Damos las gracias al laboratorio de Ulrich Schwarz para el software de seguimiento de cálculo utilizados en la cuantificación de las fuerzas de tracción celular (Sabass et al., 2008). Este trabajo fue apoyado por una concesión de carrera Burroughs Wellcome y el premio el director del NIH de Pioneer (DP10D00354) a ML Gardel y Médico Investigador Científico del Premio Nacional de Servicio (5 T32 GM07281) de invierno SP.

<ol>
	<li>Damljanovic, V., Lajerholm, B. C., Jacobson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+and+micropatterned+conjugation+of+extracellular+matrix+proteins+to+characterized+polyacrylamid+substrates+for+cell+mechanotransduction+assays.">Bulk and micropatterned conjugation of extracellular matrix proteins to characterized polyacrylamid substrates for cell mechanotransduction assays.</a> Biotechniques. 39 (6), 847-851 (2005).</li><li>Engler, A., Bacakova, L. N. e. w. m. a. n., Hategan, C., Griffin, A., M, D. D. i. s. c. h. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+compliance+versus+ligand+in+cell+on+gel+responses.">Substrate compliance versus ligand in cell on gel responses.</a> Biophys J. 86 ((1 Pt 1)), 617-628 (2004).</li><li>Gardel, M. L., Sabass, B., Ji, L., Danuser, G., Schwarz, U. S., Waterman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traction+stress+in+focal+adhesions+correlates+biphasically+with+actin+retrograde+flow+speed.">Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed.</a> J Cell Biol. 183, 999-1005 (2008).</li><li>Rajagopalan, P., Marganski, W. A., Brown, X. Q., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+comparison+of+the+spread+area,+contractility,+and+migration+of+balb/c+3T3+fibroblasts+adhered+to+fibronectin-+and+RGD-modified+substrata.">Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata.</a> Biophys J. 87 (4), 2818-2827 (2004).</li><li>Reinhart-King, C. A., Dembo, M., Hammer, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+and+mechanics+of+endothelial+cell+spreading.">The dynamics and mechanics of endothelial cell spreading.</a> Biophys J. 89, 676-689 (2005).</li><li>Stricker, J., Sabass, B., Schwarz, U. S., Gardel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+traction+force+microscopy+for+micron-sized+focal+adhesions.">Optimization of traction force microscopy for micron-sized focal adhesions.</a> J. Phys: Condensed Matter. 22, 194104-194114 (2010).</li><li>Sabass, B., Gardel, M. L., Waterman, C. M., Schwarz, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+traction+force+microscopy+based+on+experimental+and+computational+advances.">High resolution traction force microscopy based on experimental and computational advances.</a> Biophys J. 94, 207-220 (2008).</li><li>Yeung, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+substrate+stiffness+on+cell+morphology,+cytoskeletal+structure,+and+adhesion.">Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.</a> Cell Motil Cytoskeleton. 60 (1), 24-34 (2005).</li><li>Waterman-Storer, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule/organelle+motility+assays.">Microtubule/organelle motility assays.</a> Curr Protoc Cell Biol. , 13.1.1-13.1.21 (1998).</li></ol>

El procedimiento descrito aquí para la instalación de un microscopio de fuerza de tracción (TFM) experimento, junto con la implementación de rutinas de seguimiento de cálculo (Sabass et al., 2008), permite la cuantificación de las fuerzas de celulares con escala micrométrica resolución espacial. Para optimizar el protocolo experimental, es fundamental para formar un sustrato de gel puro y uniforme con capa uniforme de ligando ECM. Se discuten posibles escollos a continuación: ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>3-aminopropyltrimethyoxysilane</td>
<td>&nbsp;</td>
<td>Aldrich</td>
<td>28, 177-8</td>
<td>&nbsp;</td>
<tr>
<td>40% Acrylamide</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>161-0140</td>
<td>&nbsp;</td>
<tr>
<td>2% Bis-acrylamide</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP1404</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Fisher BioReagents</td>
<td>BP 150-20</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium persulfate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP179</td>
<td>&nbsp;</td>
<tr>
<td>40nm fluorescent micro-spheres</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F8789</td>
<td>&nbsp;</td>
<tr>
<td>Sulfo-SANPAH</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>22589</td>
<td>&nbsp;</td>
<tr>
<td>Confocal imaging chamber (RC-30)</td>
<td>&nbsp;</td>
<td>Warner Instruments</td>
<td>64-0320</td>
<td>&nbsp;</td>
<tr>
<td>Coverslip spinner</td>
<td>&nbsp;</td>
<td>Home-built</td>
<td>NA</td>
<td>&nbsp;</td>
<tr>
<td>Ultraviolet lamp CL1000</td>
<td>&nbsp;</td>
<td>UVP</td>
<td>95-0228-01</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel rack</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72239-04</td>
<td>&nbsp;</td>
<tr>
<td>acryloyl-X, SE (6-((acryloyl)amino)hexanoic acid)</td>
<td>&nbsp;</td>
<td>Invitrogen
</td>
<td>A-20770</td>
<td>&nbsp;</td>
<tr>
<td>Hydrazine hydrate</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>225819</td>
<td>&nbsp;</td>
<tr>
<td>Sodium meta-periodate</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>20504</td>
<td>&nbsp;</td>
<tr>
<td>Isopropanol</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A416-4</td>
<td>&nbsp;</td>
<tr>
<td>Fibronectin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F2006</td>
<td>&nbsp;</td>
<tr>
<td>Collagen</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (#1.5)</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940&#x2010;224</td>
<td>&nbsp;</td>
<tr>
<td>Glutaraldehyde</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>16120</td>
<td>&nbsp;</td>
<tr>
<td>Rain-X</td>
<td>&nbsp;</td>
<td>SOPUS Products</td>
<td>www.rainx.com</td>
<td>&nbsp;</td>
<tr>
<td>Acetic Acid</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>64-19-7</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

preparation of complaint matrices for quantifying cellular contraction

La regulación de la adhesión celular a la matriz extracelular (MEC) es esencial para la migración celular y la remodelación de ECM. Adhesiones focales son conjuntos macromoleculares que la pareja contráctil F-actina del citoesqueleto a la matriz extracelular. Esta conexión permite la transmisión intracelular de las fuerzas mecánicas a través de la membrana celular al sustrato subyacente. Trabajos recientes han demostrado las propiedades mecánicas de la ECM regular de adhesión focal y la morfología de la F-actina, así como numerosos procesos fisiológicos, como la diferenciación celular, la división, proliferación y migración. Por lo tanto, el uso de sustratos de cultivo celular se ha convertido en un método cada vez más frecuente para controlar con precisión y modulan las propiedades mecánicas de ECM. Para cuantificar las fuerzas de tracción en las adhesiones focales en células adherentes, sustratos compatibles se utilizan en combinación con imágenes de alta resolución y las técnicas de cómputo en un método llamado microscopio de fuerza de tracción (TFM). Esta técnica se basa en la medición de la magnitud local y dirección de las deformaciones inducidas por la contracción del sustrato celular. En combinación con la alta resolución de la microscopía de fluorescencia de las proteínas de marcado con fluorescencia, es posible correlacionar la organización del citoesqueleto y remodelación con las fuerzas de tracción. Aquí se presenta un protocolo experimental detallado para la preparación de dos dimensiones, matrices cumplen con el propósito de crear un sustrato de cultivo de células con un bien caracterizado, la rigidez mecánica ajustable, lo que es adecuado para medir la contracción celular. Estos protocolos incluyen la fabricación de hidrogeles de poliacrilamida, capa de proteínas ECM en tales geles, las células de revestimiento de gel, de alta resolución y microscopía confocal utilizando una cámara de perfusión. Además, ofrecemos una muestra representativa de los datos que demuestran la ubicación y magnitud de las fuerzas celular utilizando protocolos citados TFM.

Watch this Scientific Journal Video about Preparation of Complaint Matrices for Quantifying Cellular Contraction at JoVE.com

Preparation of Complaint Matrices for Quantifying Cellular Contraction

En este vídeo, que muestran las técnicas experimentales utilizadas para la fabricación de cumplimiento, la matriz extracelular (ECM) sustratos recubiertos adecuadas para el cultivo celular, y que son susceptibles de microscopía de fuerza de tracción y observando los efectos de la rigidez de ECM en el comportamiento celular.

Preparación de las matrices de demanda para la cuantificación de la contracción celular

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

preparation-of-complaint-matrices-for-quantifying-cellular-contraction

Research

JoVE Journal

Bioengineering

17.7K vistas.  University of Chicago. En este vídeo, que muestran las técnicas experimentales utilizadas para la fabricación de cumplimiento, la matriz extracelular (ECM) sustratos recubiertos adecuadas para el cultivo celular, y que son susceptibles de microscopía de fuerza de tracción y observando los efectos de la rigidez de ECM en el comportamiento celular.

Video: Preparation of Complaint Matrices for Quantifying Cellular Contraction

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Análisis preparación y fotoacústica de Vehículos celulares que contienen oro Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Investigación

Bioingeniería

Neonatal Cardiac Scaffolds: Novel Matrices for Regenerative Studies

The only definitive therapy for end stage heart failure is orthotopic heart transplantation. Each year, it is estimated that more than 100,000 donor hearts are needed for cardiac transplantation procedures in the United States1-2. Due to the limited numbers of donors, only approximately 2,400 transplants are performed each year in the U.S.2. Numerous approaches, from cell therapy studies to implantation of mechanical assist devices, have been undertaken, either alone or in combination, in an attempt to coax the heart to repair itself or to rest the failing heart3. In spite of these efforts, ventricular assist devices are still largely used for the purpose of bridging to transplantation and the utility of cell therapies, while they hold some curative promise, is still limited to clinical trials. Additionally, direct xenotransplantation has been attempted but success has been limited due to immune rejection. Clearly, another strategy is required to produce additional organs for transplantation and, ideally, these organs would be autologous so as to avoid the complications associated with rejection and lifetime immunosuppression. Decellularization is a process of removing resident cells from tissues to expose the native extracellular matrix (ECM) or scaffold. Perfusion decellularization offers complete preservation of the three dimensional structure of the tissue, while leaving the bulk of the mechanical properties of the tissue intact4. These scaffolds can be utilized for repopulation with healthy cells to generate research models and, possibly, much needed organs for transplantation. We have exposed the scaffolds from neonatal mice (P3), known to retain remarkable cardiac regenerative capabilities,5-8 to detergent mediated decellularization and we repopulated these scaffolds with murine cardiac cells. These studies support the feasibility of engineering a neonatal heart construct. They further allow for the investigation as to whether the ECM of early postnatal hearts may harbor cues that will result in improved recellularization strategies.

In these studies, we provide methodology for novel, neonatal, murine cardiac scaffolds for use in regenerative studies.

Los andamios neonatal cardíaca: Matrices novedosos para Estudios regenerativos

The only definitive therapy for end stage heart failure is orthotopic heart transplantation.  Each year, it is estimated that more than 100,000 donor ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Matrices 2D y 3D para estudiar la formación y la actividad invadosoma lineal

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Automated Contraction Analysis of Human Engineered Heart Tissue for Cardiac Drug Safety Screening

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these procedures in basic research and preclinical drug development, it is important to develop protocols for automated generation and analysis under standardized conditions. Here, we present a technique to generate engineered heart tissue (EHT) from cardiomyocytes of different species (rat, mouse, human). The technique relies on the assembly of a fibrin-gel containing dissociated cardiomyocytes between elastic polydimethylsiloxane (PDMS) posts in a 24-well format. Three-dimensional, force-generating EHTs constitute within two weeks after casting. This procedure allows for the generation of several hundred EHTs per week and is technically limited only by the availability of cardiomyocytes (0.4-1.0 x 106/EHT). Evaluation of auxotonic muscle contractions is performed in a modified incubation chamber with a mechanical interlock for 24-well plates and a camera placed on top of this chamber. A software controls a camera moved on an XYZ axis system to each EHT. EHT contractions are detected by an automated figure recognition algorithm, and force is calculated based on shortening of the EHT and the elastic propensity and geometry of the PDMS posts. This procedure allows for automated analysis of high numbers of EHT under standardized and sterile conditions. The reliable detection of drug effects on cardiomyocyte contraction is crucial for cardiac drug development and safety pharmacology. We demonstrate, with the example of the hERG channel inhibitor E-4031, that the human EHT system replicates drug responses on contraction kinetics of the human heart, indicating it to be a promising tool for cardiac drug safety screening.

Here, we show the generation of human engineered heart tissue from induced pluripotent stem cells (hiPSC)-derived cardiomyocytes. We present a method to analyze contraction force and exemplary alteration of contraction pattern by the hERG channel inhibitor E-4031. This method shows high level of robustness and suitability for cardiac drug screening.

Análisis automatizado contracción de ingeniería tisular para la Detección del Corazón Humano cardíaca Drug Safety

Cardiac tissue engineering describes techniques to constitute three dimensional force-generating engineered tissues. For the implementation of these ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

División de célula mamífera en Matrices 3D mediante microscopia de reflexión Confocal cuantitativa

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Gel-seq: Un método para la preparación de biblioteca simultánea de secuenciación de DNA y RNA utilizando Matrices de hidrogel

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Fabricación de una Matriz microambiente celular Artificial multiplexados

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

Novedoso proceso para la impresión 3D Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

Análisis 3D de las respuestas multicelulares a los degradados Quimioattractant

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

Preparación, administración y evaluación de la captación celular in vivo específica del tejido de liposomas marcados con colorante fluorescente

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...