1. Establishing Three-dimensional Co-cultures
<ol>
	<li>The day before establishing the co-cultures, remove the Matrigel from the freezer and thaw on ice in a 4 &deg;C refrigerator overnight.</li>
	<li>On the day of the experiment, prepare the medium for co-culture, which corresponds to the medium for the epithelial cells to be used (HMEC medium in this example: DME/F12 medium with 10% bovine calf serum, 5 &mu;g/mL insulin, 1 &mu;g/mL hydrocortisone, and 1 ng/mL EGF). Keep the medium on ice at least 1 hour prior to mixing with Matrigel.</li>
	<li>To dilute the thawed Matrigel, determine the total desired quantity of Matrigel:medium (300 &mu;L per well for 24-well plates) and add half that volume of ice-cold HMEC medium to a sterile tube on ice. Then add the same half-total volume of thawed Matrigel to the tube to achieve a 1:1 dilution. Keep the tube on ice.</li>
	<li>Place 50 &mu;L of Matrigel:medium mixture mid-well in designated wells of a 24-well plate and incubate for 10 min in a 37 &deg;C incubator with humidified air and 5% CO2.</li>
	<li>Add an additional 450 &mu;L of Matrigel:medium mixture to the wells and incubate for another 60 minutes.</li>
	<li>During the incubation, prepare a 1:1 suspension of fibroblasts (here h-TERT-immortalized Reduction Mammary Fibroblasts expressing GFP) and epithelial cells (here h-TERT-immortalized Human Mammary Epithelial Cell line expressing RFP) at a concentration of 0.5 x 105 cells each in 0.5 ml of HMEC medium.</li>
	<li>After the 60-minute incubation step, gently drop the cell suspension onto the top of the solidified gel, and return culture to the incubator.</li>
	<li>Do not disturb the cultures for 2 days. After this, medium can be changed every 2-3 days by very gently aspirating medium from the side of the well and gently replacing with fresh HMEC medium.</li>
	<li>Monitor spheroid formation daily under microscopy. Cultures are generally mature after 10-14 days. Image as appropriate using light or fluorescent microscopy. Spheroid size and projection length can be measured under light microscopy.</li>
</ol>
2. Isolation of Cells from Spheroid Co-cultures
<ol>
	<li>Very gently, pipette the culture up and down 10x using a sterile 2-mL glass pipette. Alternatively, cut off the tip of a 1000 microliter plastic pipette tip using scissors sterilized by autoclaving or immersion in 70% ethanol in order to pipette the culture. Transfer the culture with either the glass pipette or the cut plastic pipette tip to a sterile 15 ml centrifuge tube and centrifuge for 5 min at 2000 rpm at room temperature. The cellular spheroids will sediment at the bottom.</li>
	<li>Use a sterile Pasteur pipette tip to gently remove the disrupted Matrigel from the top of the centrifuged tube.</li>
	<li>Add 1 ml HMEC medium to the pelleted spheroids, pipette up and down 2-3 times with a sterile 2 mL glass pipette, and transfer the culture to a well in a 24-well tissue culture plate.</li>
	<li>Allow the spheroids to attach to the dish for at least 48 hours prior to changing the medium. Over time the cells will leave the spheroidal structures and grow as monolayers.</li>
	<li>Passage the cell cultures when cells reach 50% confluence. First expand the culture to a 35 mm dish (approximately 2-4 days), and then to a 100 mm dish (approximately 3-5 days). Immediately prior to each passage step, use a sterile Pasteur pipette to manually remove as many residual "ghost spheroids" as possible.</li>
	<li>Analyze cells by flow cytometry for population purity using appropriate cell surface markers and further separate different cell populations by fluorescence cell sorting if necessary. The isolated cells are now ready for further analysis as desired.</li>
</ol>
3. Representative Results
In three-dimensional spheroid co-cultures, fibroblasts with engineered Tiam1 silencing induce increased invasion into matrix by mammary epithelial cells (Figure 1, compare D, E, F to A, B, C). Fibroblasts with engineered over-expression of Tiam1 induce decreased matrix invasion by the breast cancer cell line SUM159 (Figure 1, compare J, K, L to G, H, I).
Once co-cultured spheroids have been established, cell populations can be isolated from spheroids by re-plating in a two-dimensional context, as shown in Figure 2. Depending on the relative growth rates of the cells tested, pure populations may emerge through serial passage (as shown in Figure 3) or can be sorted using appropriate cell markers. After isolation from 3D co-culture, epithelial cells retain the properties conferred by co-culture with fibroblasts over extended periods of time, allowing ample opportunity for analysis. In this example, exposure to Tiam1-deficient fibroblasts induced increased migration in co-cultured HMECs that persisted for weeks after isolation from 3D co-culture (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/3760/3760fig1.jpg" /> 
Figure 1. Imaging of established spheroid co-cultures under light and fluorescence microscopy. Spheroid cultures are established with mammary epithelial and fibroblast cell lines and allowed to grow in culture for 10-14 days. A-F: epithelial = HMEC-mCherry cells. G-L: epithelial cells = SUM159-mCherry cells. A-C, G-I: fibroblast = control RMF-GFP cells. D-F: fibroblast = RMF-GFP with Tiam1 silencing. J-L: fibroblast = RMF-GFP with Tiam1 over-expression. Arrows indicate epithelial projections invading into matrix.
<img alt="Figure 2" src="/files/ftp_upload/3760/3760fig2.jpg" /> 
Figure 2. Serial imaging of cultures under light and fluorescence microscopy after isolation from Matrigel culture. Day 0 represents imaging immediately after isolation; days 1, 2 and 5 indicate days after isolation. The corona of cells leaving the spheroid increases over time. Eventually a "ghost" spheroid with very few cells remains (arrow). Very few spheroids remain after ghost removal and first passage.
<img alt="Figure 3" src="/files/ftp_upload/3760/3760fig3.jpg" /> 
Figure 3. Flow cytometry analysis of cells after isolation from Matrigel co-culture. After spheroid co-cultures have matured, cells are isolated from the 3D co-culture with pipetting and serial passaging. Sample aliquots are analyzed by flow cytometry (in this example using green fluorescence and red fluorescence to identify GFP-expressing fibroblasts and mCherry-expressing epithelial cells respectively) to determine relative populations of epithelial and fibroblast cells.
<img alt="Figure 4" src="/files/ftp_upload/3760/3760fig4.jpg" /> 
Figure 4. Transwell migration of HMECs persists after isolation from Matrigel co-culture. Once pure populations have been obtained through culture or fluorescence cell sorting, cells can be analyzed as desired. Here migration of HMECs co-cultured with either control (C) or Tiam1 deficient (shT) fibroblasts across perforated transwells was assessed at 2, 4 and 8 weeks after isolation from co-culture.

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li><li>Kim, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+tissue+culture+models+in+cancer+biology.">Three-dimensional tissue culture models in cancer biology.</a> Semin. Cancer Biol. 15, 365-377 (2005).</li><li>Li, H., Fan, X., Houghton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microenvironment:+the+role+of+the+tumor+stroma+in+cancer.">Tumor microenvironment: the role of the tumor stroma in cancer.</a> J. Cell Biochem. 101, 805-815 (2007).</li><li>Tse, J. C., Kalluri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+metastasis:+epithelial-to-mesenchymal+transition+and+contribution+of+tumor+microenvironment.">Mechanisms of metastasis: epithelial-to-mesenchymal transition and contribution of tumor microenvironment.</a> J. Cell Biochem. 101, 816-829 (2007).</li><li>Bhowmick, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+in+fibroblasts+modulates+the+oncogenic+potential+of+adjacent+epithelia.">TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia.</a> Science. 303, 848-851 (2004).</li><li>Bhowmick, N. A., Moses, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-stroma+interactions.">Tumor-stroma interactions.</a> Curr. Opin. Genet. Dev. 15, 97-101 (2005).</li><li>Havlickova, B., Biro, T., Mescalchin, A., Arenberger, P., Paus, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+optimization+of+an+organotypic+assay+system+that+imitates+human+hair+follicle-like+epithelial-mesenchymal+interactions.">Towards optimization of an organotypic assay system that imitates human hair follicle-like epithelial-mesenchymal interactions.</a> Br. J. Dermatol. 151, 753-765 (2004).</li><li>Kilani, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+cytotoxicity+of+gemcitabine+in+bladder+cancer+cell+lines.">Selective cytotoxicity of gemcitabine in bladder cancer cell lines.</a> Anticancer Drugs. 13, 557-566 (2002).</li><li>Seidl, P., Huettinger, R., Knuechel, R., Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+fibroblast-tumor+cell+interaction+causes+downregulation+of+RACK1+mRNA+expression+in+breast+cancer+cells+in+vitro.">Three-dimensional fibroblast-tumor cell interaction causes downregulation of RACK1 mRNA expression in breast cancer cells in vitro.</a> Int. J. Cancer. 102, 129-136 (2002).</li><li>Silzle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+fibroblasts+recruit+blood+monocytes+into+tumor+tissue.">Tumor-associated fibroblasts recruit blood monocytes into tumor tissue.</a> Eur. J. Immunol. 33, 1311-1320 (2003).</li><li>Bartling, B., Demling, N., Silber, R. E., Simm, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferative+stimulus+of+lung+fibroblasts+on+lung+cancer+cells+is+impaired+by+the+receptor+for+advanced+glycation+end-products.">Proliferative stimulus of lung fibroblasts on lung cancer cells is impaired by the receptor for advanced glycation end-products.</a> Am. J. Respir. Cell Mol. Biol. 34, 83-91 (2006).</li><li>Kunz-Schughart, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+fibroblasts+to+regulate+the+formation+of+three-dimensional+vessel-like+structures+from+endothelial+cells+in+vitro.">Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro.</a> Am. J. Physiol. Cell Physiol. 290, C1385-C1398 (2006).</li><li>Liu, T., Lin, B., Qin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinoma-associated+fibroblasts+promoted+tumor+spheroid+invasion+on+a+microfluidic+3D+co-culture+device.">Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device.</a> Lab Chip. 10, 1671-1677 (2010).</li><li>Zengel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimodal+therapy+for+synergic+inhibition+of+tumour+cell+invasion+and+tumour-induced+angiogenesis.">Multimodal therapy for synergic inhibition of tumour cell invasion and tumour-induced angiogenesis.</a> BMC Cancer. 10, 92-92 (2010).</li><li>Kunz-Schughart, L. A., Heyder, P., Schroeder, J., Knuechel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heterologous+3-D+coculture+model+of+breast+tumor+cells+and+fibroblasts+to+study+tumor-associated+fibroblast+differentiation.">A heterologous 3-D coculture model of breast tumor cells and fibroblasts to study tumor-associated fibroblast differentiation.</a> Exp. Cell Res. 266, 74-86 (2001).</li><li>Djordjevic, B., Lange, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cell+interactions+in+spheroids+maintained+in+suspension.">Cell-cell interactions in spheroids maintained in suspension.</a> Acta Oncol. 45, 412-420 (2006).</li><li>Hirschhaeuser, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> J. Biotechnol. 148, 3-15 (2010).</li><li>Hoevel, T., Macek, R., Swisshelm, K., Kubbies, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reexpression+of+the+TJ+protein+CLDN1+induces+apoptosis+in+breast+tumor+spheroids.">Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids.</a> Int. J. Cancer. 108, 374-383 (2004).</li><li>Paduch, R., Kandefer-Szerszen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitamin+D,+tamoxifen+and+beta-estradiol+modulate+breast+cancer+cell+growth+and+interleukin-6+and+metalloproteinase-2+production+in+three-dimensional+co-cultures+of+tumor+cell+spheroids+with+endothelium.">Vitamin D, tamoxifen and beta-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium.</a> Cell. Biol. Toxicol. 21, 247-256 (2005).</li><li>Timmins, N. E., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+multicellular+tumor+spheroids+by+the+hanging-drop+method.">Generation of multicellular tumor spheroids by the hanging-drop method.</a> Methods Mol. Med. 140, 141-151 (2007).</li><li>Foty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Hanging+Drop+Cell+Culture+Protocol+for+Generation+of+3D+Spheroids.">A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids.</a> J. Vis. Exp. (51), e2720-e2720 (2011).</li><li>Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+fibroblast+Tiam1+in+tumor+cell+invasion+and+metastasis.">The role of fibroblast Tiam1 in tumor cell invasion and metastasis.</a> Oncogene. 29, 6533-6542 (2010).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Il metodo qui presentato rappresenta un approccio semplice per analizzare gli effetti di fibroblasti stromali sulle cellule epiteliali, comprese le cellule tumorali. Esso consente diretto cellula-cellula interazione all&#39;interno di un contesto tridimensionale supportato da matrice extracellulare membrana basale, pur consentendo una facile estrazione delle celle della matrice per ulteriori analisi. Questo può essere applicato allo studio del tumore-associato stroma, come linee di fibroblasti possono essere manipolati...

<table border="1"><tbody><tr><td> Nome di reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> BD Matrigel </td><td> BD Biosciences </td><td> 356237 </td><td> Fenolo-rosso senza </td></tr><tr><td> Hyclone Media Classical liquido: DMEM F12 </td><td> ThermoScientific </td><td> SH30023.01 </td><td></td></tr><tr><td> Hyclone Bovine Calf Serum </td><td> ThermoScientific </td><td> SH3007303 </td><td></td></tr><tr><td> Insulina </td><td> EMD Chemicals </td><td> 407709 </td><td> Sciogliere in 0.005N HCl; 0,22 uM filtro </td></tr><tr><td> Idrocortisone </td><td> Sigma-Aldrich </td><td> H4001 </td><td> Sciogliere in 50% EtOH; 0,22 uM filtro </td></tr><tr><td> EGF </td><td> Sigma-Aldrich </td><td> E9644 </td><td> Sciogliere in 10mM acido acetico; 0,22 uM filtro </td></tr></tbody></table>

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

Mentre enormi sforzi sono andati ad identificare vie di segnalazione e le molecole coinvolte in comportamenti di cellule normali e maligne 1-2, gran parte di questo lavoro è stato effettuato utilizzando classici bidimensionali modelli di coltura cellulare, che consentono la manipolazione cellulare facile. E &#39;diventato chiaro che vie di segnalazione che sono influenzati da forze extracellulari, tra cui dimensionalità e tensione superficiale delle cellule 3-4. Molteplici approcci sono state prese per sviluppare modelli tridimensionali che rappresentano il tessuto più accuratamente l&#39;architettura biologica 3. Mentre questi modelli incorporano multidimensionalità e sollecitazioni architettoniche, lo studio degli effetti conseguenti sulle cellule è meno facile rispetto a bidimensionale coltura tissutale a causa dei limiti dei modelli e la difficoltà di estrazione di cellule per la successiva analisi. L&#39;importante ruolo del microambiente intorno tumori nella tumorigenesi e il comportamento del tumore is sempre più riconosciuta 4. Stroma tumorale è composta da più tipi di cellule e molecole extracellulari. Durante lo sviluppo del tumore sono segnali bidirezionali tra cellule tumorali e cellule stromali 5. Sebbene alcuni fattori che partecipano stroma del tumore-co-evoluzione sono stati identificati, vi è ancora la necessità di sviluppare tecniche semplici per identificare sistematicamente e studiare la gamma completa di questi segnali 6. I fibroblasti sono il tipo cellulare più abbondante nei tessuti normali stromali o tumore-associato, e contribuire alla deposizione e alla manutenzione della membrana basale e fattori di crescita paracrini 7. Molti gruppi hanno utilizzato tre sistemi di coltura tridimensionale per studiare il ruolo dei fibroblasti su varie funzioni cellulari, compresa la risposta del tumore alle terapie, il reclutamento delle cellule immunitarie, le molecole di segnalazione, la proliferazione, l&#39;apoptosi, l&#39;angiogenesi e l&#39;invasione 8-15. Abbiamo ottimizzato un metodo semplice per assessing gli effetti di fibroblasti mammarie su cellule epiteliali mammarie utilizzando un modello disponibile in commercio matrice extracellulare per creare tridimensionali colture miste di popolazioni cellulari (co-colture) 16-22. Con continua co-coltura delle cellule formano sferoidi con il raggruppamento fibroblasti all&#39;interno e le cellule epiteliali ampiamente sulla parte esterna degli sferoidi e formando multicellulari sporgenze nella matrice. Manipolazione dei fibroblasti che porta alla alterato l&#39;invasività delle cellule epiteliali possono essere facilmente quantificato dai cambiamenti del numero e la lunghezza delle proiezioni epiteliali 23. Inoltre, abbiamo sviluppato un metodo per isolare le cellule epiteliali di tridimensionale co-cultura che facilita l&#39;analisi degli effetti dell&#39;esposizione fibroblasti sul comportamento epiteliale. Abbiamo trovato che gli effetti della co-coltura persistere per settimane dopo l&#39;isolamento delle cellule epiteliali, consentendo tempo sufficiente per effettuare saggi multipli. Questo metodo è adattabile a cellulevariabile potenziale maligno e non richiede attrezzature specializzate. Questa tecnica permette di valutazione rapida dei modelli in vitro di cellule in condizioni diverse, ed i corrispondenti risultati possono essere confrontati con tessuto in modelli animali in vivo così come campioni di tessuti umani.

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

Un semplice metodo è descritto per analizzare gli effetti di fibroblasti su tessuti associati cellule epiteliali. La combinazione di questo metodo e tridimensionale coltura tissutale può facilitare l&#39;analisi di cellule dopo l&#39;isolamento da 3D. La tecnica è applicabile a celle di varia potenziale maligno, consentendo studio sistematico di effetti di tumore-associato stroma sulle cellule tumorali.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid 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

Research

JoVE Journal

Biology

16.0K Views.  Tufts Medical Center. Un semplice metodo è descritto per analizzare gli effetti di fibroblasti su tessuti associati cellule epiteliali. La combinazione di questo metodo e tridimensionale coltura tissutale può facilitare l'analisi di cellule dopo l'isolamento da 3D. La tecnica è applicabile a celle di varia potenziale maligno, consentendo studio sistematico di effetti di tumore-associato stroma sulle cellule tumorali.

Video: 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.

Topo cellule epiteliali mammarie forma Mammospheres durante la differenziazione lattogenica

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

Ricerca

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.

Microfluidica Co-coltura di cellule epiteliali e batteri per le indagini solubile del segnale mediato da Interazioni

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.

Procedura di trapianto epiteliali mammarie

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 la coltura di cellule staminali epiteliali da pelle umana

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 di cromatina Purificazione RNA (disturbo)

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.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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 della Dental epiteliali cellule staminali del topo adulto Incisor

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.

Valutazione delle proprietà angiogenetiche delle cellule staminali del cancro ovarico utilizzando il sistema di co-coltura tridimensionale, 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.

Colorazione e imaging ad alta risoluzione di modelli organoidi e sferoidi tridimensionali

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 delle cellule epiteliali dal follicolo dentale umano

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