אנו מודים לד&quot;ר גרנט סאמידה לקריאה ביקורתית של כתב היד. עבודה זו נתמכה על ידי פרס החוקר הצעיר Beckman (ש&quot;י), משפחה חדשה הלמן הפקולטה פרס (ש&quot;י), EUREKA-NIH, מאוניברסיטת קליפורניה לחקר הסרטן ועדת התיאום.

<ol>
	<li>Shih, W., Yamada, S., Myosin, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dependent+retrograde+flow+drives+3D+cell+migration.">A dependent retrograde flow drives 3D cell migration.</a> Biophys. J. 98 (8), L29-L31 (2010).</li><li>O'Brien, L. E., Yu, W., Tang, K., Jou, T. S., Zegers, M. M., Mostov, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+biochemical+analysis+of+Rac1+in+three-dimensional+epithelial+cell+cultures.">Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.</a> Methods Enzymol. 406, 676-691 (2006).</li><li>Legant, W. R., Miller, J. S., Blakely, B. L., Cohen, D. M., Genin, G. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+mechanical+tractions+exerted+by+cells+in+three-dimensional+matrices.">Measurement of mechanical tractions exerted by cells in three-dimensional matrices.</a> Nat. Methods. 7 (12), 969-971 (2010).</li><li>Del Alamo, J. C., Meili, R., Alonso-Latorre, B., Rodriguez-Rodriguez, J., Aliseda, A., Firtel, R. A., Lasheras, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+analysis+of+eurkaryotic+cell+motility+by+improved+force+cytometry.">Spatio-temporal analysis of eurkaryotic cell motility by improved force cytometry.</a> Proc. Natl. Acad. Sci. U.S.A. 104 (33), 13343-13348 (2007).</li><li>Lippincott-Schwartz, J., Snapp, E., Kenworthy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+protein+dynamics+in+living+cells.">Studying protein dynamics in living cells.</a> Nat. Rev. Mol. Cell Biol. 2 (6), 444-456 (2001).</li><li>Phair, R. D., Misteli, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+modeling+approaches+to+in+vivo+imaging.">Kinetic modeling approaches to in vivo imaging.</a> Nat. Rev. Mol. Cell Biol. 2 (12), 898-907 (2001).</li><li>Giepmans, B. N., Adams, S. R., Ellisman, M. H., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+toolbox+for+assessing+protein+location+and+function.">Fluorescent toolbox for assessing protein location and function.</a> Science. 312 (5771), 217-224 (2006).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Methods. 2, 905-909 (2005).</li><li>Cukierman, E., Pankov, R., Stevens, D. R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taking+cell-matrix+adhesions+to+the+third+dimension.">Taking cell-matrix adhesions to the third dimension.</a> Science. 294 (5547), 1708-1712 (2001).</li></ol>

אין ניגודי אינטרסים הכריז.

כאן אנו מתארים שיטה לשימוש לחיות תאים הדמיה ללמוד את המכניזם של נדידת תאים במטריצה ​​תלת מימדי. ההצלחה של טכניקה זו תלויה בהשגת &quot;טוב&quot; שיבוטים ביציבות להביע GFP-tagged חלבונים. רמה נמוכה של חלבון ה-GFP ידרוש חשיפה עירור עודף פשרות בריאות התא, ואילו רמה גבוהה מדי של GFP יה...

<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> קולגן, שור, סוג I </td><td> BD Biosciences </td><td> 354231 </td><td> מלאי הוא כ 3 מ&quot;ג / מ&quot;ל </td></tr><tr><td> 3-aminopropyltrimethoxysilane </td><td> סיגמא אולדריץ&#39; </td><td> 281778 </td><td> מדולל במים </td></tr><tr><td> glutaraldehyde </td><td> סיגמא אולדריץ&#39; </td><td> 340855 </td><td> מדולל ב PBS </td></tr><tr><td> 1M Hepes </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> Fluospheres פוליסטירן microspheres 1 מיקרומטר, אדום פלואורסצנטי (580/605) </td><td> Invitrogen </td><td> F13083 </td><td></td></tr><tr><td> Geneticin (G418) </td><td> Invitrogen </td><td> 11811-031 </td><td></td></tr&gt; <tr><td colspan="4"> תרבות התקשורת מרכיבים: </td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 31600-034 </td><td></td></tr><tr><td> סרום שור עוברית </td><td> אטלנטה הביולוגיים </td><td> S115500 </td><td></td></tr><tr><td> פניצילין / סטרפטומיצין </td><td> Invitrogen </td><td> 15140-122 </td><td></td></tr><tr><td> Kanamycin </td><td> Invitrogen </td><td> 15160-054 </td><td></td></tr></tbody></table>

live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix

באופן מסורתי, נדידת תאים נחקרה על דו מימדי, משטחי פלסטיק נוקשה. עם זאת, במהלך תהליכים ביולוגיים חשובים כגון ריפוי פצעים, התחדשות רקמות, גרורות סרטן, התאים חייבים לנווט רקמות מורכבות תלת ממדי, תאית. כדי להבין טוב יותר את המנגנונים מאחורי אלה תהליכים ביולוגיים, חשוב לבחון את תפקידם של החלבונים האחראיים על נהיגה נדידת תאים. כאן, אנו מתאר פרוטוקול ללמוד את המכניזם של נדידת תאים באמצעות שורת תאים אפיתל (MDCK), וכן תלת ממדי, סיבי, עצמית polymerizing מטריקס כמערכת מודל. זה תאי מטריקס ברור אופטית ניתנת בקלות לחיות תאים לימודי הדמיה טוב יותר מחקה את הסביבה הפיזיולוגית, רקמות רכות. דו&quot;ח זה מדגים טכניקה ישירות חזותי לוקליזציה חלבון דינמיקה, ו דפורמציה של מטריקס תלת ממדי שמסביב. בחינהביעור לוקליזציה של חלבונים ודינמיקה במהלך תהליכים תאיים מספק תובנה מפתח לתוך פונקציות חלבון. תגי פלורסנט מקודדים גנטית לספק שיטה ייחודית התבוננות לוקליזציה חלבון ודינמיקה. באמצעות טכניקה זו, אנו יכולים לנתח את הצטברות subcellular של מפתח, כוח להפקת רכיבים cytoskeletal בזמן אמת כמו תמרונים התא באמצעות המטריצה. בנוסף, באמצעות תגי פלורסנט עם מספר אורכי גל שונים, אנו יכולים לבחון את לוקליזציה של חלבונים רבים בו זמנית, ובכך מאפשר לנו לבחון, למשל, אם חלבונים שונים יש תפקידים דומים או מסתעף. יתר על כן, הדינמיקה של החלבונים המתויגים fluorescently ניתן לכמת באמצעות שחזור פלורסנט לאחר photobleaching (FRAP) ניתוח. זה מבחני המדידה ניידות חלבון ואיך קשור ביציבות החלבונים הם לרשת cytoskeletal. על ידי שילוב של תאים חיים הדמיה בטיפול של מעכבי תפקוד החלבון,אנו יכולים לבחון בזמן אמת את השינויים בהתפלגות של חלבונים מורפולוגיה של תאים נודדים. יתר על כן, אנו משלבים גם לחיות תאים הדמיה עם שימוש של חלקיקים נותב פלורסנט מוטבע בתוך המטריצה ​​כדי להמחיש את העיוות מטריקס במהלך נדידת תאים. לפיכך, אנו יכולים לדמיין כיצד תא נודדות מפיצה כוח להפקת חלבונים, שבו כוחות המתיחה הם הפעילו את המטריצה ​​שמסביב. באמצעות טכניקות אלה, אנו יכולים לקבל תובנה חשובה התפקידים של חלבונים ספציפיים תרומתם המנגנונים של נדידת תאים.

Watch this Scientific Journal Video about Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix at JoVE.com

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

תהליכים תאיים כגון נדידת תאים מסורתי נחקרו על דו מימדי, משטחי פלסטיק נוקשה. דו&quot;ח זה מתאר טכניקה ישירות חזותי לוקליזציה חלבון וניתוח הדינמיקה חלבון בתאי נודדות בתוך מטריצה ​​יותר רלוונטי מבחינה פיזיולוגית תלת מימדי,.

Live-תא הדמיה של תאים מעבר הבעת חלבונים fluorescently-מתויג מטריקס תלת מימדי

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

live-cell-imaging-migrating-cells-expressing-fluorescently-tagged

Research

JoVE Journal

Bioengineering

13.0K Views.  University of California, Davis. תהליכים תאיים כגון נדידת תאים מסורתי נחקרו על דו מימדי, משטחי פלסטיק נוקשה. דו"ח זה מתאר טכניקה ישירות חזותי לוקליזציה חלבון וניתוח הדינמיקה חלבון בתאי נודדות בתוך מטריצה ​​יותר רלוונטי מבחינה פיזיולוגית תלת מימדי,.

Video: Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

תלת ממדית ברזולוציה אופטית photoacoustic מיקרוסקופית

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Planar and Three-Dimensional Printing of Conductive Inks

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

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

הדפסה מישוריים תלת ממדי של דיו מוליך

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

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

יוניברסל כף היד תלת ממדי Optoacoustic הדמיה בדיקה לרקמות עמוקות אנגיוגרפיה אדם ופונקציונליים פרה-קליניים מחקרים בזמן אמת

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

ניתוח של נדידת תאים בתוך קולגן מטריקס תלת ממדי

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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 ...

Live Cell Imaging during Mechanical Stretch

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in their capability for measuring responses in real time in live cells or viable tissue. A protocol was developed with the use of a cell actuator to distend live cells grown on or tissues attached to an elastic substrate while imaging with confocal and atomic force microscopy (AFM). Preliminary studies show that tonic stretching of human bronchial epithelial cells caused a significant increase in the production of mitochondrial superoxide. Moreover, using this protocol, alveolar epithelial cells were stretched and imaged, which showed direct damage to the epithelial cells by overdistention simulating one form of lung injury in vitro. A protocol to conduct AFM nano-indentation on stretched cells is also provided.

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

הדמיה של תא חי במהלך מכאני למתוח

There is currently a significant interest in understanding how cells and tissues respond to mechanical stimuli, but current approaches are limited in ...

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, ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

להמיס Electrospinning הכתיבה של פיגומים Poly(ε-caprolactone) תלת מימדי עם מורפולוגיות לשליטה עבור יישומי הנדסת רקמות

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Culturing בתרבית של תאים בפפטיד תלת מימדי פיגומים

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

שלושה תלת-ממד של מטריקס-Adipocyte מודל תרבות המודל ללימוד תא מטריקס-מטבולית הסלולר

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...