המחברים מבקשים להודות ברוק Hjelm עבור המומחיות הטכנית שלה, אנדרו לארסן לניתוח החלבון שלו. עבודה זו מומנה בחלקה על ידי חלופות מחקר ופיתוח (MMHK) גרנט NIH מחלות המועברות במגע מיני NIAID ו microbicides מקומיים שיתופית מרכז המחקר IU19 AI062150-01 (MMHK). אנו בתודה להכיר ביולוגיה של רבייה לשימוש חוזר של דמויות.

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החוקרים אין לי מה לחשוף.

ניצול הטכנולוגיה bioreactor RWV המוצג כאן עשוי לספק לחוקרים את היכולת לקדם את התרבות המערכת הנוכחית שלהם התא מודל רלוונטי יותר מבחינה פיזיולוגית תא organotypic התרבות. תא bioreactor RWV מערכת התרבות מספקת microenvironment גזירה נמוך המאפשר לתאים ליצירת אגרגטים הסלולר 3-D עם in vivo, כמו מאפי?...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td><td> תגובות </td></tr><tr><td> Alexa פלואוריד 488 </td><td> Invitrogen </td><td> A21131 </td><td> השתמשו ב דילול 1:500 </td></tr><tr><td> FACSDiva </td><td> BD </td><td></td><td> תזרים cytometer </td></tr><tr><td> β-tublin נוגדן </td><td> Calbiochem </td><td> 654162 </td><td> השתמשו בדילול 1:5000 </td></tr><tr><td> Bio-Plex 2000 </td><td> BioRad </td><td> 171-000205 </td><td> V5 תוכנה </td></tr><tr><td> Bioreactor ורכיבים </td><td> Synthecon </td><td> RCCS-4 </td><td></td></tr><tr><td> תא מסננת </td><td> BD פלקון </td><td> 352340 </td><td> גודל הנקבוביות 40μm </td></tr><tr><td> צינור חרוטי (50 מ&quot;ל) </td><td> קורנינג </td><td> 5-538-60 </td><td></td></tr><tr><td> Coverslips </td><td> VWR </td><td> 48366067 </td><td></td></tr><tr><td> חרוזים ציטוקינים מערך ערכות </td><td> BioRad </td><td> ערכת האדם מותאם אישית </td><td></td></tr><tr><td> Cytodex חרוזים </td><td> סיגמא </td><td> C3275 </td><td></td></tr><tr><td> DPBS </td><td> Gibco </td><td> 14190 </td><td></td></tr><tr><td> EDTA </td><td> סיגמא </td><td> ED-500 גרם </td><td> Ethylenediaminetetraacetic חומצה </td></tr><tr><td> נוגדן ספציפי אפיתל (ESA) </td><td> Chemicon </td><td> CBL251 </td><td> השתמשו בדילול 1:50 </td></tr><tr><td> העובר שור סרום (FBS) </td><td> Gibco </td><td> 10438 </td><td> מומת חום </td></tr><tr><td> הארוו (חד פעמי) </td><td> Synthecon </td><td> D-405 </td><td></td></tr><tr><td> מימן כלורי </td><td> סיגמא </td><td> 258148 </td><td> 37% </td></tr><tr><td> Involucrin נוגדן </td&gt; <td> סיגמא </td><td> אני 9018 </td><td></td></tr><tr><td> מיקרוסקופ שקופיות </td><td> VWR </td><td> 16004-368 </td><td></td></tr><tr><td> MTT מגיב </td><td> MP Biomedicals, LLC </td><td> 194592 </td><td> 3 - (4,5-Dimethylthiazolyl 1-2) -2,5-Diphenyl ברומיד Tetrazolium </td></tr><tr><td> MUC1 נוגדנים (מיקרוסקופ) </td><td> סנטה קרוז </td><td> SC-7313 </td><td> השתמשו בדילול 1:50 </td></tr><tr><td> MUC1 נוגדנים (cytometry זרימה) </td><td> BD Pharmingen </td><td> 559774 </td><td> נקרא גם CD227, השתמש 20μL לכל מבחן </td></tr><tr><td> Paraformaldehyde </td><td> מיקרוסקופית אלקטרונים למדעים </td><td> 15710 </td><td> מדולל ל -4% ב DPBS </td></tr><tr><td> צלחת פטרי (קטן) </td><td> BD פלקון </td><td> 353002 </td><td></td></tr><tr><td> פוליסטירן צינור עם פילטר </td><td> BD פלקון </td><td> 352235 </td><td></td></tr><tr><td&gt; זרימת פוליסטירן צינור </td><td> BD פלקון </td><td> 352058 </td><td></td></tr><tr><td> יחסי ציבור נוגדן </td><td> DAKO </td><td> M3569 </td><td> השתמשו ב דילול 1:100 </td></tr><tr><td> להאריך זהב </td><td> Invitrogen </td><td> P36931 </td><td> התקשורת גובר עם DAPI </td></tr><tr><td> ערכת RNeasy מיני </td><td> Qiagen </td><td> 74903 </td><td></td></tr><tr><td> Dodecyl נתרן גופרתי </td><td> סיגמא </td><td> 71725 </td><td></td></tr><tr><td> עיקור כיס </td><td> VWR </td><td> 11213-035 </td><td></td></tr><tr><td> והברזים למיניהם (חד כיווני) </td><td> Medex </td><td> MX5061L </td><td></td></tr><tr><td> מזרק (10 מ&quot;ל) </td><td> BD </td><td> 309604 </td><td> Luer נעילת קצה </td></tr><tr><td> מזרק (5 מ&quot;ל) </td><td> BD </td><td> 309603 </td><td> Luer נעילת קצה </td></tr><tr><td> Trypan כחול </td><td> Invitrogen </td><td>T10282 </td><td></td></tr><tr><td> Vp5 נוגדן </td><td> סנטה קרוז </td><td> SC-13525 </td><td> HSV-2 Clone נוגדנים 6F10, המשמש בדילול 1:5000 </td></tr></tbody></table>

culturing and applications of rotating wall vessel bioreactor derived 3d epithelial cell models

תאים ורקמות של הגוף את הניסיון תנאים סביבתיים המשפיעים על הארכיטקטורה שלהם, תקשורת אינטר, פונקציות הכוללות. עבור בתרבות מודלים חוץ גופית סלולריים לחקות במדויק את הרקמה של עניין, את הסביבה הצמיחה של התרבות היא היבט חשוב לשקול. נפוץ קונבנציונאלי תרבות להפיץ מערכות תא מתאי אפיתל שטוחים על משטחים בלתי חדירים דו מימדי (2-D). למרות שדברים רבים נלמדו ממערכות התרבות תאים רגילים, ממצאים רבים אינם לשחזור ניסויים קליניים בבני אדם או explants רקמות, פוטנציאל כתוצאה מחוסר microenvironment רלוונטי מבחינה פיזיולוגית. כאן אנו מתארים מערכת תרבות מתגבר רבים של מצב גבולות התרבות של 2-D בתרביות תאים, באמצעות כלי חדשני מסתובב הקיר (RWV) טכנולוגיה bioreactor. אנחנו ואחרים הראו כי organotypic RWV הנגזרות מודלים יכול לסכם structur, פונקציה E, תגובות אנושי אמיתי לגירויים חיצוניים דומה לרקמות explant אדם 1-6. Bioreactor RWV היא תרבות ההשעיה מערכת המאפשרת צמיחה של תאים אפיתל תחת תנאים גזירה נמוכים פיסיולוגיות נוזל. Bioreactors מגיעים בשני פורמטים שונים, כלי גבוהה היבט מסתובב (הארוו) או כלי שיט איטי מפנה לרוחב (STLV), שבו הם נבדלים זה מזה על ידי מקור אוורור שלהם. בתאי אפיתל מתווספים bioreactor הבחירה בשילוב עם נקבוביים, קולגן מצופים חרוזים microcarrier (איור 1 א). התאים לנצל את החרוזים כמו צמיחה פיגום בסתיו חינם קבוע bioreactor (1B איור). Microenvironment מסופק על ידי bioreactor מאפשר לתאים כדי ליצור אגרגטים תלת ממדי (3-D) הצגת in vivo, כמו מאפיינים בדרך כלל לא נצפתה על פי תקן 2-D התנאים תרבות 1D (איור). מאפיינים אלה כוללים צמתים הדוקים, MUCלנו ייצור, הפסגה / הבסיס אוריינטציה, בלוקליזציה חלבון vivo, וגם נוספות אפיתל לתא מסוג מאפיינים ספציפיים. התקדמות מ monolayer של תאים אפיתל לצבור 3-D הבדיל באופן מלא משתנה בהתאם לסוג התא 1, 7-13. הדגימה מעת לעת bioreactor מאפשרת ניטור של היווצרות המצרפי אפיתל, סמנים התמיינות ועל הכדאיות (1D איור). לאחר התמיינות היווצרות המצרפי הוא הוקם, התאים נקצרים מן bioreactor, ואת מבחני דומה שבוצעה על 2-D תאים יכול להיות מיושם על המצרפים 3-D עם כמה שיקולים (איור 1E-G). בעבודה זו אנו מתארים שלבים מפורטים של איך תרבות 3-D אגרגטים תא אפיתל במערכת bioreactor RWV ועוד מגוון של מבחני וניתוחים פוטנציאליים כי ניתן לבצע עם 3-D אגרגטים. ניתוחים אלה כוללים, אך לא רק, מבני / מ &#39;ניתוח orphological (confocal, סריקה מיקרוסקופית אלקטרונים הילוכים), ציטוקינים / chemokine הפרשת ו איתות תא (מערך חרוז cytometric וניתוח כתם המערבי), ביטוי גנים ניתוח (בזמן אמת PCR), טוקסיקולוגית / סמים או ניתוח אינטראקציות בין מאכסן לפתוגן. ניצול של מבחני אלה להגדיר את הבסיס מחקרים מפורטים יותר ורחבה כמו metabolomics, transcriptomics, proteomics ועוד מגוון יישומים מבוססי. המטרה שלנו היא להציג לא קונבנציונאלי באמצעות תאים אנושיים culturing אפיתל לייצר organotypic 3-D מודלים לשחזר האנושי ברקמה vivo, במערכת קליל וחזק לשימוש על ידי חוקרים בעלי אינטרסים מדעיים שונים.

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Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

תרבות מסתובב בתא מערכת המאפשר לתאי האפיתל לצמוח בתנאים פיסיולוגיים וכתוצאה מכך היווצרות 3 D-המצרפי הסלולרי המתואר. מצרפים את התצוגה באופן<em&gt; In vivo</em&gt; כמו מאפיינים לא נצפתה מודלים תרבות קונבנציונליים ולשמש מערכת מדויקת יותר מודל organotypic עבור מספר רב של מחקרים מדעיים.

Culturing ויישומים של סיבוב bioreactor וול כלי נגזר מודלים 3D תא אפיתל

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. ...

culturing-applications-rotating-wall-vessel-bioreactor-derived-3d

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

26.3K Views.  University of Arizona College of Medicine - Phoenix. תרבות מסתובב בתא מערכת המאפשר לתאי האפיתל לצמוח בתנאים פיסיולוגיים וכתוצאה מכך היווצרות 3 D-המצרפי הסלולרי המתואר. מצרפים את התצוגה באופן In vivo כמו מאפיינים לא נצפתה מודלים תרבות קונבנציונליים ולשמש מערכת מדויקת יותר מודל organotypic עבור מספר רב של מחקרים מד?...

Video: Culturing and Applications of Rotating Wall Vessel Bioreactor Derived 3D Epithelial Cell Models

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

סיבוב תרבית תאים מערכות התרבות תאים אנושיים: תאים Trophoblast האדם כמודל

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Bioengineering

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

שבב מבוסס תלת ממדי נייד תרבות Micro-bioreactors perfused

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

ריאקטורים סיבים חלולים עבור<em&gt; In vivo</em&gt; תרבות הרקמות היונקת-כמו

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

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., ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

הנדסה תלת מימדי רקמות אפיתל Embedded בתוך מטריקס

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

רשת כלי הדם perfusable עם דגם רקמה במכשיר Microfluidic

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation and Quantitative Characterization of Functional and Polarized Biliary Epithelial Cysts

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the context of liver diseases, 3-dimensional (3D) models based on cholangiocytes have emerged such as cysts, spheroids, or tube-like structures to mimic tissue topology for organogenesis, disease modeling, and drug screening studies. These structures have been mainly obtained by embedding cholangiocytes in a hydrogel. The main purpose was to study self-organization by addressing epithelial polarity, functional, and morphological properties. However, very few studies focus on cyst formation efficiency. When this is the case, the efficiency is often quantified from images of a single plane. Functional assays and structural analysis are performed without representing the potential heterogeneity of cyst distribution arising from hydrogel polymerization heterogeneities and side effects. Therefore, the quantitative analysis, when done, cannot be used for comparison from one article to another. Moreover, this methodology does not allow comparisons of 3D growth potential of different matrices and cell types. Additionally, there is no mention of the experimental troubleshooting for immunostaining cysts. In this article, we provide a reliable and universal method to show that the initial cell distribution is related to the heterogeneous vertical distribution of cyst formation. Cholangiocyte cells embedded in hydrogel are followed with Z-stacks analysis along the hydrogel depth over the time course of 10 days. With this method, a robust kinetics of cyst formation efficiency and growth is obtained. We also present methods to evaluate cyst polarity and secretory function. Finally, additional tips for optimizing immunostaining protocols are provided in order to limit cyst collapse for imaging. This approach can be applied to other 3D cell culture studies, thus opening the possibilities to compare one system to another.

Three-dimensional (3D) cellular systems are relevant models for investigating organogenesis. A hydrogel-based method for biliary cysts production and their characterization is proposed. This protocol unravels the barriers of 3D characterization, with a straightforward and reliable method to assess cyst formation efficiency, sizes, and to test their functionality.

ייצור ואפיון כמותי של ציסטות אפיתל מרה פונקציונליות ומקוטבות

Cholangiocytes, the epithelial cells that line up the bile ducts in the liver, oversee bile formation and modification. In the last twenty years, in the ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

הדפסה ביולוגית ישירה של ספירואידי שד רב-תאיים תלת-תאיים ברשתות אנדותל

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

ריבוי תאים ואיברים דנטליים ונשימתיים במיקרו-כבידה

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

<meta charset="utf8"></head><body>מערכת תרבית משותפת תלת-ממדית דו-שכבתית מתקדמת לתאי אפיתל ופיברובלסטים

Establishing a Three-Dimensional Coculture Module of Epithelial Cells Using Nanofibrous Membranes

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods can enhance cell culture efficiency. This study introduces an advanced two-layered culture system intended to cultivate epithelial cells as tissue-like layers with the culture of fibroblasts within a 3D environment. Polyvinyl alcohol (PVA) and poly(&#949;-caprolactone) (PCL) nanofibrous membranes (NMs) were fabricated via electrospinning and utilized as a physiologically relevant extracellular matrix for the culture of epithelial cells and fibroblasts, respectively. In the upper insert wells, lung epithelial cells were cultivated on the PVA NM, and in the lower chambers, fibroblasts were cultured on the PCL NM. This configuration eliminates direct cell-cell contact and facilitates the examination of paracrine signaling mediated by soluble factors. Confocal microscopy was employed to analyze the distribution, growth pattern, and expression of intracellular proteins, including zona occludens in epithelial cells. Z-stacking techniques enabled detailed 3D reconstructions, providing precise insights into the integrity of tight junctions and spatial organization within the epithelial layer. Scanning electron microscopy (SEM) assessed the morphological characteristics of cell types on the nanofibrous membranes. SEM imaging revealed intricate cell surface structures and interactions with the nanofibers, offering a comprehensive perspective on cellular architecture and cell interaction with nanofibrous structure. The Cell Counting Kit-8 (CCK-8) assay is a simple method for measuring epithelial cell and fibroblast growth rates over time. It provides the proliferative behaviors and potential synergistic effects of coculturing these cells. These findings highlight the effectiveness of a simple insert co-culture system for simultaneous culture of fibroblasts and epithelial cells, which is crucial in various physiological and pharmacological contexts, including epithelial tissue regeneration, tumor microenvironment with endothelial, immune, and other stroma cells, toxicity assay, and drug activity test.

<meta charset="utf8"></head><body>מחבר זרקור: קידום מערכות קוקולטורה תלת-ממדית עם PVA-PCL ממברנות ננו-סיביות

Here, we demonstrate how epithelial cells cultured with fibroblasts in the nanofibrous membrane-based two-layer system can stably adhere to and grow on the membrane.

הקמת מודול קוקולטורה תלת מימדי של תאי אפיתל באמצעות ממברנות ננו-סיביות

Technical hurdles in a culture of epithelial cells include dedifferentiation and loss of function. Biomimetic three-dimensional (3D) cell culture methods ...