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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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>17-&#946;-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 <img alt="Equation 1" src="/files/ftp_upload/66873/66873eq01.jpg" style="margin: auto;" />BSC</td>
			<td>Aseptic operations, making culture dishes, aliquoting reagents, etc</td>
		</tr>
		<tr>
			<td>CO2 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&#160;</td>
			<td>SIGMA</td>
			<td>V900833</td>
			<td>Reagent grade, powder&#160;</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&#160;</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

603 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

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

克里斯托弗休斯：在体外血管生成的研究模型（面试）

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

科研

生物学

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

大鼠心脏中植入的工程组织

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

An In Vitro Skin Irritation Test (SIT) using the EpiDerm Reconstructed Human Epidermal (RHE) Model

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. The EpiDerm SIT utilizes the 3D in vitro reconstructed human epidermal (RHE) model EpiDerm. The procedure described in this protocol allows for discrimination between irritants of GHS category 2 and non-irritants (6). The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay. After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37&deg;C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium. After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours. At the end of the entire 42h post-incubation (day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations (7).

An In Vitro Skin Irritation Test SIT using the EpiDerm Reconstructed Human Epidermal RHE Model

In this video, we demonstrate the EpiDerm Skin Irritation test (EpiDerm SIT) developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients.

一个<em在体外皮肤刺激测试（SIT），使用的表皮重建人表皮（RHE）型号

The EpiDerm Skin Irritation test (EpiDerm SIT) was developed (1,2,3) and validated (4,5) for in vitro skin irritation testing of chemicals, including ...

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

一个<em在体外 FluoroBlok肿瘤侵袭实验

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

一个 EX - OVO鸡胚胎培养系统适用于成像和显微外科的应用

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

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 ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

胞外酸性 pH 培养体系的建立

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Implantation of Electrospun Vascular Grafts with Optimized Structure in a Rat Model

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta replacement. The electrospun vascular grafts often possess relatively small pores, which limit cell infiltration into the grafts and hinder the regeneration and remodeling of the neo-arteries. In this study, PCL vascular grafts with thicker fibers (5 - 6 &#181;m) and larger pores (~30 &#181;m) were fabricated by using a modified processing technique. The long-term performance of the graft was evaluated by implantation in a rat abdominal aorta model. Ultrasound analysis showed that the grafts remained patent without aneurysm or stenosis occurring even after 12 months of implantation. Macroporous structure improved the cell ingrowth and thus promoted tissue regenerated at 3 months. More importantly, there was no sign of adverse remodeling, such as calcification within the graft wall after 12 months. Therefore, electrospun PCL vascular grafts with modified macroporous processing hold potential to be an artery substitute for long-term implantation.

Here, we present a modified electrospinning method to fabricate PCL vascular grafts with thick fibers and large pores, and describe a protocol to evaluate the in vivo performance in a rat model of abdominal aorta replacement.

优化结构静电纺丝血管移植在大鼠模型中的植入

Here, we present a protocol to fabricate macroporous PCL vascular graft and describe an evaluation protocol by using a rat model of abdominal aorta ...