支持我们的工作是由美国国立卫生研究院拨款NS048447和AG017216to科威特第纳尔。刘丽想感谢他的监督和支持，在原有协议的发展科瓦莱宁Tanila（博士，芬兰的库奥皮奥，库奥皮奥大学）。

拉玻璃毛细管 <ol><li>从萨特仪器公司（硼硅玻璃，B100 - 75 - 10）的玻璃毛细管购买。 </li><li>拉毛细管，在萨特的P - 87火焰山微量拉拔器，设置热指数在300和330压力指数。 </li><li>用剪刀修剪的玻璃毛细管中的一角，使锥尖有一个约0.5毫米的内径。 </li></ol> 枕大池脑脊液穿刺技术 脑脊液标本均取自枕大池使用，此前公布的1的方法（图1） 。 <img src="/files/ftp_upload/old/960_2008_10_13_2/960_Figure-01.png" alt="图1" width="432" height="184" /> 图1 1。 anesthesized氯胺酮（100mg/kg）和甲苯噻嗪（10mg/kg的），腹腔注射的老鼠。在麻醉诱导时，老鼠都保持在37℃培养箱。 2。颈部的皮肤被剃光，鼠标，然后放置俯卧于脑立体定位仪与直接接触加热垫。直肠温度探头插入直肠，使产生的热量加热垫在体温的变化进行调整。头是头适配器担保。手术部位是用10％ 碘伏擦洗，其次是 7 0％乙醇（重复3次），矢状切口的皮肤是劣质枕部。 3。解剖显微镜下，皮下组织和肌肉（M. biventer cervicis和M.直肌头癣实蝇主要 ）是分开的钝性分离钳。一个对microretractors除了用来存放肌肉。 4。放下鼠标，使头部形成一个近135 o与身体的角度。 5。解剖显微镜下，枕大池硬脑膜出现延髓和主要血管（动脉实蝇spinalis），脑脊液空间是可见的（图2）作为一个闪闪发光的和明确的反向三角形。 <img src="/files/ftp_upload/old/960_2008_10_13_3/960_Figure-02.png" alt="图2" width="304" height="307" /> 图2 6。硬脑膜抹杀用无菌棉签擦干。毛细管渗透到citerna麦格纳通过硬脑膜，外侧足背动脉spinalis（图2）。电阻插入毛细管的一个显着的变化，脑脊液流入毛细管。 7。小心取出毛细管，并通过内部直径1毫米的聚乙烯管材连接3毫升注射器。脑脊液注入到一个预先标记的0.5 mL Eppendorf管，并立即冻结管干冰，然后转移到一个-80 ° C冰箱。 8。脑脊液取样后，肌肉重新对齐，和皮肤缝合（4-0，爱惜康，强生公司）。约1毫升0.9％氯化钠溶液皮下注射，防止脱水。鼠标是保存在孵化器，以保持体温，直到它恢复，监视鼠标的重量是1天，并在手术后1周。 结果与讨论 我们描述了一个高度可靠的协议，从小鼠脑脊液串行采样，没有检测到的血浆污染。 1。整个过程通常需要10分钟（包括麻醉）每鼠。获得脑脊液量是依赖于小鼠品系。的PS / APP双，我们用了2 Tg的小鼠，平均体积约5μL（3至7μL），而P301L（JNPL3）小鼠3产量约10μL，15μL。串行采样，最多7-8μL，可以安全地在每次间隔2-3个月。 2。在手术过程中，它的重要位置上的立体框架鼠标的头部和身体正常，可以充分暴露硬脑膜，使枕大池。小心避免血管穿透硬脑膜毛细管血浆蛋白，可在脑脊液样本中载脂蛋白B（ApoB）4免疫监测，以防止污染。 3。最大限度地减少组织损伤手术在每个重要的是，组织粘连，可以增加诱发出血以下取样的难度。 4。需要手术过程中保持体温，因为麻醉造成的低温可显着地影响大脑中的磷酸化tau水平非常迅速5。 5。抗体酶联免疫吸附协议和抗体，我们用中详细介绍了Refolo等。6。脑脊液μL的PS / APP小鼠会产生令人满意的结果，在稀释1:50-1:60。对于头ELISA法，4-5微升脑脊液P301L（JNPL3）小鼠，将足以为检测总头或头 荧光粉在苏氨酸ylated 231（包从生物源购买）。 6。这种技术可以应用到其他神经系统疾病，脑脊液体积小，其中一个是所需检测的满意的小鼠模型。

<ol>
	<li>Liu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+observation+on+CSF+Abeta42+levels+in+young+to+middle-aged+amyloid+precursor+protein/presenilin-1+doubly+transgenic+mice.">Longitudinal observation on CSF Abeta42 levels in young to middle-aged amyloid precursor protein/presenilin-1 doubly transgenic mice.</a> Neurobiol Dis. 17, 516 (2004).</li><li>Holcomb, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+Alzheimer-type+phenotype+in+transgenic+mice+carrying+both+mutant+amyloid+precursor+protein+and+presenilin+1+transgenes.">Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.</a> Nat Med. 4, 97 (1998).</li><li>Lewis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibrillary+tangles,+amyotrophy+and+progressive+motor+disturbance+in+mice+expressing+mutant+(P301L)+tau+protein.">Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein.</a> Nat Genet. 25, 402 (2000).</li><li>DeMattos, R. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plaque-associated+disruption+of+CSF+and+plasma+amyloid-beta+(Abeta)+equilibrium+in+a+mouse+model+of+Alzheimer's+disease.">Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimer's disease.</a> J Neurochem. 81, 229 (2002).</li><li>Planel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+leads+to+tau+hyperphosphorylation+through+inhibition+of+phosphatase+activity+by+hypothermia.">Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.</a> J Neurosci. 27, 3090 (2007).</li><li>Refolo, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cholesterol-lowering+drug+reduces+beta-amyloid+pathology+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">A cholesterol-lowering drug reduces beta-amyloid pathology in a transgenic mouse model of Alzheimer's disease.</a> Neurobiol Dis. 8, 890 (2001).</li></ol>

我们描述了一个高度可靠的协议，从小鼠脑脊液串行采样，没有检测到的血浆污染。 1。整个过程通常需要10分钟（包括麻醉）每鼠。获得脑脊液量是依赖于小鼠品系。的PS / APP双我们used2的Tg小鼠，平均体积约5μL（3至7μL），而P301L（JNPL3）小鼠3产量约10μL，15μL。串行采样，最多7-8μL，可以安全地在每次间隔2-3个月。 2。在手术过程中，它是很重要的位置上的立体框架鼠标的头部和身体正常，可以充分...

a technique for serial collection of cerebrospinal fluid from the cisterna magna in mouse 

阿尔茨海默氏病（AD）是一种渐进性神经退行性疾病，是由β-淀粉样肽（Aβ）和磷酸化tau蛋白的intraneuronal积累外沉积病理特征。由于脑脊液（CSF）是在与外脑的空间直接接触，它提供了一种在大脑中的反应病理过程中的生化变化的反映。 AD患者脑脊液减少Aβ（Aβ42）的42个氨基酸的形式，并在总tau蛋白和磷酸化tau蛋白的增加，但负责这些变化的机制还没有完全理解。转基因（TG）AD小鼠模型提供了一个极好的机会，探讨如何以及为什么脑脊液中的Aβ或tau蛋白水平变化，随着病情的发展。在这里，我们展示了一个精致脑脊液从鼠标的采样枕大池穿刺技术。这种极其温柔的采样技术可在2-3个月的时间间隔相同的鼠标，从而大大降低鼠之间的Aβ或tau蛋白水平的变化混杂的效果，使得它可以检测到细微的改变，随着时间的推移得到串行脑脊液标本。在与Aβ和tau蛋白的ELISA法的结合，这种技术将被用于旨在探讨在AD小鼠模型中的大脑的脑脊液Aβ42和tau蛋白的水平之间的关系，和他们的新陈代谢的研究。脑脊液的Aβ或头水平的潜力可作为监测疾病的进展生物标志物的Tg小鼠的研究可以提供重要的验证，并监测治疗干预的效果。作为可以牺牲的老鼠的大脑可用于生化或组织学变化研究，脑脊液变化的内在机制，可以更好地进行评估。这些数据可能被解释为人类AD脑脊液变化信息。

Watch this Scientific Journal Video about A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse at JoVE.com

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

转基因（TG）AD小鼠模型提供了一个极好的机会，探讨如何以及为什么脑脊液中的Aβ或tau蛋白水平变化的疾病在人类患者进展。在这里，我们展示了一个串行鼠标脑脊液采样精枕大池穿刺技术。

一个串行收集脑脊液从池鼠标麦格纳技术

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of &#946;-amyloid ...

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Research

JoVE Journal

Biology

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse 

100.9K 次观看.  Columbia University. 转基因（TG）AD小鼠模型提供了一个极好的机会，探讨如何以及为什么脑脊液中的Aβ或tau蛋白水平变化的疾病在人类患者进展。在这里，我们展示了一个串行鼠标脑脊液采样精枕大池穿刺技术。 

视频: A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

科研

生物学

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取：核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

渗透离心技术质外体中提取植物液使用叶<em&gt;菜豆</em&gt;为例

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

大会和“剪切环”中的应用：一种新型内皮模型轨道，单向和定期流体流动和剪应力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

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 ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

从人骨关节炎膝关节收集和RNA提取

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

从成人小鼠腭中分离和培养原代口腔角质形成细胞

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...