首先介绍小鼠囊胚获取和人子宫内膜腺癌细胞 （Ishikawa） 植入制剂，然后是小鼠胚胎和 Ishikawa 细胞的共培养方法。最后，我们进行了一项研究，以评估不同浓度的 17-β-雌二醇 （E2） 和孕酮 （P4） 对基于该模型的胚胎粘附率的影响。我们的研究结果表明，高浓度的 E2 显着降低了胚胎粘附，而添加黄体酮可以恢复粘附率。该模型提供了一个简单快速的平台，用于评估和筛选参与粘附过程的分子，例如细胞因子、药物和控制着床和子宫内膜容受性的转录因子。

在辅助生殖技术过程中，受控卵巢过度刺激 （COH） 是关键步骤，与自然周期相比，导致患者的雌激素水平显着升高 10-20 倍以上11。鉴于此，我们询问了高血清雌激素浓度是否会影响新鲜胚胎移植过程中的胚胎着床。基于这个胚胎植入模型，我们研究了不同浓度的 E2 和 P4 对胚胎着床率的影响12。饥饿处理后，将 Ishikawa 细胞与 E2 （0、0.1、 1、10 ?...

作者聚焦:通过分析胚胎-子宫内膜相互作用推进提高妊娠率的治疗策略

该协议描述了建立简单有效的胚胎植入 体外 模型的方法，用于评估影响胚胎植入过程的相关分子。

体外胚胎植入模型的建立

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

establishment-of-an-embryo-implantation-model-in-vitro

Research

JoVE Journal

Biology

Establishment of an Embryo Implantation Model In Vitro

821 次观看.  Fudan University. 胚胎植入是成功怀孕的第一步，对于了解其生物学基础和应对不孕症的挑战至关重要。本研究的目的是建立一个一致的平台，用于在受控条件下分析胚胎子宫内膜相互作用。基于这个体外模型，我们发现超高浓度的雌激素显着降低了胚胎着床的风险，而黄体酮补充剂可以依赖地减轻这些负面影响。本研究为改善 IVF 控制性卵巢刺激期间新鲜胚胎?...

视频: 作者聚焦:通过分析胚胎-子宫内膜相互作用推进提高妊娠率的治疗策略

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

科研

发育生物学

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

利用活体中鸡胆膜研究妇科和泌尿癌

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

癌症研究

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. ...

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

Pregnancy establishment is a dynamic process involving complex embryo and uterine crosstalk. The precise contributions of the maternal uterine environment to these processes remain an active area of investigation. Here, detailed protocols are provided to aid in designing in vivo animal models to address these research questions.

研究卵巢切除小鼠模型中子宫对妊娠建立的贡献的方法

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation ...

Utero-tubal Embryo Transfer and Vasectomy in the Mouse Model

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects of epigenetic alterations originated during preimplantation development on subsequent fetal development and adult health. The use of an effective and consistent embryo transfer technique is crucial to enhance the generation of genetically modified animals and to determine the effect of different treatments on implantation rates and survival to term. Embryos at the blastocyst stage are usually transferred by uterine transfer, performing a puncture in the uterine wall to introduce the embryo manipulation pipette. The orifice performed in the uterus does not close after the pipette has been withdrawn, and the embryos can outflow to the abdominal cavity due to the positive pressure of the uterus. The puncture can also produce a hemorrhage that impairs implantation, blocks the transfer pipette and may affect embryo development, especially when embryos without zona are transferred. Consequently, this technique often results in very variable and overall low embryo survival rates. Avoiding these negative effects, utero-tubal embryo transfer take advantage of the utero-tubal junction as a natural barrier that impedes embryo outflow and avoid the puncture of the uterine wall. Vasectomized males are required for obtaining pseudopregnant recipients. A technique to perform vasectomy is described as a complement to the utero-tubal embryo transfer.

Utero-tubal embryo transfer uses the utero-tubal junction as a barrier to prevent the embryo outflow that may occur when performing uterine transfer. Vasectomized males are required to obtain pseudopregnant recipients for embryo transfer. Both techniques are discussed.

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

The transfer of preimplantation embryos to a surrogate female is a required step for the production of genetically modified mice or to study the effects ...

生物学

Single Cell Collection of Trophoblast Cells in Peri-implantation Stage Human Embryos

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a critical yet enigmatic biological event that has been historically difficult to study due to technical and ethical limitations. Implantation is initiated by the development of the trophectoderm to early trophoblast and subsequent differentiation into distinct trophoblast sublineages. Aberrant early trophoblast differentiation may lead to implantation failure, placental pathologies, fetal abnormalities, and miscarriage. Recently, methods have been developed to allow human embryos to grow until day 13 post-fertilization in vitro in the absence of maternal tissues, a time-period that encompasses the implantation period in humans. This has given researchers the opportunity to investigate human implantation and recapitulate the dynamics of trophoblast differentiation during this critical period without confounding maternal influences and avoiding inherent obstacles to study early embryo differentiation events in vivo. To characterize different trophoblast sublineages during implantation, we have adopted existing two-dimensional (2D) extended culture methods and developed a procedure to enzymatically digest and isolate different types of trophoblast cells for downstream assays. Embryos cultured in 2D conditions have a relatively flattened morphology and may be suboptimal in modeling in vivo three-dimensional (3D) embryonic architectures. However, trophoblast differentiation seems to be less affected as demonstrated by anticipated morphology and gene expression changes over the course of extended culture. Different trophoblast sublineages, including cytotrophoblast, syncytiotrophoblast and migratory trophoblast can be separated by size, location, and temporal emergence, and used for further characterization or experimentation. Investigation of these early trophoblast cells may be instrumental in understanding human implantation, treating common placental pathologies, and mitigating the incidence of pregnancy loss.

Here, we describe a method for warming vitrified human blastocysts, culturing them through the implantation period in vitro, digesting them into single cells and collecting early trophoblast cells for further investigation.

近植入阶段人类胚胎中特罗福细胞的单细胞集合

Human implantation, the apposition and adhesion to the uterine surface epithelia and subsequent invasion of the blastocyst into the maternal decidua, is a ...

<meta charset="utf8"></head><body>用于研究胚胎植入机制的 3D 打印支架模型

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

<meta charset="utf8"></head><body>作者聚焦:研究上皮-基质和胚胎相互作用的体外子宫内膜模型

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

使用两种贴壁细胞系的共培养模型

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

体外胚胎植入模型的建立

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体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

利用活体中鸡胆膜研究妇科和泌尿癌

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

基于微注射的免疫胚胎心肌细胞体内植入系统

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养

研究卵巢切除小鼠模型中子宫对妊娠建立的贡献的方法

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

近植入阶段人类胚胎中特罗福细胞的单细胞集合

使用两种贴壁细胞系的共培养模型

使用两种贴壁细胞系的共培养模型

体外胚胎植入模型的建立

副本

摘要

探索更多视频

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

利用活体中鸡胆膜研究妇科和泌尿癌

家兔模型最佳胚胎阶段微创胚胎移植与胚胎玻璃化

基于微注射的免疫胚胎心肌细胞体内植入系统

前子宫 从前肠胚形成到晚期器官生成的小鼠胚胎培养

研究卵巢切除小鼠模型中子宫对妊娠建立的贡献的方法

子宫 - 输卵管胚胎移植和输精管结扎术在小鼠模型

近植入阶段人类胚胎中特罗福细胞的单细胞集合

使用两种贴壁细胞系的共培养模型

使用两种贴壁细胞系的共培养模型

前子宫从前肠胚形成到晚期器官生成的小鼠胚胎培养