אנו מודים מתקן המכון Wistar בעלי חיים, מתקן מיקרוסקופית, מתקן Histotechnology ואספקה ​​מרכז המחקר. המחקר מומן בחלקו על ידי מענקים מהמוסד הלאומי לבריאות ע&quot;א 076674, קליפורניה 098101, 025874 ו - CA CA 10815.

<ol>
	<li>Sun, T., Jackson, S., Haycock, J. W., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+skin+cells+in+3D+rather+than+2D+improves+their+ability+to+survive+exposure+to+cytotoxic+agents.">Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents.</a> J Biotechnol. 122, 372-381 (2006).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev Biol Anim. 42, 242-247 (2006).</li><li>Sun, T., Norton, D., McKean, R. J., Haycock, J. W., Ryan, A. J., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+3D+cell+culture+system+for+investigating+cell+interactions+with+electrospun+fibers.">Development of a 3D cell culture system for investigating cell interactions with electrospun fibers.</a> Biotechnol Bioeng. 97, 1318-1328 (2007).</li><li>Kremer, M., Lang, E., Berger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypical+engineering+of+differentiated+composite-skin+equivalents+of+human+keratinocytes+in+a+collagen-GAG+matrix+(INTEGRA+Artificial+Skin)+in+a+perfusion+culture+system.">Organotypical engineering of differentiated composite-skin equivalents of human keratinocytes in a collagen-GAG matrix (INTEGRA Artificial Skin) in a perfusion culture system.</a> Langenbecks Arch Surg. 386, 357-363 (2001).</li><li>Esc&#225;mez, M. J., Garc&#237;, M., Larcher, F., Meana, A., Mu&#241;oz, E., Jorcano, J. L., Del R&#237;o, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+model+of+wound+healing+in+genetically+modified+skin-humanized+mice.">An in vivo model of wound healing in genetically modified skin-humanized mice.</a> J Invest Dermatol. 123, 1182-1191 (2004).</li><li>Li, L., Fukunaga-Kalabis, M., Yu, H., Xu, X., Kong, J., Lee, J. T., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J Cell Sci. 123, 853-860 (2010).</li><li>Meier, F., Nesbit, M., Hsu, M. Y., Martin, B., Van Belle, P., Elder, D. E., Schaumburg-Lever, G., Garbe, C., Walz, T. M., Donatien, P., Crombleholme, T. M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoma+progression+in+skin+reconstructs:+biological+significance+of+bFGF.">Human melanoma progression in skin reconstructs: biological significance of bFGF.</a> Am J Pathol. 156, 193-200 (2000).</li><li>Yu, H., McDaid, R., Lee, J., Li, L., Kumar, S. M., Elder, D. E., Van Belle, P., Gimotty, P., Guerra, M., Hammond, R., Nathanson, K. L., Dalla Palma, M., Herlyn, M., Xu, X. <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+BRAF+mutation+and+p53+inactivation+during+transformation+of+a+subpopulation+of+primary+human+melanocytes.">The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.</a> Am J Pathol. 174, 2367-2377 (2009).</li><li>Lee, J. T., Li, L., Brafford, P. A., van den Eijnden, M., Halloran, M. B., Sproesser, K., Haass, N. K., Smalley, K. S., Tsai, J., Bollag, G., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLX4032,+a+potent+inhibitor+of+the+B-Raf+V600E+oncogene,+selectively+inhibits+V600E-positive+melanomas.">PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas.</a> Pigment Cell Melanoma Res. 23, 820-827 (2010).</li><li>Tsai, J., Lee, J. T., Wang, W., Zhang, J., Cho, H., Mamo, S., Bremer, R., Gillette, S., Kong, J., Haass, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+selective+inhibitor+of+oncogenic+B-Raf+kinase+with+potent+antimelanoma+activity.">Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.</a> Proc Natl Acad Sci U S A. 105, 3041-3046 (2008).</li></ol>

אין ניגודי אינטרסים הכריז.

תיארנו יצירת עור 3D משחזר עם מלנוציטים אדם נורמלי, לתאי גזע עורי ותאי מלנומה. כאשר גדל בתרבות monolayer, תאים melanocytic להציג מורפולוגיה דומה (שטוח, כישור או יותר דנדריטים בצורת), ללא קשר למוצאם של השלב הקליני. לעומת זאת, עור 3D משחזר לשחזר הבמה מאפיינים ספציפיים של תאים מלנומה. מלנוציטים אדם רגיל מתגוררים בבית ממברנות במרתף בין שכבות האפידרמיס ואת עורי כמו תאים בודדים. תאים RGP מלנומה ראשונית לגדול בקבוצות קטנות לאורך הממברנה במרתף בעוד יותר אגרסיביים תאים VGP מלנומה לגדול ככל אשכולות גדולים שבירת דרך הממברנה אל תוך מרתף בדרמיס. בתאי מלנומה גרורתית לגדול לכל הכיוונים לפלוש עמוק לתוך הדרמיס כמו תאים או אשכולות יחיד. ניתוח כמותי יכול להתבצע על המודל הזה על ידי מדידת עומק הפלישה והיקף התפוצה. בנוסף ואפיון של תאים ממאירים melanocytic נורמלי, באמצעות מודל שאנחנו יכולים לגרום ישירות התמיינות בתאי גזע עורי לתוך מלנוציטים התבגר, אשר בבית אל שכבת הבסיס של האפידרמיס ליצור תקשורת בתום לב עם קרטינוציטים דרך upregulation של E-cadherin ( 6). באמצעות וקטורים ויראליים, אנו מסוגלים גם להפעיל או להשבית פונקציה גן הבנה טובה יותר של הדינמיקה ואת משמעות תפקודית של גנים לידי ביטוי כל סוג תא העור משחזר, אשר מבטיח להיות מודל יעיל ללמוד לא רק על מנגנוני טרנספורמציה של מלנוציטים , אלא גם התקדמות של מלנומה (8). לטווח ארוך של התבוננות פנוטיפים הסלולרי הפך אפשרי באמצעות השתלת עור משחזר לבעלי חיים immunodeficient. אדם כזה העור עכבר הכימרה היא כלי מחקר מעולה ללמוד melanomagenesis וגרורות מלנומה. העור משחזר יכול להיות גם פלטפורמה יעילה להערכת תרופות. תרופות רבות אשר לחסל תאים סרטניים בתנאי תרבות 2D לעיתים קרובות יש השפעה מועטה ביישומים הניסוי הקליני. כפי שניתן לראות בגידול vivo, בתאי מלנומה בתרבויות 3D הם לעתים קרובות עמידים לתרופות אשר התאים בתרביות 2D להגיב, מה שמראה כי microenvironment מודולציה מסלולי איתות מלנומה. לגילוי סמים מוצלח, העור לשחזר מודל 3D הוא כלי אידיאלי פרה לחזות את ההשפעות של תרכובות in vivo (9,10). לסיכום, העור לשחזר מודלים לגשר על הפער בין במבחנה במחקרים vivo. הם יובילו להבנה טובה יותר של אילו גנים מעורבים טרנספורמציה וכיצד בתאי גזע לתרום השינוי.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> שם מגיב </th><th> חברה </th><th> מספר קטלוגי </th></tr><tr><td> 10x EMEM </td><td> ביו וויטאקר </td><td> 12-684F </td></tr><tr><td> L-גלוטמין </td><td> Gibco </td><td> 25030-081 </td></tr><tr><td> סרום שור עוברית </td><td> Hyclone </td><td> SH-30,071.02 </td></tr><tr><td> גיד שור חומצה חולץ קולגן </td><td> Organogenesis </td><td> 200/50 </td></tr><tr><td> תרביות רקמה של 6 גם מגשים עם תוספות </td><td> Organogenesis </td><td> 9285 </td></tr><tr><td> Keratinocyte בינונית חינם סרום </td><td> Gibco </td><td> 17005042 </td></tr><tr><td> Dialyzed עגל עוברית סרום </td><td> Hyclone </td><td> SH-30,079.03 </td></tr><tr><td> רקומביננטי גזע אנושיים Cell Factor </td><td> פיצג&#39;רלד תעשיות </td><td> RDI-307-255X </td></tr><tr><td> יסוד FGF </td><td> פיצג&#39;רלד תעשיות </td><td> 30R-AF015 </td></tr><tr><td> אנדותלין-3, אדם </td><td> האמריקאי פפטיד ושות </td><td> 88-5-10 </td></tr><tr><td> סידן כלוריד </td><td> סיגמא </td><td> C-7902 </td></tr><tr><td> DMEM </td><td> JRH מדעי החיים </td><td> 56430-10L </td></tr><tr><td> F12 (HAM של) </td><td> Gibco </td><td> 11765-54 </td></tr><tr><td> הידרוקורטיזון </td><td> סיגמא </td><td> H-0888 </td></tr><tr><td> אינסולין, transferrin, Ethanolamine, סלניום (ITES) </td><td> BioWhittaker </td><td> 17839Z </td></tr><tr><td> O-Phosphorylethanolamine </td><td> סיגמא </td><td> P0503 </td></tr><tr><td> אדנין </td><td> סיגמא </td><td> A9795 </td></tr><tr><td> הפרוגסטרון </td><td> סיגמא </td><td> P-8783 </td></tr><tr><td> Triiodothyronine </td><td> סיגמא </td><td> T-5516 </td></tr><tr><td> סרום סובך של יילוד </td><td> Hyclone </td><td> SH3011802 </td></tr><tr><td> חיץ 10% פורמלין </td><td> Surgipath </td><td> 00600 </td></tr><tr><td> טמפרטורה אופטימלית חיתוך קפוא התקשורת (אוקטובר) </td><td> סאקורה </td><td> 4583 </td></tr><tr><td> Multi קלטות </td><td> Surgipath </td><td> 02293-BX </td></tr><tr><td> TBS ביופסיה ניירות </td><td> משולש למדעי ביו </td><td> BP-B </td></tr><tr><td> TBS ביופסיה ניירות </td><td> משולש למדעי ביו </td><td> BP-B </td></tr><tr><td> מאגר תבניות חד פעמיות </td><td> דיג </td><td> 15-182-501C </td></tr><tr><td> הבנזואין מתחם תמיסת </td><td> Disposables Professional, Inc, </td><td> S42450 </td></tr><tr><td> מטליות אלכוהול Prep </td><td> בית המרקחת CVS </td><td></td></tr><tr><td> משי שחורה קלועה 5-0 </td><td> Ethicon </td><td> 682G </td></tr><tr><td> גזה ספוגים (סטרילי) </td><td> בית המרקחת CVS </td><td></td></tr><tr><td> מצבטים </td><td> Roboz </td><td> RS5070 </td></tr><tr><td> איריס מספריים </td><td> Roboz </td><td> RS5913 </td></tr><tr><td> כירורגי להבי </td><td> נוצה </td><td> 2976 </td></tr><tr><td> EZ הרדמה </td><td> Euthanex קורפ </td><td></td></tr><tr><td> SCID עכבר outbred שיער </td><td> צ&#39;ארלס הנהר </td><td></td></tr></tbody></table>

the three-dimensional human skin reconstruct model: a tool to study normal  skin and melanoma progression

רוב במבחנה בביולוגיה העור ניסיוני נעשו monocultures 2 ממדי (2D), תוך צבירת הראיות עולה כי התאים מתנהגים באופן שונה כאשר הם גדלו בתוך מטריצה ​​חוץ תאית 3D וגם אינטראקציה עם תאים אחרים (1-5) . מודלים עכבר כבר נוצלו בהרחבה ללמוד המורפוגנזה רקמה in vivo. עם זאת העכבר העור האנושי יש הבדלים משמעותיים בארכיטקטורה הסלולר פיזיולוגיה, אשר מקשה להסיק מחקרים העכבר לבני אדם. מאז מלנוציטים בעור עכבר מרוכזים בעיקר בזקיקי השיער, יש להם מאפיינים ביולוגיים שונים מאלה של בני אדם, אשר לאתר בעיקר בשכבת הבסיס של האפידרמיס. ההתפתחות האחרונה של העור האנושי לשחזר מודלים 3D אפשרה את השדה כדי לחקור תאים מטריקס תאים תאים אינטראקציות בין תאים מסוגים שונים. משחזר מורכב &quot;הדרמיס&quot; עם fibroblasts מוטבע קולגן אני מטריקס, &quot;האפידרמיס&quot;, אשר מורכבת, מרובדת קרטינוציטים מבודל קרום במרתף פונקציונלי, המפריד בין הדרמיס לאפידרמיס. קולגן מספק פיגומים, אספקת חומרי מזון, ואת הפוטנציאל לאינטראקציה התא אל התא. העור מודלים 3D המשלב תאים melanocytic לשחזר את המאפיינים הטבעיים של הומאוסטזיס מלנוציט והתקדמות מלנומה בעור האדם. כפי in vivo, מלנוציטים בעור משוחזר מרוכזים בבית קרום המרתף וביניהם קרטינוציטים שכבת הבסיס. בתאי מלנומה התערוכה אותם מאפיינים המשקף את שלב הגידול המקורי (RGP, VGP ותאי מלנומה גרורתית) in vivo. לאחרונה, בתאי גזע עורי זוהו בדרמיס אדם (6). אלה רב עוצמה בתאי גזע יכול לעבור האפידרמיס להבדיל כדי מלנוציטים.

Watch this Scientific Journal Video about The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression at JoVE.com

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

בדו&quot;ח זה, אנו מתארים את העור תלת ממדי לשחזר מודל אשר מחקה העור האנושי באדריכלות הרכב. הפיזיולוגיה מלנוציט, התקדמות מלנומה גורל בתאי גזע עורי נחקרו באמצעות העור לשחזר מודל. המודל שימושית גם ככלי קליני להערכת תרופות.

עור תלת ממדי האדם לשחזר דגם: ככלי מחקר עור רגיל התקדמות מלנומה

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

the-three-dimensional-human-skin-reconstruct-model-tool-to-study

Research

JoVE Journal

Bioengineering

49.2K Views.  The Wistar Institute. בדו"ח זה, אנו מתארים את העור תלת ממדי לשחזר מודל אשר מחקה העור האנושי באדריכלות הרכב. הפיזיולוגיה מלנוציט, התקדמות מלנומה גורל בתאי גזע עורי נחקרו באמצעות העור לשחזר מודל. המודל שימושית גם ככלי קליני להערכת תרופות.

Video: The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

הדפסה מישוריים תלת ממדי של דיו מוליך

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

שיטה משופרת להכנת סוג I קולגן מעור

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

חקירת הפרדת הזרימה התלת מימדית הנגרמת על ידי מודל פוליפ קיפול ווקאלי

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

ייצור רקמת סחוס אנושי באמצעות שלושה ממדי הזרקת דיו טכנולוגיית הדפסה

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

עור מלא פגמים דגם להערכת כלי דם של Biomaterials<em&gt; בVivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

דור של עור בעובי מלא תלת ממדי Equivalent ואוטומטי פציעה

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

פיתוח תאיים תלת ממד Organotypic דגם של אדם המעיים הריריים Grown במיקרוגרבטיציה

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

במודל חוץ גופית של עורית האדם יפרטרופית צלקות באמצעות הצפיפות Macromolecular

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

טיפוח אפידרמיס אנושי תלת מימדי משוחזר בקנה מידה גדול

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

דור של שווה ערך לעור אנושי בהרכבה עצמית

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...