טרייסי ג Grikscheit, אריק ר Barthel, ופרדריק ג&#39;סאלה נתמכת על ידי המכון בקליפורניה הרגנרציה רפואה (CIRM), מספרי המענק RN2-00946-1 (TCG) וTG2-01168 (ארב, מדרשה). אליסון ל שפר הוא חברה של אוניברסיטת מנתחי Ethicon חוקר. Yashuhiro Torashima ממומן על ידי המלגה של בית החולים לילדים בלוס אנג&#39;לס סבן מכון מחקר פיתוח הקריירה.

1. הכנת יחידות Organoid <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> המכשירים מתאימים לנתיחת עכבר (מספריים ומלקחיים) צריכים להיות מעוקרים על ידי חיטוי. </li><li style=";text-align:right;direction:rtl"> הומני להרדים את עכבר התורם על פי פרוטוקולי IACUC מקומיים. ודא כי בעל החיים מתים לפני שימשיכו. </li><li style=";text-align:right;direction:rtl"> לעשות חתך קו אמצע כדי לקבל גישה לחלל הצפק. שולי עור יכולים להשתקף כנדרשים כדי לשפר את החשיפה. </li><li style=";text-align:right;direction:rtl"> כסח את המעי הדק ומחלק אותו רק דיסטלי לרצועות של Treitz. הפרד את המעי הדק מלפדרו באמצעות נתיחה בוטה חדה ועדינה. זהה את צומת ileocecal ולחלק את 5 מ&quot;מ המעי הדק הפרוקסימלי לזה. </li><li style=";text-align:right;direction:rtl"> בעזרת מספריים, פתח את מעי אורכו לאורך גבול antimesenteric בצלחת פטרי עם 10 מ&quot;ל 4 מעלות צלזיוס, מלוח &quot;סטרילי הנקס נאגר (HBSS, Invitrogen, Carlsbad CA) / 1X אנטיביוטיקה נגד פטריות (אנטי אנטי, Invitrogen) פתרון. נקה את הצואה מהמעי פתח עםתסיסה עדינה לאחר מכן להעביר מעי נפתח לצינור צנטריפוגה 15 מ&quot;ל עם 10 מ&quot;ל של 4 מעלות צלזיוס, HBSS סטריליים / 1X אנטי אנטי. </li><li style=";text-align:right;direction:rtl"> שטוף את המעי נפתח שלוש פעמים ב10 מ&quot;ל של 4 מעלות צלזיוס, HBSS / 1X סטריליים אנטי אנטי במבחנה. כל שטיפה ניתן לבצע עם רעד קל של צינור המ&quot;ל 15 למשך 30 שניות. אחרי שלחץ, רקמת המעי שוקע לתחתית של התחתית. השלך חומרים צפים, שהיא פסולת mesenchymal. הסר את פתרון הכביסה בזהירות עם pipet. </li><li style=";text-align:right;direction:rtl"> ברר במעי השטוף בצלחת פטרי עם 10 מ&quot;ל של 4 מעלות צלזיוס, HBSS סטריליים / 1X אנטי אנטי פחות מ 1 מ&quot;מ ריבועים באמצעות מספריים. אסוף את החומר הטחון עם pipet אוטומטי ולמקם אותו לתוך מבחנה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה הצינור ב 500 סל&quot;ד לדקות 8. בטל supernatant, המכיל שומן וmesenchyme. </li><li style=";text-align:right;direction:rtl"> לעכל את החומר טחון, שטוף עם 10 מ&quot;ל של HBSS סטריליים / תוספת אנטי אנטי 1X 0.125 מ&quot;ג / המ&quot;ל dispase (Invitrogen) ו800 יחידות / מ &#39;סוג l collagenase 1 (וורטינגטון, לייקווד ניו ג&#39;רזי). כדי להכין 40 מ&quot;ל של תמיסת העיכול, לשקול את 5 מ&quot;ג dispase, 142 מ&quot;ג של collagenase, ולהוסיף עד HBSS סטריליים לנפח של 40 מ&quot;ל. הכן את הפתרון זה עתה את כל יחידות דיור organoid זמן מוכנות, ולשמור ב 4 ° C עד מוכן לשימוש. הוסף פתרון העיכול ישירות לגלולה מצעד 1.8. </li><li style=";text-align:right;direction:rtl"> דגירת מבחנה המכילה את החומר טחון, שטוף עם פתרון העיכול על 37 מעלות צלזיוס למשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> תחזר את המבחנה ועוד לשבש את רקמת עיכול על ידי טחינה דקה עם pipet מ&quot;ל 10. חזור על בין 20 ל 50 פעמים עד שמראה אחיד מתקבל. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה המבחנה למשך 5 דקות ב 800 סל&quot;ד. בטל supernatant, המכיל תאים בודדים. </li><li style=";text-align:right;direction:rtl"> עצור את תגובת העיכול עם 10 מ&quot;ל של 4 מעלות צלזיוס שינוי הנשר הבינוני של סטרילי Dulbecco (DMEM, Invitrogen) בתוספת 10% בסרום, v / v חום מומת עוברי שור (HI-FBS, Invitrogen). Resuspend פלהt ולנער את הצינור. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה המבחנה למשך 5 דקות ב 800 סל&quot;ד. הסר את supernatant זהירות עם pipet אוטומטי עד הטיפין האחרונות. השתמש pipet פלסטיק חד פעמי לטיפין האחרונות, כדי למנוע resuspending הגלולה. </li></ol> 2. טעינה של Polyglycolic חומצת הפיגום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> טופס, פיגומי 2-מ&quot;מ ארוך 5-מ&quot;מ קוטר חיצוני גליליים מחומצת polyglycolic הארוגה (עובי 2-מ&quot;מ גיליון, 60 מ&quot;ג צפיפות סנטימטר-3 בתפזורת; נקבוביים%&gt; 95, סיבי קונקורדיה, RI קובנטרי) כפי שמתוארת במס. 4. </li><li style=";text-align:right;direction:rtl"> לקצץ 2 מ&quot;מ הדיסטלי של pipet microliter הפנוי 1000 קצה עם מספריים מוכנים עם אתנול 70% במים מזוקקים. </li><li style=";text-align:right;direction:rtl"> הנח את הפיגום לתוך צלחת תרבות 4-כן. טען את יחידות organoid גבי הפיגום עם pipet microliter 1000, קודם ללום ואז על פני השטח החיצוניים. השתמש במלקחיים כדי להבטיח ציפוי של לומן. אל תפגעו או לפרוץ צורה הגלילית של הפולימר. </li&gt; </ol> 3. השתלה לעכבר מארח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> השתמש בעכבר המארח syngeneic באותו הרקע כתורם אם הוא זמין. אחרת, להעסיק סוכרתי nonobese immunocompromised / חמור בשילוב בעלי חי immunodeficient או NOD / SCID (ג&#39;קסון מעבדות, סקרמנטו קליפורניה). </li><li style=";text-align:right;direction:rtl"> לגרום להרדמה כללית עם isoflurane. גילוח, הכנה ולעטוף בבטנו של העכבר. </li><li style=";text-align:right;direction:rtl"> לעשות חתך קו אמצע 5 מ&quot;מ כדי לזכות בכניסה לחלל הצפק. זיהוי וכסח omentum יותר בזהירות. הנח את הפולימר הטעון על גבי omentum ולעטוף אותו ברקמות. אל תקרע omentum. </li><li style=";text-align:right;direction:rtl"> אבטח את הפולימר לomentum עם תפר 5-0 monocryl. מחזיר בעדינות את omentum עם הפולימר העטוף בתנוחה אנטומית שלה. </li><li style=";text-align:right;direction:rtl"> סגור את החתך בבטן בשכבות באמצעות תפרי vicryl 4-0. הפעל את סגירת השרירים ולדאוג שלא לפגוע באיברים הפנימיים בבטן מתחת לחתך. השתמש תפרים לעור. </li><li style=";text-align:right;direction:rtl"> כאבים לאחר ניתוח לנהל עם 2 מ&quot;ג / קילו ketoprofen (Ketofen, בריאות בעלי חי Fort Dodge) במים סטריליים כwheal סמוך לחתך תת עורי. בעלי חיים צריכים להיות מוערכים מדי יום ואם בעל החיים הוא מפגינים סימנים של כאב או מצוקה, מנה נוספת של ketoprofen יכולה להינתן ביום ניתוח לאחר 2. ביום שלאחר הניתוח השלישי, בעלי החיים צריכים להיות התאוששו לחלוטין ללא עדות לכאב או מצוקה. אם כאב או מצוקה ממשיך ביום ניתוח לאחר 3, זה נחשב נורמלי ויש להתייחס בהתאם לפרוטוקולי מתקן טיפול IACUC ובעלי חיים. </li><li style=";text-align:right;direction:rtl"> אפשר העכבר כדי להתאושש ומעי רקמה המהונדס לגדול במשך ארבעה שבועות. תן גישה כרצונך חיה למכרסם צ&#39;או (המעבדה דיאט 5001, מדד מנהלי רכש תזונה, סנט לואיס מיזורי) ומים עם מ&quot;ג Septra 200 מ&quot;ג / 5 מ&quot;ל לכל 40 (היי טק Pharmacal, Amityville ניו יורק) בשעת 1:100 דילול. </li></ol> 4. קציר <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הומאנלי להרדימו מארח ארבעה שבועות לאחר השתלה. </li><li style=";text-align:right;direction:rtl"> פתח מחדש את החתך המקורי ומשקף את עור cephalad כדי להקל על גישה לחלל הצפק. </li><li style=";text-align:right;direction:rtl"> פתח את שכבת השריר ולזהות את מבנה רקמות המהונדסות כעולם של רקמה. </li><li style=";text-align:right;direction:rtl"> קח את ההידבקויות למבנה מהקרביים intraabdominal באמצעות נתיחה חדה. </li><li style=";text-align:right;direction:rtl"> תקן ההבניה בפורמלין לפרפין מאוחר הרכבה, או להשתמש ברקמה הטריה למבחנים ביוכימיים כגון PCR בזמן אמת או בידוד חלבון. </li></ol>

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אין ניגודי האינטרסים הכריזו.

אנו מציגים פרוטוקול לייצור רקמת מעי מהונדס בעכבר באמצעות organoid גישת יחידות על פיגום. השלבים הקריטיים ביותר הם אלה של הכנת יחידות organoid. יש להקפיד לנקות כראוי ומכאני לעבד את הרקמה, אבל טיפול שווה יש להקפיד שלא לoverdigest או overtriturate יחידות organoid לאחר העיכול מתבצע (שלב 1.11). אם זה...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות (אופציונלי) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> אנטיביוטיקת Antimycotic 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> סוג collagenase 1 </td><td> וורטינגטון </td><td> LS004194 </td><td></td></tr><tr><td> גלוקוז גבוהה DMEM 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> FBS המומת חום </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100% PGA </td><td> קונקורדיה רפואית </td><td> FELT01-1005 </td><td> להכנת פולימר כמו במס.4 </td></tr><tr><td> חומצת פולי-L-לקטית </td><td> Durect </td><td> B6002-1 </td><td> להכנת פולימר כמו במס. 4 </td></tr><tr><td> סוג אני קולגן, זנב חולדה </td><td> סיגמה אולדריץ </td><td> C3867-1VL </td><td> להכנת פולימר כמו במס. 4 </td></tr><tr><td> Ketoprofen 100 מ&quot;ג / מ&quot;ל </td><td> לבריאות בעלי חי דודג&#39; פורט </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 המכרסם צ&#39;או </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200/40 מ&quot;ג מ&quot;ג לכל 5 מ&quot;ל, USP </td><td> היי טק Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> , USP isoflurane </td><td> תרופות פניקס </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

מעי דק רקמה מהונדסת (Tesi) בהצלחה שמש כדי להציל את לואיס חולדות לאחר כריתת מעי דק מסיבית, וכתוצאה מכך חזר למשקלים לפני ניתוח תוך 40 יום. 1 בבני אדם, כריתת מעי דק מסיבית יכולה לגרום לתסמונת מעי קצרה, malabsorptive פונקציונלי המדינה שמעניקה תחלואה משמעותית, תמותה ועלויות שירותי בריאות, כולל תלות Parenteral תזונה, אי ספיקת כבד ושחמת כבד, ואת הצורך בהשתלת איברי multivisceral. 2 במאמר זה, אנו מתארים ולתעד בפרוטוקול שלנו ליצירת רקמות מהונדסות מעי במודל של עכברים עם גישה רבה תאית organoid יחידות על פיגום. יחידות Organoid הן אגרגטים תאיים הנגזרים מהמעי המכילים אלמנטים הן ריריים וmesenchymal, 3 קשר בין המשמר את הנישה של תאי גזע במעי. 4 במחקר מתמשך ובעתיד, את המעבר שלנו לטכניקההעכבר יאפשר חקירה של התהליכים המעורבים בהיווצרות Tesi על ידי ניצול הכלים המהונדסים הזמינים במין זה. 5 הזמינות של זני עכברי מדוכאי חיסון גם תאפשר לנו ליישם את הטכניקה לרקמת מעי אנושית ולייעל את היווצרות Tesi האנושי xenograft עכבר לפני המעבר שלו לבני אדם. השיטה שלנו מעסיקה (GMP) ריאגנטים וחומרים שכבר אושרו לשימוש בחולים אנושיים, ולכן מציעים יתרון משמעותי על פני גישות המסתמכות על רקמות חי decellularized ייצור נאות. המטרה הסופית של שיטה זו היא התרגום שלה לבני אדם כאסטרטגיה טיפולית רפואת רגנרטיבית לתסמונת מעי קצרה.

איור 1 מציג סכמה כללית לפרוטוקול המתועד כאן. התוצאה הסופית של פרוטוקול זה היא מבנה כדורי של גלובוס או מעי עכברי רקמות מהונדס עם לומן, ריריות, submucosa, ומקיף 2A muscularis. איור מראה גלובוס אופייני בהשוואה לפיגום פולימרים. 2B איור התחלה מציגה ...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

מאמר זה ולהציג את הווידאו הנלווה הפרוטוקול שלנו ליצירת רקמות מהונדסות מעי בעכבר, באמצעות גישת organoid יחידות על פיגום.

הנדסת רקמות של המעי בדגם Murine

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K Views.  Keck School of Medicine of the University of Southern California. מאמר זה ולהציג את הווידאו הנלווה הפרוטוקול שלנו ליצירת רקמות מהונדסות מעי בעכבר, באמצעות גישת organoid יחידות על פיגום.

Video: Tissue Engineering of the Intestine in a Murine Model

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction עיבוד Fibres Electrospun עבור הנדסת רקמות

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

רקמות שריר שלד הנדסה מתאי Murine היצמדות מולידו ויישום של גירוי חשמלי

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

עיצוב של Bioreactor טוען מכונות Biaxial להנדסת רקמות

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

הנדסת רקמות של 3D אדם<em&gt; במבחנה</emמערכת מבחן גידול&gt;

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

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 ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

ייצור, אפיון ופוטנציאל שימושים של הוושט רירי דגם אדם מהונדס-רקמות 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

הנדסת ג&#39;ל קולגן Cellularized 3D לשחזור רקמות וכלי דם

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

הנדסת רקמות ידי עצמותי כלי דם בתוך<em&gt; In vivo</em&gt; הנדסת רקמות קאמרית

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

Arteriovenous (AV) Loop במודל בעלי חיים קטנים לחקר אנגיוגנזה והנדסת רקמות כלי דם

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

טיפול של רצועות ליגמנט עם תרגיל ממוזג בסרום: רקמות תרגום הנדסה דגם

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...