1。建立三维共培养<ol><li>建立联合培养的前一天，从4℃冰箱中冷冻和解冻冰基底膜一夜之间。 </li><li>在当天的实验，准备共同的文化，它对应的上皮细胞，可用于介质（在这个例子中的三菱电梯中等介质：DME/F12介质与10％小牛血清，5微克/毫升胰岛素1微克/毫升氢化可的松，1毫微克/毫升表皮生长因子）。保持冰至少1小时前与基底膜混合介质。 </li><li>冲淡了解冻的基底膜，确定总的基底膜所需数量：中型（300μL，每口井的24孔板），并添加冰冷的三菱电梯中等量的一半，上冰的无菌管。然后加入相同的半解冻基底膜总量管达到1:1稀释。管置于冰上。 </li><li> 50μL基底膜：中等混合中旬在指定的24孔板井井孵育10分钟在37℃孵化器与湿润的空气和5％的CO 2。 </li><li>添加一个额外的基底膜450微升：中等混合井和孵育60分钟。 </li><li>孵化期间，准备在浓度为0.5×10的成纤维细胞（H-叔永生减少乳腺成纤维细胞表达GFP）和上皮细胞（H-叔永生化人类乳腺上皮细胞系表达利福平）1:1暂停5细胞每0.5毫升三菱电梯中等。 </li><li>经过60分钟的孵化步骤，轻轻地放到固化凝胶的顶部细胞悬液，并返回文化的孵化器。 </li><li>请勿打扰2天的文化。在此之后，媒体可以改变每2-3天非常轻轻地吸气和更换新鲜的三菱电梯介质轻轻从一侧的媒介。 </li><li>每天监测显微镜下的球体形成。文化一般经过10-14天成熟。形象适当使用光或荧光显微镜。光学显微镜下可测椭球的大小和投影长度。 </li></ol> 2。从球体共培养的细胞的分离<ol><li>非常轻柔，吸取文化和使用无菌的2 mL的玻璃吸管下降10倍。另外，切断了1000微升塑料吸管尖端使用高压灭菌或70％的乙醇浸泡消毒的剪刀，以吸取文化的一角。转让与玻璃吸管或无菌的15 mL离心管切割塑料枪头，在2000年，在室温下转离心5分钟的文化。细胞球体将在底部的沉积物。 </li><li>使用无菌巴斯德枪头轻轻地清除破坏基底膜的离心管顶部。 </li><li>加入1毫升三菱电梯中等颗粒球体，吸取2毫升无菌玻璃吸管的2-3倍，在24孔和转让，以良好的文化组织培养板。 </li><li>允许的球体附加到盘前至少48小时，以改变介质。随着时间的推移，细胞会离开的球状结构，并成长为单层。 </li><li>通过细胞培养，当细胞达到50％汇合。首先，扩大文化，以35毫米的菜（约2-4天），然后到100毫米菜（约3-5天）。紧接每个通道步骤，使用无菌巴斯德吸管许多残余的“鬼球体”尽可能手动删除。 </li><li>流动人口使用适当的细胞表面标志物，并进一步区分不同细胞群的纯度，如果有必要的荧光细胞分选仪分析细胞。孤立的细胞现在准备需要作进一步的分析。 </li></ol> 3。代表结果在三维球体的共同文化，与工程Tiam 1 mRNA沉默的成纤维细胞诱导成马增加入侵乳腺上皮细胞基质（ 图1，比较D，E，F到一，乙，丙类）。与成纤维细胞工程的Tiam 1 mRNA的过度表达诱导的乳腺癌细胞株SUM159（ 图1，比较G，H，J，K L来）减少矩阵入侵。 一旦已经建立了合作培养球体，细胞群可以被孤立在一个二维的情况下再镀球体， 如图2所示。根据测试细胞的相对增长率，纯种群可能会出现通过串行通道（ 如图3所示），或可以使用适当的细胞标记进行排序。从3D共培养分离后，上皮细胞保留了长时间的成纤维细胞共培养赋予的属性，允许充分的机会进行分析。在这个例子中，暴露Tiam 1 mRNA的缺陷成纤维细胞诱导合作培养HMECs的移民增加，坚持几个星期1fter隔离3D共培养（ 图4）。 <img alt="图1" src="/files/ftp_upload/3760/3760fig1.jpg" /> 图1。成像光镜和荧光显微镜下建立球体的共同文化。球体文化的建立与乳腺上皮细胞和成纤维细胞系，并允许在文化成长为10-14天。自动对焦：上皮=三菱电梯mCherry细胞。 GL：上皮细胞= SUM159-mCherry细胞的。交流，GI：成纤维细胞=控制旋转磁场-GFP的细胞。 DF：成纤维细胞= RMF的绿色荧光蛋白与Tiam 1 mRNA沉默。 JL：成纤维细胞= RMF的绿色荧光蛋白与Tiam 1 mRNA的过度表达。箭头表示成矩阵入侵上皮预测。 <img alt="图2" src="/files/ftp_upload/3760/3760fig2.jpg" /> 图2。光，荧光显微镜从基底膜文化隔离后下的文化序列成像。 0天表示成像后，立即隔离; 1天，25表明天隔离后。离开球体随着时间的推移增加细胞日冕。最后一个“鬼”有极少数细胞球体仍然（箭头）。很少球体后仍然鬼拆除和第一段。 <img alt="图3" src="/files/ftp_upload/3760/3760fig3.jpg" /> 图3。流流式细胞仪分析细胞从基底膜共培养后的隔离。后球体的共同的文化已经成熟，从移液器3D共培养和串行传代细胞分离。流式细胞仪检测样品等分（在这个例子中使用绿色荧光和红色荧光标识的GFP分别表达mCherry表达的成纤维细胞和上皮细胞的），以确定相对人口的上皮细胞和成纤维细胞。 <img alt="图4" src="/files/ftp_upload/3760/3760fig4.jpg" /> 图4。Transwell小室迁移的HMECs后仍然isolati从基底膜合作的文化。一旦纯种群已获得通过文化或荧光细胞分选，细胞可以分析的需要。在这里，无论是对照组（C）或Tiam 1 mRNA缺陷（SHT）跨穿孔transwells的成纤维细胞共培养HMECs迁移隔离共培养2，4和8周后评估。

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没有利益冲突的声明。

这里介绍的方法代表着一个简单的方法分析，包括肿瘤细胞的上皮细胞间质成纤维细胞的影响。它允许直接的细胞内细胞外基底膜基质的三维背景的互动，作进一步的分析，同时使基质细胞容易提取。这可以应用于研究肿瘤相关基质，成纤维细胞系可以被操纵，影响信号通路的选择和上皮线，可以在不同的侵入性的潜力，如在图1所示。三维共培养模型，使细胞侵袭的变化已经引起视?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>屋宇署基底膜</td><td> BD公司</td><td> 356237 </td><td>酚红免费</td></tr><tr><td>胎牛血清古典液体培养基：DMEM培养基F12键</td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td>小牛血清胎牛血清牛</td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td>胰岛素</td><td>电解二氧化锰化学品</td><td> 407709 </td><td>溶于盐酸0.005N; 0.22微米过滤器</td></tr><tr><td>氢化可的松</td><td> Sigma-Aldrich公司</td><td> H4001 </td><td>溶于50％乙醇; 0.22微米过滤器</td></tr><tr><td>表皮生长因子</td><td> Sigma-Aldrich公司</td><td> E9644 </td><td> 10mm的醋酸中溶解; 0.22微米过滤器</td></tr></tbody></table>

isolation of mammary epithelial cells from three-dimensional mixed-cell spheroid co-culture

虽然已经进入了巨大的努力识别信号通路和分子参与正常细胞和恶性行为1-2，使用传统的二维细胞培养模型，这项工作已完成，允许细胞操纵方便。它已成为明确的细胞内信号通路的影响外的力量，包括维和细胞的表面张力3-4。已采取多种途径，开发三维模型，更准确地反映生物组织架构3。虽然这些模型纳入多维度和建筑讲，由此产生的对细胞影响的研究是比在两维模型的局限性和困难，在随后的分析中提取细胞组织培养的简便。 重要作用，肿瘤周围的微环境在肿瘤发生的行为我之所以成为越来越认识4。肿瘤基质是由多种细胞类型和细胞外分子。在肿瘤的发展有肿瘤细胞和基质细胞之间的双向信号。虽然参与肿瘤基质协同进化的一些因素已经确定，仍然有必要制定简单的技术，系统地识别和研究这些信号6阵列。成纤维细胞是最丰富的细胞类型，在正常或肿瘤相关间质组织，促进基底膜和旁分泌生长因子7的沉积和维护。 许多团体已经使用三维培养系统研究成纤维细胞的各种细胞功能的作用，包括肿瘤反应治疗，免疫细胞招聘，信号分子，细胞增殖，凋亡，血管生成，侵袭8-15。我们已经优化了屁股的简单方法对乳腺上皮细胞，使用市售的细胞外基质的模型来创建三维混合细胞群（联合培养）16-22文化essing乳腺成纤维细胞的影响。续共培养细胞的形成与球体内部的聚类成纤维细胞和上皮细胞很大程度上取决于外部的球体，形成多细胞的预测，到矩阵。操纵改变上皮细胞的侵袭，导致成纤维细胞可以很容易量化的数量和长度23上皮预测的变化。此外，我们已经制定为隔离上皮细胞三维共培养，促进成纤维细胞的对上皮行为暴露的影响分析的方法。我们已经找到了合作文化的影响，持续数周后上皮细胞的分离，允许充裕的时间来执行多个检测。这种方法是适应细胞不同恶性潜能，不需要专门的设备。这种技术允许多个条件下，在体外细胞模型的快速评估和相应的结果，可以在动物体内组织模型以及人体组织样本。

Watch this Scientific Journal Video about Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture at JoVE.com

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

被形容为一个简单的方法，分析相关的上皮细胞组织成纤维细胞的影响。这种方法和三维组织培养相结​​合，可以方便从3D隔离后的细胞分析。该技术适用于不同恶性潜能的细胞，使系统的研究对肿瘤细胞的肿瘤相关基质的影响。

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

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JoVE Journal

Biology

16.0K 次观看.  Tufts Medical Center. 被形容为一个简单的方法，分析相关的上皮细胞组织成纤维细胞的影响。这种方法和三维组织培养相结​​合，可以方便从3D隔离后的细胞分析。该技术适用于不同恶性潜能的细胞，使系统的研究对肿瘤细胞的肿瘤相关基质的影响。

视频: Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

科研

生物学

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

微流体调查可溶性信号介导的相互作用的上皮细胞和细菌的共同文化

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

乳腺上皮移植手术

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA纯化的染色质隔离（CHIRP）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

使用三维共培养系统NICO-1评估卵巢癌干细胞样细胞的血管遗传特性

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

三维类器官和球状体模型的染色和高分辨率成像

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...

Isolation of Epithelial Cells from Human Dental Follicle

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was performed on the day of DF harvest. The DF was washed three times with DPBS and then dissected with tissue scissors until the tissue had a pulpy or squishy consistency. Single-cell populations were pelleted by centrifugation and washed with keratinocyte serum-free medium. Heterogeneous cell populations were distributed in a culture dish. Keratinocyte serum-free medium was used to select the epithelial cells. The culture medium was changed daily until no floating debris or dead cells were observed. Epithelial cells appeared within 7-10 days after cell population distribution. Epithelial cells survived in serum-free medium, while &#945;-modification minimal essential medium supplemented with 10% fetal bovine serum allowed the proliferation of mesenchymal-type cells. The DF is a tissue source for the isolation of dental epithelial cells.
The purpose of this study was to establish a method for the isolation of epithelial cells from human DF. Periodontal ligament (PDL) was used for the isolation of human dental epithelial cells. Procuring epithelial cells from human PDL is not always successful due to the small tissue volume, leading to low numbers of epithelial cells. DF has a larger volume than PDL and contains more cells. DF can be a tissue source for the primary culture of human dental epithelial cells. This protocol is easier and more efficient than the isolation method using PDL. Procuring human dental epithelial cells may facilitate further studies of dental epithelial-mesenchymal interactions.

The dental follicle contains an epithelial population and mesenchymal cells. The epithelial population was selected from the heterogeneous dental follicle cell population by providing a distinct culture medium. Epithelial cells survived and formed colonies in a serum-free medium.

从人牙囊中分离上皮细胞

The dental follicle (DF) was harvested during the removal of an impacted third molar by an oral maxillofacial surgeon. Epithelial cell isolation was ...