עבודה זו מומנה על ידי NIH מענק R01AI077610.

כל הנהלים בוצעו בהתאם מדריך לטיפול ושימוש בחיות מעבדה של המכון הלאומי לבריאות (NIH) ואושרו על ידי ועדת טיפול בבעלי חיים באוניברסיטת ג&#39;ונס הופקינס ושימוש (JHUACUC). הנהלים הספציפיים בוצעו תחת MO13M292 פרוטוקולי ACUC המאושרות MO13M370. קציר עור תורם 1. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר התורם עם isoflurane (מאדה אינדוקציה ב 4% אחזקה ל 1 - 2% דרך חרוט העכבר). השתמש רפלקס נסיגת קמצוץ בוהן כדי לפקח על עומק ההרדמה. </li><li style=";text-align:right;direction:rtl"> באמצעות מכונת גילוח חשמלית, לגלח את הגב של החיה ולחטא אותו ביוד povidone 10% (למשל, בבטאדין). </li><li style=";text-align:right;direction:rtl"> בעזרת מספרי סטריליות, כפפות, וטכניקה ספטית, עור בחזרה תורם קציר ממותן עד הצוואר, עם דיסקציה קהה ברמה של רקמת חיבור areolar. </li><li style=";text-align:right;direction:rtl"> להרדימו על ידי נקע בצוואר הרחם לאחר קצירתשתל עור. </li><li style=";text-align:right;direction:rtl"> תחת מיקרוסקופ, להפריד את רקמת חיבור, רקמת שומן, מִרפָּד carnosus מהעור בחזרה באמצעות מספרי tenotomy בסדר. Carnosus מִרפָּד הוא שריר דק, שקוף אחראי תנועות מתעוות עור. </li><li style=";text-align:right;direction:rtl"> באמצעות טכניקה בכלים סטריליים, לגזור שתלי 15 מ&quot;מ x 15 מ&quot;מ מהעור בחזרה במשך 10 מ&quot;מ 10 מ&quot;מ x 15 מ&quot;מ x המיטה שתל 15 מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> אחסן את השתלים על gauzes ספוגה פוספט שנאגר המלוח סטרילית (PBS) בצלחת פטרי על קרח. הערה: 8 - 10 שתלים ניתן להשיג עכבר תורם אחד. </li></ol> השתלת 2. נמען עור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להרדים את העכבר הנמען עם isoflurane (מאדה אינדוקציה ב 4% אחזקה ל 1 - 2% דרך חרוט העכבר). השתמש רפלקס נסיגת קמצוץ בוהן כדי לפקח על עומק ההרדמה. </li><li style=";text-align:right;direction:rtl"> נהל 0.02 מ&quot;ג / ק&quot;ג BW של עצירות להקלה על הכאב לאחר הניתוח. </li><li style=";text-align:right;direction:rtl"> לגלח את הצד של החלק האחורי של החיה שבו wi שתלll להיות מוכנס ולחטא עם יוד povidone 10%. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים, לחתוך 10 מ&quot;מ x 10 מ&quot;מ עד 15 מ&quot;מ x 15 מ&quot;מ מרובע של העור. גודל הפגם צריך להיות מעט גדול (10%) מאשר את השתל. גזור באופן שטחי ככל האפשר. תשמור על עצמך כדי לשמר את מִרפָּד carnosus וכלי. Carnosus מִרפָּד ניתן להבחין מן fascia הבסיסית על ידי הניידות שלה ואת כלי הדם שמופעלים שטחי אליו. </li><li style=";text-align:right;direction:rtl"> מקם את השתל על מיטת השתל, הימנעות קפלים בשולים. </li><li style=";text-align:right;direction:rtl"> מניח 8 תפרים על הפינות על האמצע של כל קצה. עבור כל תפר, להעביר את המחט דרך השתל ולאחר מכן דרך carnosus מִרפָּד של השתל למיטה מתחת לעור הנמען שמסביב. </li><li style=";text-align:right;direction:rtl"> הסר את מסכת ההרדמה ולתת החיה להתאושש חלקית מן ההרדמה לפני החלת פלסטר. </li><li style=";text-align:right;direction:rtl"> עוטף את עכבר נמען תחבושת דבקה עם גזה מקופלת על השתל. הפוך את התחבושת על ידי שילוב של שתי תחבושות, חיתוךהחלק הדבק של אחד והעמיד יחד שתי רפידות סופגות. </li><li style=";text-align:right;direction:rtl"> צג את העכבר מקרוב במהלך שחזור כדי להבטיח את התחבושת אינה הגבלת טיול חזה ונושמת. הסר את התחבושת אם קצב הנשימה פוחת או החיה מתחילה להשתנק או נשימה שטחית. </li></ol> 3. טיפול לאחר הניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נהל Enrofloxacin 5 מ&quot;ג / ק&quot;ג לאחר ניתוח טיפול מונע זיהום. </li><li style=";text-align:right;direction:rtl"> מניח את העכבר המושתל בתוך כלוב נקי מעל כרית חימום במיקרוגל עד שהוא מתאושש לחלוטין מן ההרדמה. </li><li style=";text-align:right;direction:rtl"> שים את העכבר עבור שעה 1 לאחר ניתוח לפני החזרת אותו למוקד הדיור. </li><li style=";text-align:right;direction:rtl"> שבעה ימים לאחר הניתוח, להרדים את העכבר כמו בשלב 2.1. הסר את התחבושת על ידי חיתוך רק דרך צד הגחון של התחבושת. </li><li style=";text-align:right;direction:rtl"> להתבונן למשש את השתל למחרת סימנים של scabbing, התכווצות, או קשיות. אם הווה, את השתל אולי לא נחל vasculariza הנאהtion וצריך להיחשב כשל טכני. </li><li style=";text-align:right;direction:rtl"> צג יום סימנים של דחייה. שקול שתלי דחה כאשר ≥90% של רקמת שתל הופך נמקי. </li><li style=";text-align:right;direction:rtl"> להרדים את החיות דחו ולקצור אותם לניתוח. </li></ol>

<ol><li>Furtmuller, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Orthotopic+Hind+Limb+Transplantation+in+the+Mouse%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Orthotopic Hind Limb Transplantation in the Mouse.</a> J Vis Exp. , (2016).</li>
<li>Oh, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Microsurgical+Model+for+Heterotopic%2C+En+Bloc+Chest+Wall%2C+Thymus%2C+and+Heart+Transplantation+in+Mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice.</a> J Vis Exp. , (2016).</li>
<li>Oberhuber, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+cervical+heart+transplantation+model+using+a+modified+cuff+technique%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Murine cervical heart transplantation model using a modified cuff technique.</a> J Vis Exp. , e50753(2014).</li>
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<li>Medawar, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+behaviour+and+fate+of+skin+autografts+and+skin+homografts+in+rabbits%3A+A+report+to+the+War+Wounds+Committee+of+the+Medical+Research+Council%5BTitle%5D)+AND+%22J+Anat%22%5BJournal%5D)">The behaviour and fate of skin autografts and skin homografts in rabbits: A report to the War Wounds Committee of the Medical Research Council.</a> J Anat. 78, 176-199 (1944).</li>
<li>Billingham, R. E., Brent, L., Medawar, P. B., Sparrow, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+studies+on+tissue+transplantation+immunity.+I.+The+survival+times+of+skin+homografts+exchanged+between+members+of+different+inbred+strains+of+mice%5BTitle%5D)+AND+%22Proc+R+Soc+Lond+B+Biol+Sci%22%5BJournal%5D)">Quantitative studies on tissue transplantation immunity. I. The survival times of skin homografts exchanged between members of different inbred strains of mice.</a> Proc R Soc Lond B Biol Sci. 143, 43-58 (1954).</li>
<li>Garrod, K. R., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Murine+skin+transplantation%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Murine skin transplantation.</a> J Vis Exp. , (2008).</li>
<li>Schmaler, M., Broggi, M. A., Rossi, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+tail+skin+to+study+allogeneic+CD4+T+cell+responses+in+mice%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Transplantation of tail skin to study allogeneic CD4 T cell responses in mice.</a> J Vis Exp. , e51724(2014).</li>
<li>Bergstresser, P. R., Toews, G. B., Gilliam, J. N., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unusual+numbers+and+distribution+of+Langerhans+cells+in+skin+with+unique+immunologic+properties%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Unusual numbers and distribution of Langerhans cells in skin with unique immunologic properties.</a> J Invest Dermatol. 74, 312-314 (1980).</li>
<li>Chen, H. D., Silvers, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Influence+of+Langerhans+cells+on+the+survival+of+H-Y+incompatible+skin+grafts+in+rats%5BTitle%5D)+AND+%22J+Invest+Dermatol%22%5BJournal%5D)">Influence of Langerhans cells on the survival of H-Y incompatible skin grafts in rats.</a> J Invest Dermatol. 81, 20-23 (1983).</li>
<li>Chong, A. S., Alegre, M. L., Miller, M. L., Fairchild, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lessons+and+limits+of+mouse+models%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Med%22%5BJournal%5D)">Lessons and limits of mouse models.</a> Cold Spring Harb Perspect Med. 3, a015495(2013).</li>
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החוקרים אין לי מה לחשוף.

מאז השקתו ידי מךוואר, ראשון במחקרים בבני אדם ולאחר מכן בארנבות ועכברים, השתלת עור כבר מודל יסולא בפז לחקר תגובות חיסוניות אלוגנאית 6,7. בכתב היד הזה, אנו מציגים מודל של בקנה מידה גדול, הלא vascularized, השתלת עור בעובי מלא באמצעות העור הגב העליון והתחתון. שיטות חלופיות ש...

השתלת איברים היא טיפול הבחירה בחולים עם כשל איברים סופנית, ותוצאות השתפרו פלאים עם ההתקדמות ניתוחים ופרוטוקולים דיכוי חיסוני. עם זאת, דיכוי מערכת חיסוני לטווח ארוך קשור לתופעות לוואי משמעותיות, לבין ההתפתחות של אסטרטגיות חדשות המקדמות סובלנות נשארת מטרת מחקר השתלה המודרנית. במודלים של בעלי חיים רבים פותחו למחקר בסיסי בהשתלות, כדי לחקור את המנגנונים של דחייה של השתל וכדי לבחון גישות דיכוי חיסוני למניעת דחיית שתל ו לקידום סובלנות לטווח ארוך 1-3. מודלי העכבר הפכו התווך של מחקר האימונולוגי בשל הזמינויות הבלעדיות מכריעות של נוגדני אבחון וטיפול ומוגדרים היטב זנים טהורים ו מהונדסים. השתלת עור היא הליך פשוט שאינו דורש מיומנויות microsurgical מיוחדות והוא יכולבקלות להיות במעקב אחרי הניתוח. יחדיו, השתלת עור העכבר כבר כלי יוצא דופן ללמוד היבטים רבים המעורבים בתגובה alloimmune, כולל משלוח אנטיגן, סחר התא, והרס רקמות במהלך דחיית שתל 4,5. כאן, אנו מציגים את הצעד-אחר-צעד הליך השתלת עור בעובי מלא תוך שימוש במודל העכבר, אנו מתארים את הצעדים החשובים הכרחיים עבור engraftment מוצלח של העור המושתל.

<table><tbody><tr><td>Straight micro forceps</td><td>Sigma</td><td>F4017</td><td></td></tr><tr><td>Curved micro forceps</td><td>Aesculap</td><td>BD333R</td><td></td></tr><tr><td>Curved Stevens tenotomy scissors</td><td>Aesculap</td><td>BC905R</td><td></td></tr><tr><td>Mayo dissecting scissors</td><td>Sigma</td><td>S3146</td><td></td></tr><tr><td>Micro needle holder</td><td>Aesculap</td><td>BM563R</td><td></td></tr><tr><td>Sterile gauze</td><td>Covidien</td><td>441218</td><td></td></tr><tr><td>6-0 Nylon suture</td><td>MWI</td><td>31849</td><td></td></tr><tr><td>Plastic Strips Band&amp;#45;Aid</td><td>Johnson &amp;amp; Johnson</td><td></td><td>Obtained from pharmacy</td></tr><tr><td>10 cm Petri dish</td><td>Fisherbrand</td><td>FB0875712</td><td></td></tr><tr><td>PBS</td><td>Quality Biological</td><td>119069131</td><td></td></tr><tr><td>Buprenorphine</td><td></td><td></td><td>DEA Number required; Obtained from hospital pharmacy</td></tr><tr><td>Enrofloxacin</td><td>Bayer Health Care</td><td>186599</td><td></td></tr></tbody></table>

murine full-thickness skin transplantation

השתלת עור בעובי מלאת Murine היא מודל in vivo ומבוסס ללמוד alloimmune תגובת דחיית שתל. למרות היישום המוגבל שלה לבני אדם, השתלת עור בעכברים הועסקה נרחבת למחקר השתלה. הנוהל הוא קל ללמוד ולבצע, וזה אינו דורש טכניקות מייקרו עדינות ולא הכשרה מקיפה. יתר על כן, דחיית שתל במודל זה מתרחשת תגובה אימונולוגית מאוד לשחזור מנוטר בקלות על ידי בדיקה ישירה ומישוש. בנוסף, השתלת עור משני עם שתלים בהתאמה או צד שליש עור יכולה להתבצע על מודלי השתלה מורכבים יותר כמו שיטה חלופית ולא מסובכת להעריך סובלנות תורם ספציפי. הסיבוכים נמוכים והם בכלל מוגבלים יתר הרדמה או מצוקה נשימתית לאחר ההליך. לכישלון השתל, מצד שני, בדרך כלל מתרחשת כתוצאה הכנה לקויה של גרמיצוב ומאחור, שגוי במיטת השתל, או מיקום לא ראוי של התחבושת. במאמר זה, אנו מציגים פרוטוקול להשתלת עור בעובי מלא בעכברים ולתאר הצעדים החשובים דרושי הליך מוצלח.

המיקום של התחבושת על עכבר הנמען הוא צעד חשוב של ההליך. שתל עור ממוקם על גזע הנמען, בין הכתף, הירך ועמוד השדרה (איור 1). התחבושת הוא עשה עם גזה מקופלת ושילוב של שתי תחבושות דבקות פלסטיק. עכבר הנמען מושם עם השתל למטה על הגזה על המרכז את התחבושת. שי...

Watch this Scientific Journal Video about Murine Full-thickness Skin Transplantation at JoVE.com

Murine Full-thickness Skin Transplantation

השתלת עור בעובי מלאת Murine היא מודל ומבוסס ללמוד דחייה בסביבת alloimmune. כאן, אנו מספקים הדרכה של כל שלב המעורב בביצוע BALB / ג - השתלת עור&gt; C57BL / 6 בעובי מלא.

השתלת עור בעובי מלא Murine

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

murine-full-thickness-skin-transplantation

Research

JoVE Journal

Immunology and Infection

20.1K Views.  Johns Hopkins University School of Medicine.  השתלת עור בעובי מלאת Murine היא מודל ומבוסס ללמוד דחייה בסביבת alloimmune. כאן, אנו מספקים הדרכה של כל שלב המעורב בביצוע BALB / ג - השתלת עור> C57BL / 6 בעובי מלא. 

Video: Murine Full-thickness Skin Transplantation

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

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

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 ...

Bioengineering

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

השתלת עור Murine

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Biology

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 ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

מודל עכברי של ריפוי פצע

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

רקמות טחונות ב הדחוס קולגן: A Biotransplant המכיל נייד עבור תיקון Reconstructive המבוים יחיד

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

השתלה של עור זנב לחקר תגובות תא Allogeneic T מסוג CD4 בעכברים

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

דור של תא צורב עור תלת-ממד של כבל דם-derived המושרה בתאי גזע Pluripotent

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Developmental Biology

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

מודל Xenograft לעור למניפולציה של תגובות חיסוניות אנושיות in vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

מודל מורנה של פצע צריבה שוחזר עם שתל העור Allogeneic

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

A Technique for Human Skin Grafting in a Mouse Model

Source: Moss, M. I. et al., <a href="https://app.jove.com/v/64040">Xenograft Skin Model to Manipulate Human Immune Responses&#160;In Vivo.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates a method for grafting human skin onto an immunodeficient mouse with a suppressed neutrophil response. The method involves applying human split-thickness skin to the mouse&#39;s graft bed, sealing it, and covering it for recovery. Eventually, the human skin adheres to the mouse&#39;s skin, establishing vascularization and circulation, resulting in a successful skin xenograft.

טכניקה להשתלת עור אנושי במודל עכבר

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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