מחקר ב Spagnoli המעבדה. ממומן על ידי אגודת הלמהולץ, FP7-IRG-2008-ENDOPANC המענק וגרנט HEPATOPANCREATIC ERC-2009 החל-.

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	<li>Slack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+biology+of+the+pancreas.">Developmental biology of the pancreas.</a> Development. 121, 1569-1580 (1995).</li><li>Pan, F., Wright, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreas+organogenesis:+from+bud+to+plexus+to+gland.">Pancreas organogenesis: from bud to plexus to gland.</a> Dev. Dyn. 240, 530-565 (2011).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+Plasticity+within+the+Pancreas-+Lessons+Learned+from+Development.">Cellular Plasticity within the Pancreas- Lessons Learned from Development.</a> Developmental Cell. 18, 342-356 (2010).</li><li>Spagnoli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+endoderm+to+pancreas:+a+multistep+journey.">From endoderm to pancreas: a multistep journey.</a> Cell. Mol. Life Sci. 64, 2378-2390 (2007).</li><li>Hick, A. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+primitive+duct+formation+in+the+pancreas+and+submandibular+glands:+a+role+for+SDF-1.">Mechanism of primitive duct formation in the pancreas and submandibular glands: a role for SDF-1.</a> BMC Dev. Biol. 9, 1-17 (2009).</li><li>Villasenor, A., Chong, D., Henkemeyer, M., Cleaver, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+dynamics+of+pancreatic+branching+morphogenesis.">Epithelial dynamics of pancreatic branching morphogenesis.</a> Development. 137, 4295-4305 (2010).</li><li>Kesavan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc42-Mediated+Tubulogenesis+Controls+Cell+Specification.">Cdc42-Mediated Tubulogenesis Controls Cell Specification.</a> Cell. 139, 791-801 (2009).</li><li>Zhou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multipotent+Progenitor+Domain+Guides+Pancreatic+Organogenesis.">A Multipotent Progenitor Domain Guides Pancreatic Organogenesis.</a> Developmental Cell. 13, 103-114 (2007).</li><li>Percival, A., Slack, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+pancreatic+development+using+a+cell+lineage+label.">Analysis of pancreatic development using a cell lineage label.</a> Exp. Cell Res. 247, 123-132 (1999).</li><li>Miralles, F., Czernichow, P., Ozaki, K., Itoh, N., Scharfmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+through+fibroblast+growth+factor+receptor+2b+plays+a+key+role+in+the+development+of+the+exocrine+pancreas.">Signaling through fibroblast growth factor receptor 2b plays a key role in the development of the exocrine pancreas.</a> Proc. Natl. Acad. Sci. U.S.A. 96, 6267-6272 (1999).</li><li>Puri, S., Hebrok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+embryonic+pancreas+development+using+real-time+imaging.">Dynamics of embryonic pancreas development using real-time imaging.</a> Dev. Biol. 306, 82-93 (2007).</li><li>Magenheim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, 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> Manipulating the Mouse Embryo: A Laboratory Manual. , (2003).</li><li>Horb, L. D., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+cell+division+in+branching+morphogenesis+and+differentiation+of+the+embryonic+pancreas.">Role of cell division in branching morphogenesis and differentiation of the embryonic pancreas.</a> Int. J. Dev. Biol. 44, 791-796 (2000).</li><li>Muzumdar, M., Tasic, B., Miyamichi, K., Li, L., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+global+double-fluorescent+Cre+reporter+mouse.">A global double-fluorescent Cre reporter mouse.</a> Genesis. 45, 593-605 (2007).</li></ol>

אין ניגודי האינטרסים הכריזו.

ברגע שגורל לבלב מצוין, ותאי לבלב לעבור התפשטות נרחבת, בידול והמורפוגנזה סופו של דבר ליצירת איבר בוגר ותפקודי 2,4. נכון לעכשיו, איך ההסתעפות מתרחשת בלבלב ואיך זה קשור לריבוי מולידו ובידול היא במידה רבה לא ידוע. תרבויות explant לבלב מייצגות מערכת אידיאלית להבהיר תהליכ...

<table border="1" style=";text-align:right;direction:rtl"><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> נוגדנים: Carboxypeptidase E-cadherin ה-F-תקטין גלוקגון אינסולין β1-integrin Pdx1 Pdx1 Phospho-היסטון H3 </td><td> עבד Serotec Invitrogen בדיקות מולקולריות ImmunoStar Millipore Millipore Abcam היברידומה בנק איתות תא </td><td> 1810-0006 13-1900 -12373 20076 4011-01 MAB1997 ab47267 F109-D12 9706 </td><td></td></tr><tr><td> אסאל בינוני נשר (BME) </td><td> סיגמא </td><td> B1522-500 מ&quot;ל </td><td> המשיך בתנאים סטריליים </td></tr><tr><td> מי כיתת תרבית תאים </td><td> Paa </td><td> S15-012 </tד&gt; <td> המשיך בתנאים סטריליים </td></tr><tr><td> מנות תרבות (בעלת קרקעית זכוכית), 35-מ&quot;מ </td><td> מאטק תאגיד </td><td> P35G-0-20-C </td><td></td></tr><tr><td> דונקי סרום </td><td> Chemicon </td><td> S30-100 מ&quot;ל </td><td></td></tr><tr><td> עגל עוברי סרום זהב </td><td> Paa </td><td> A15-151 </td><td> המשיך בתנאים סטריליים </td></tr><tr><td> פיברונקטין </td><td> Invitrogen </td><td> 330100-8 </td><td> מניית סול. 1 מ&quot;ג / מ&quot;ל ​​במי כיתת תרבית תאים </td></tr><tr><td> גנטמיצין </td><td> Invitrogen </td><td> 15750-037 </td><td> המשיך בתנאים סטריליים </td></tr><tr><td> גלוטמין </td><td> Invitrogen </td><td> 25030-024 </td><td> המשיך בתנאים סטריליים </td></tr><tr><td> Multidishes 4-כן </td><td> Nunc </td><td> 176740 </td><td></td></tr><tr><td> מיקרוסקופים: Confocal מיקרוסקופ ההפוך (LSM 700) סטראו (גילוי V12) </td><td> Zeiss Zeiss </td><td></td><td> מטרות: C-Apochromat 10X / 0.45 W M27 (work. dist 1.8 מ&quot;מ;. עומק הדמיה ~ 100 מ&quot;מ); C-Apochromat 40X / 1.2 W הקור M27 (work. 0.28 מ&quot;מ dist;. ~ עומק ההדמיה 50 מיקרומטר) קוף מלמטה ותאורת סיבים אופטיים מלמעלה </td></tr><tr><td> Paraformaldehyde </td><td> רוט </td><td> 0,335.3 </td><td> פתרון מנייה 20% </td></tr><tr><td> Pipet פסטר (זכוכית), 150 מ&quot;מ </td><td> VWR </td><td> HECH567 / 1 </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין </td><td> Paa </td><td> P11-010 </td><td> המשיך בג סטריליonditions </td></tr><tr><td> צלחות פטרי, 60 מ&quot;מ </td><td> גריינר ביו אחד </td><td> 628102 </td><td></td></tr><tr><td> צלחות פטרי, 35 מ&quot;מ </td><td> גריינר ביו אחד </td><td> 627161 </td><td></td></tr><tr><td> 1X PBS, pH7.4 </td><td> Paa </td><td> H15-002 </td><td> המשיך בתנאים סטריליים </td></tr><tr><td> להב מספרי 8 מ&quot;מ האביב מעוקל </td><td> כלי מדע פיין </td><td> 15023-10 </td><td></td></tr><tr><td> טריטון-x100 </td><td> רוט </td><td> 3051.3 </td><td></td></tr><tr><td> של שען foreceps דומון # 5 </td><td> רוט </td><td> K342.1 </td><td></td></tr></table>

a system for ex vivo culturing of embryonic pancreas

הלבלב שולט בתפקודים חיוניים של הגוף שלנו, כולל ייצור של אנזימי עיכול ופיקוח על רמות סוכר בדם 1. אמנם בעשור האחרון מחקרים רבים תרמו ליסודות מוצקים להבנת organogenesis לבלב, פערים חשובים להתמיד בידע של היווצרות לבלב המוקדמת 2 שלנו. הבנה מלאה של האירועים המוקדמים הללו תספק תובנה לתוך הפיתוח של איבר זה, אלא גם למחלות חשוכות מרפא המתמקדות בלבלב, כמו סוכרת או סרטן הלבלב. לבסוף, את המידע הזה יפיק תכנית לפיתוח טיפולים להחלפת תא בהקשר של סוכרת. במהלך התפתחות עוברת, הלבלב נובע מאוטופיות צמחה עובריות שונות של האנדודרם המעי הקדמי גב וגחון ביום עוברי (E) 9.5 בעבר עכבר 3,4. שני האוטופיות צמח evaginate לmesenchyme מקיף כepithelia מוצקניצני הליטר, שעוברים התפשטות, מסתעף ובידול לייצר איבר בגרות מלאה 2,5,6. העדויות האחרונות הראו כי צמיחה והתבדלות של תאי לבלב שושלות, כולל את β-תאים המייצרים אינסולין-, תלויות ברקמות ארכיטקטורת שיפוץ ראוי, אפיתל ומיצוב תא בתוך אפיתל לבלב הסתעפות 7,8. עם זאת, איך מסתעף המורפוגנזה מתרחשת ומתואם עם ריבוי ובידול בלבלב הוא ידוע ברובם. זה בחלקו בשל העובדה שהידע נוכחי על תהליכים התפתחותיים אלה הסתמך כמעט אך ורק על ניתוח של דגימות קבועות, תוך אירועים המוךפו&quot;גנטי הם דינמיים מאוד. כאן, אנו מדווחים שיטה לvivo culturing העכבר העוברי לבלב הניצנים לשעבר במנות תחתית זכוכית, המאפשרות הדמיה ישירה של לבלב הפיתוח (איור 1) ולנתח. כת זומערכת יור פתח באופן אידיאלי למיקרוסקופיה confocal סריקת הליזר, ובהדמיה בפרט, חי תאים. explants לבלב יכול להיות מוכן לא רק מעוברי עכברי סוג פראי, אלא גם מזני עכבר מהונדס גנטי (למשל מהונדס או נוקאאוט), המאפשר לימודים בזמן אמיתי של פנוטיפים מוטציה. יתר על כן, מערכת תרבות vivo לשעבר זה יקר כדי לחקור את ההשפעות של תרכובות כימיות על התפתחות לבלב, ומאפשר לקבל מידע כמוני על שגשוג וצמיחה, התארכות, הסתעפות, tubulogenesis והבחנה. לסיכום, הפיתוח של שיטת vivo לשעבר לבלב explant תרבות בשילוב עם הדמיה ברזולוציה גבוהה מספק פלטפורמה חזקה להתבוננות אירועים המוךפו&quot;גנטי ובידול כפי שהם מתרחשים בתוך עובר העכבר המתפתח.

Watch this Scientific Journal Video about A System for ex vivo Culturing of Embryonic Pancreas at JoVE.com

A System for ex vivo Culturing of Embryonic Pancreas

כאן, אנו מתארים שיטה לבידוד, תרבות ומניפולציה של לבלב העוברי של עכבר. זה מייצג מצוין<em&gt; Vivo לשעבר</em&gt; מערכת ללימוד היבטים שונים של פיתוח לבלב, כולל המורפוגנזה, התמיינות וגדילה. explants ניצן הלבלב יכול להיות מתורבת במשך כמה ימים ולהשתמש במגוון של יישומים שונים, כולל כל הר-immunofluorescence והדמיה חייה.

מערכת ל<em&gt; Vivo לשעבר</em&gt; Culturing של לבלב עוברי

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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A System for ex vivo Culturing of Embryonic Pancreas

13.5K Views.  Max-Delbrück-Center for Molecular Medicine. כאן, אנו מתארים שיטה לבידוד, תרבות ומניפולציה של לבלב העוברי של עכבר. זה מייצג מצוין Vivo לשעבר מערכת ללימוד היבטים שונים של פיתוח לבלב, כולל המורפוגנזה, התמיינות וגדילה. explants ניצן הלבלב יכול להיות מתורבת במשך כמה ימים ולהשתמש במגוון של יישומים שונים, כולל ?...

Video: A System for ex vivo Culturing of Embryonic Pancreas

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

שיטת לתרבות מוקדם של עוברים צ&#39;יק לשעבר vivo (תרבות חדש)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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

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

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

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

לשעבר אובו העובר עוף התרבות מתאים ליישומים הדמיה microsurgery מערכת

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

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

מערכת culturing איריס תאים פיגמנט אפיתל לחקר התחדשות עדשה של ניוט

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

מערכת 3D עבור chondrocytes culturing האדם במפרק של נוזל סינוביאלי

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

הכנת עובריים של עכברים תאים פיברובלסטים מתאים האדם culturing תאים עובריים pluripotent המושרה בתאי גזע

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

רומן<em&gt; Ex vivo</em&gt; שיטת תרבות ללב העכבר העוברית

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

שיטה לCulturing עוברי<em&gt; ג elegans</em&gt; תאים

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

אקס תרבות Vivo של עור העכבר עוברי וLive-הדמיה של הגירת Melanoblast

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

<em&gt; Ex vivo</em&gt; מערכת תרבות ללמוד התפתחות בלוטת התריס

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

במבחנה הלבלב Organogenesis ממפוזר אבות עכבר עובריים

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...