1. Estabelecer tridimensionais Co-culturas <ol><li> No dia anterior, que estabelece os co-culturas, remover o Matrigel do congelador e descongelamento em gelo em um 4 ° C frigorífico durante a noite. </li><li> No dia da experiência, preparar o meio de co-cultura, o que corresponde à média para as células epiteliais para ser utilizados (HMEC médio neste exemplo: DME/F12 meio com 10% soro de vitelo, 5 ug / mL de insulina , 1 ug / mL de hidrocortisona, e 1 ng / mL EGF). Manter o meio em gelo horas, pelo menos, 1 antes da mistura com Matrigel. </li><li> Para diluir o Matrigel descongelado, determinar a quantidade total desejado de Matrigel: médio (300 uL por poço de placas de 24 poços) e adicionar metade do volume que de gelado médio HMEC para um tubo estéril em gelo. Em seguida, adicionar o volume meia-mesmo total de Matrigel descongelado para o tubo para atingir uma diluição de 1:1. Manter o tubo em gelo. </li><li> UL lugar 50 de Matrigel: mistura médio meados de bem em poços designadas de uma placa de 24 poços eincubar durante 10 min em um 37 ° C incubadora com ar humidificado e 5% de CO 2. </li><li> Adicionar uma uL 450 adicional de Matrigel: mistura meio aos poços e incubar durante mais 60 minutos. </li><li> Durante o período de incubação, preparar uma suspensão de 1:1 de fibroblastos (aqui Fibroblastos h-TERT-imortalizadas redução mamária que expressam GFP) e as células epiteliais (aqui h-TERT-imortalizada linha celular humana mamária epitelial expressando RFP) com um concentração de 0,5 x 10 5 células cada, em 0,5 ml de meio de HMEC. </li><li> Após o passo incubação 60 minutos, cuidadosamente gota a suspensão da célula para a superfície do gel, solidificado, e voltar a cultura para a incubadora. </li><li> Não perturbar as culturas de 2 dias. Depois disto, meio pode ser trocado a cada 2-3 dias por meio de aspiração muito suavemente a partir do lado do poço e suavemente substituindo com meio HMEC fresco. </li><li> Monitorar formação esferóide diariamente sob microscopia. As culturas são geralmente madura após 10-14 dias. Imagem comoapropriada usando luz ou microscopia de fluorescência. Tamanho esferoidal e comprimento de projecção pode ser medido por microscopia óptica. </li></ol> 2. Isolamento de Células de esferoidal co-culturas <ol><li> Muito delicadamente, pipetar a cultura cima e para baixo 10x utilizando uma agulha estéril 2-mL pipeta de vidro. Alternativamente, cortou a ponta de uma ponta de pipeta de plástico 1000 microlitro com tesoura esterilizados por autoclave ou imersão em etanol a 70%, a fim de pipetar a cultura. Transferir a cultura quer com o vidro pipeta ou a ponta de pipeta de plástico cortada para um tubo de centrífuga estéril de 15 ml e centrifugar durante 5 min a 2000 rpm à temperatura ambiente. Os esferóides celular vai sedimento no fundo. </li><li> Usar uma ponta de pipeta Pasteur estéril para remover suavemente o Matrigel interrompido a partir do topo do tubo centrifugada. </li><li> Adicionar 1 ml de meio HMEC para os esferóides peletizadas, pipetar cima e para baixo 2-3 vezes com uma solução estéril de 2 mL de vidro pipeta, e transferir a cultura para um bem em um de 24 poçosplaca de cultura de tecido. </li><li> Permita que os esferóides para anexar ao prato por pelo menos 48 horas antes de mudar o meio. Ao longo do tempo das células vai deixar as estruturas esferoidais e crescer como monocamadas. </li><li> Passagem da célula culturas quando as células chegam confluência de 50%. Primeiro expandir a cultura para um prato de 35 mm (aproximadamente 2-4 dias), e depois para um prato de 100 mm (aproximadamente 3-5 dias). Imediatamente antes de cada etapa passagem, use uma pipeta de Pasteur estéril para remover manualmente como muitos residuais &quot;esferóides fantasmas&quot; quanto possível. </li><li> Analise as células por citometria de fluxo para a pureza população usando marcadores de células apropriadas de superfície e separar ainda mais diferentes populações de células por separação de células por fluorescência, se necessário. As células isoladas são agora pronto para posterior análise, como desejado. </li></ol> 3. Os resultados representativos Em tridimensional esférica co-culturas, fibroblastos com silenciamento Tiam1 engenharia induzir invasão aumentou em matrix por células epiteliais mamárias (Figura 1, compare D, E, F para A, B, C). Fibroblastos com engenharia a sobre-expressão de Tiam1 induzir invasão da matriz diminuiu a linha celular do cancro da mama SUM159 (Figura 1, compare J, K, L e G, H, I). Uma vez que a co-cultura de esferóides foram estabelecidas, populações de células podem ser isoladas a partir de esferóides por re-plating num contexto bidimensional, como mostrado na Figura 2. Dependendo das taxas de crescimento relativo das células testadas, populações puras podem emergir através da passagem de série (como mostrado na Figura 3) ou podem ser classificados usando marcadores de células adequadas. Após o isolamento a partir de 3D co-cultura, células epiteliais reter as propriedades conferidas por co-cultura com fibroblastos ao longo de períodos de tempo prolongados, permitindo uma ampla oportunidade para análise. Neste exemplo, a exposição a Tiam1 deficientes em fibroblastos induzida o aumento da migração em co-cultivados HMECs que persistiu durante semanas umepois de isolamento a partir de 3D ​​co-cultura (Figura 4). <img alt="A Figura 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> Figura 1. Imagem de esferóide estabelecido co-culturas sob microscopia de luz e de fluorescência. Culturas esferóides são estabelecidos com mamárias linhas de células epiteliais e fibroblastos e deixadas a crescer em cultura durante 10-14 dias. AF: epiteliais = HMEC-mCherry células. GL: células epiteliais = SUM159-mCherry células. CA, GI: controle de fibroblastos = RMF-GFP células. DF: fibroblastos = RMF-GFP com Tiam1 silenciamento. JL: fibroblastos = RMF-GFP com Tiam1 sobre-expressão. As setas indicam projeções epiteliais invasoras na matriz. <img alt="A Figura 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> Figura 2. Imagiologia Serial de culturas sob microscopia de luz e de fluorescência após o isolamento a partir de cultura de Matrigel. Dia 0 representa imagem imediatamente após o isolamento; dias 1, 2 e5 indicam os dias após o isolamento. A coroa de células que saem dos aumentos esferóides ao longo do tempo. Eventualmente, um &quot;fantasma&quot; esferóide com muito poucas células, permanece (seta). Esferóides muito poucos permanecem após a remoção do fantasma e da primeira passagem. <img alt="A Figura 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> Figura 3. Análise de citometria de fluxo de células após o isolamento a partir de Matrigel co-cultura. Depois de esferóide co-culturas amadureceram, as células são isoladas a partir do 3D co-cultura com pipetagem e serial passaging. Alíquotas de amostra são analisadas por citometria de fluxo (neste exemplo, utilizando fluorescência de fluorescência e vermelho verde para identificar GFP-expressando fibroblastos e mCherry-expressando células epiteliais, respectivamente) para determinar as populações relativas de células epiteliais e fibroblastos. <img alt="A Figura 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> Figura 4. Transwell migração de HMECs persiste após isolatia partir de Matrigel co-cultura. Uma vez que populações puras foram obtidos através da cultura ou separação de células por fluorescência, as células podem ser analisadas, como desejado. Aqui a migração de HMECs co-cultivadas com quer de controlo (C) ou Tiam1 deficientes (THA) fibroblastos em toda transwells perfuradas foi avaliada em 2 semanas, 4 e 8 após o isolamento a partir de co-cultura.

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O método aqui apresentado representa uma abordagem simples para analisar os efeitos de fibroblastos estromais em células epiteliais, incluindo as células tumorais. Ela permite a interacção célula-célula directa dentro de um contexto tridimensional suportado por matriz extracelular da membrana basal, enquanto permite a extracção fácil de células a partir da matriz para análise posterior. Isto pode ser aplicada ao estudo de estroma associado ao tumor, como linhas de fibroblastos podem ser manipuladas para afec...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Fenol vermelho-livre </td></tr><tr><td> Hyclone Médio Clássica Líquido: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone soro de vitelo </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insulina </td><td> Merck Química </td><td> 407709 </td><td> Dissolve em 0.005N HCl; 0,22 filtro iM </td></tr><tr><td> Hidrocortisona </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Dissolve-se em EtOH 50%; 0,22 filtro iM </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Dissolver em ácido acético 10 mM; 0,22 mM filtro </td></tr></tbody></table>

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

Enquanto os esforços enormes ter ido para identificar vias de sinalização e moléculas envolvidos em comportamentos de células normais e malignas 1-2, muito desse trabalho tem sido feito utilizando clássicos modelos bidimensionais de cultura de células, que permitem a manipulação de células fácil. Tornou-se claro que as vias de sinalização intracelulares são afetados por forças extracelulares, incluindo dimensionalidade e tensão da superfície da célula 3-4. Várias abordagens têm sido tomadas para desenvolver modelos tridimensionais que representam mais precisamente arquitetura biológica tecido 3. Embora estes modelos incorporam multidimensionalidade e sublinha arquitetônicas, estudo de consequentes efeitos sobre células é menos fácil do que no bidimensional cultura de tecidos, devido às limitações dos modelos e da dificuldade em extrair células para análise posterior. O importante papel do microambiente em torno de tumores na tumorigênese e comportamento tumoral iEstá se tornando cada vez mais reconhecida 4. Estroma tumoral é composto por vários tipos de células e moléculas extracelulares. Durante o desenvolvimento do tumor existem sinais bidireccionais entre as células tumorais e células estromais 5. Apesar de alguns factores que participam no tumor-estroma co-evolução foram identificados, há ainda uma necessidade de desenvolver técnicas simples para identificar sistematicamente e estudar a matriz completa destes 6 sinais. Os fibroblastos são o tipo de célula mais abundante no normais ou associado ao tumor tecidos do estroma, e contribuir para a deposição e manutenção da membrana basal e factores de crescimento parácrinos 7. Muitos grupos têm utilizado sistemas de cultura de três dimensões para estudar o papel de fibroblastos em várias funções celulares, incluindo a resposta do tumor à terapias, o recrutamento de células imunitárias, as moléculas de sinalização, a proliferação, a apoptose, angiogénese e invasão 8-15. Nós otimizamos um método simples para burroessing os efeitos de fibroblastos mamarias em células epiteliais mamárias utilizando um modelo de matriz extracelular comercialmente disponível para criar tridimensionais culturas de populações de células mistas (co-culturas) 16-22. Com continuada co-cultura das células formam esferóides com a aglomeração fibroblastos no interior e as células epiteliais em grande parte, no exterior dos esferóides e formando multicelulares projecções na matriz. Manipulação dos fibroblastos que leva a capacidade de invasão de células epiteliais alteradas pode ser facilmente quantificado por alterações no número e comprimento das projecções epiteliais 23. Além disso, desenvolveram um método para isolamento de células epiteliais de tridimensional co-cultura que facilita a análise dos efeitos da exposição de fibroblastos no comportamento epitelial. Verificou-se que os efeitos da co-cultura persistir durante semanas após o isolamento das células epiteliais, permitindo tempo suficiente para realizar os ensaios múltiplos. Este método é adaptável a células devariando potencial maligno e não requer equipamento especializado. Esta técnica permite a avaliação rápida em modelos de células in vitro sob várias condições, e os resultados correspondentes podem ser comparados com modelos animais in vivo de tecidos, bem como amostras de tecidos humanos.

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

Um método simples é descrita para analisar os efeitos da fibroblastos de tecido em associadas células epiteliais. A combinação deste método eo tridimensional de cultura de tecido pode facilitar a análise das células após o isolamento a partir de 3D. A técnica é aplicável a células de diferentes potencial maligno, permitindo estudo sistemático dos efeitos de estroma associado ao tumor em células tumorais.

Isolamento de células epiteliais mamárias de tridimensional Mixed-célula esferoidal co-cultura

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

isolation-mammary-epithelial-cells-from-three-dimensional-mixed-cell

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Biology

16.0K visualizações.  Tufts Medical Center. Um método simples é descrita para analisar os efeitos da fibroblastos de tecido em associadas células epiteliais. A combinação deste método eo tridimensional de cultura de tecido pode facilitar a análise das células após o isolamento a partir de 3D. A técnica é aplicável a células de diferentes potencial maligno, permitindo estudo sistemático dos efeitos de estroma associado ao tumor em células tumorais.

Vídeo: 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.

Rato células epiteliais mamárias forma Mammospheres durante a diferenciação lactogênica

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

Biologia

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.

Microfluídicos Co-cultura de células epiteliais e bactérias para a Investigação Solúvel sinal mediada por interações

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.

Procedimento de transplante do epitélio mamário

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.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

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.

Isolamento de cromatina por purificação de RNA (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.

Indução e Análise de epitelial para mesenquimal Transição

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.

Isolamento e Cultura da Dental epiteliais células-tronco do Adulto Rato Incisivo

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.

Avaliando as propriedades angiogenéticas de células-tronco do câncer de ovário usando o Sistema de Cocultura Tridimensional, NICO-1

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.

Coloração e Imagem de Alta Resolução de Modelos Organoides e Esferoides Tridimensionais

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.

Isolamento de Células Epiteliais do Folículo Dental Humano

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