El autor desea agradecer al Dr. Jia Dongxuan de asistencia técnica. Algunas de las imágenes que aparecen en este artículo fueron en colaboración con el Dr. Malcolm S. Steinberg, del Departamento de Biología Molecular, Universidad de Princeton. El autor también desea agradecer al Departamento de Defensa, Programa de Investigación de Cáncer de Próstata (subvenciones 030.482 PC y PC-991552) y el de NCI / NIH (subvención R01CA118755) su generoso apoyo.

1. Preparación de una suspensión de células individuales <ol><li> Adherente culturas de células deben ser cultivadas a 90% de confluencia, las monocapas con lo cual se deben enjuagar dos veces con PBS. Después de drenar bien, añadir 2 ml (para placas de 100 mm) de la tripsina-1 0.05% mM EDTA y se incuba a 37 ° C hasta que las células separar. Dejar de tripsinización mediante la adición de 2 ml de medio completo y con cuidado el uso de una pipeta de 5 ml para triturar la mezcla hasta que las células están en suspensión. La transferencia de células a un tubo cónico de 15 ml. </li><li> Añadir 40 ml de un 10 acciones mg / ml DNAsa e incubar durante 5 minutos a temperatura ambiente. Vortex brevemente y centrifugar a 200 xg durante 5 minutos. </li><li> Desechar el sobrenadante y lavar pellet con 1 ml de medio completo de cultivo de tejidos. Repito, pues suspender las células en 2 ml de medio completo de cultivo de tejidos. </li><li> Contar las células con un hemocitómetro, o contador celular automático y ajustar la concentración de 2,5 x 10 6 células / ml. Para esta demostración un BioRad TC10 contador celular automático fue utilizado. </li></ol> 2. Formación de gotas colgantes. <ol><li> Quite la tapa de un plato de cultivo de tejidos de 60 mm y colocar 5 ml de PBS en el fondo del plato. Esto actúa como una cámara de hidratación. </li><li> Invertir la tapa y el uso de una pipeta de 20 l de depósito l 10 gotas en la parte inferior de la tapa. Asegúrese de que las gotas se colocan lo suficientemente separados para que no toque. Es posible colocar al menos 20 gotas por plato. </li><li> Invertir la tapa sobre el PBS cámara llena de fondo y se incuba a 37 ° C / 5% CO 2 / 95% de humedad, controlar las gotas todos los días y se incuban hasta que las hojas de células o grupos, se han formado. Un microscopio estéreo se puede utilizar para evaluar la formación de agregados. </li><li> Una vez que forman las hojas, que pueden ser transferidos a fondo redondo coctelera frascos que contienen 3 ml de medio completo y se incubaron en un baño de agua agitando a 37 º C y 5% de CO 2 hasta que se formen esferoides. </li></ol> 3. Notas <ol><li> Dependiendo del tamaño de la célula, la concentración puede ser necesario ajustar hacia arriba o hacia abajo. </li><li> Las células pueden ser teñidas con colorantes fluorescentes membrana intercalar antes de la formación de gota. </li><li> Dos diferentes tipos de células pueden ser diferenciada de colores y mezclados en una relación 1:1 (u otros) la relación antes de la formación de gotas colgantes. Estas pueden ser las células del cáncer y del estroma, tejidos de embriones o células manipuladas genéticamente para tener firmas específicas adhesivo. </li><li> Las células pueden separarse de los platos de cultivo de tejidos con 0,05% de tripsina / 2 mM de calcio para preservar la función cadherina. </li><li> Dependiendo del experimento, los tamaños de hoja pueden ser medidos, o agregados pueden ser utilizados para los ensayos mecánicos o para el análisis bioquímico y molecular. </li></ol> 4. Los resultados representativos: Hoja o la formación de esferoides se produce normalmente dentro de las 24 horas, pero puede tomar más tiempo dependiendo del tipo de célula. La figura 1 representa las imágenes capturadas después de 18 horas de incubación de tratar el cáncer de próstata LyLu MAT (MLL), las células en la cultura colgantes caída (Figura 1) o las células tratadas con 25 micras inhibidor de MEK PD98059. Tenga en cuenta que los resultados del tratamiento Meki en una compactación marcada de la capa celular. La compactación se puede cuantificar midiendo el tamaño de 10-20 (o más) que cuelgan gotas y la comparación de tamaño medio entre los grupos de tratamiento. Por lo general analizar las imágenes utilizando el software ImageJ umbral por cada imagen a continuación, convertir imágenes a modo binario, con lo cual se aplica el análisis de partículas para revelar el área total de la imagen en píxeles. Esto puede ser fácilmente convertido en micrones cuadrados. La Figura 2 representa una comparación de tamaño de las células no tratadas y Meki MLL tratados. Como se ha indicado, el tratamiento Meki reduce significativamente el tamaño de hoja, en comparación con la t de Student-test (P &lt;0,0001). Para esta comparación, 10 agregados para cada una de las células tratadas y no tratadas se midieron. La figura 3 representa un esferoide de pollo hígado embrionario formado después de 24 horas en colgar la cultura de caída y dos días en un baño de agitación. La prueba de gota también puede ser modificado para incluir más de un tipo de células para revelar el posicionamiento espacial adoptada. Por ejemplo, las células de embriones de pollo de hígado puede ser aislado, se tiñeron con un fluorescente membrana intercalante tinte y se mezclan con las células de pollo corazón embrionarias que han sido teñidas con un colorante fluorescente diferente. Figura 4 muestra que en lugar de permanecer mezclados, las células del hígado y el corazón tienden a &quot;una especie de salida&quot; de unos a los otros y adoptar una esfera dentro de una esfera de configuración en la que las células del corazón están envueltas por las células del hígado. Esta configuración puede ser explicado por diferencias en la adhesión intercelular entre células del hígado y del corazón. Este concepto está codificado en la hipótesis de adhesión diferencial que se ha utilizado para explicar el comportamiento de clasificación de células de tejido embrionario entre la capa de germen de anfibios y tres embriones de teleósteos 4, para la organización de células de los islotes pancreáticos y 5 de clasificación de células entre dos poblaciones de células idénticas in todos los demás aspectos que para los niveles de expresión del mismo tipo de cadherina (6 y Figura 4B). Este resultado es particularmente importante para demostrar cómo la ingeniería una simple diferencia de adherencia entre las poblaciones de células puede resultar en la generación de la estructura del órgano, por ejemplo, un islote del páncreas 5. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Figura 1" /> Figura 1. Imágenes de las células se incubaron en la cultura colgantes caída durante 18 horas ya sea en ausencia (A) o presencia (B) de 25 micras de los inhibidores de MEK PD98059. Las imágenes fueron capturadas con una Nikon Eclipse TE 300 microscopio de epifluorescencia y Fotometría CoolSnap cámara ES digital. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Figura 2" /> Figura 2. Cuantificación de Meki inducida por la compactación de la rata las células de cáncer de próstata MLL. Colgando culturas gota de células MLL tratados y Meki tratados se incubaron durante 18 horas después de lo cual el tamaño total se determinó como se describió anteriormente. El análisis estadístico de tamaño de los agregados fue por la t de Student prueba. El asterisco representa una diferencia estadísticamente significativa en el tamaño de los agregados (P &lt;0,0001) entre las células no tratadas y tratadas Meki y sugiere que el tratamiento con Meki en agregados significativamente más pequeños y compactos. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Figura 3" /> Figura 3. Un esferoide formado mediante la incubación de las células de embriones de pollo en el hígado colgando cultura gota durante 18 horas después en una estufa / baño de agitación durante 48 horas. Esta imagen fue tomada usando un microscopio estereoscópico Nikon SMZ800 y una cámara CCD Sony Negro. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Figura 4" /> Figura 4. Colgantes cultura caída revela la clasificación de espera entre el comportamiento de diferentes tipos de células. En el panel A, las células de embriones de pollo hígado se tiñeron con PKH-2 membrana colorante intercalante y mezclado en proporciones iguales con PKH-26 células de pollo teñidas corazón embrionario. En el panel B, dos N-cadherina-transfectadas clones de células L (LN4 y LN2a), que expresan N-cad en su superficie en una proporción de 2,4:1, se tiñeron también con los tintes fluorescentes membrana intercalando PKH-2 y PKH 26 , (Sigma-Aldrich), mezclado en proporciones iguales y se cultivan como gotas colgando 7. Después de 18 horas en cultivo, las hojas de la célula fueron trasladados a frascos de agitación y se incubó durante otras 48 horas. Los agregados fueron fijadas en paraformaldehído al 2% en tampón fosfato salino y se ve con un BioRad MRC600 sistema de escaneo láser confocal microscopio conectado a una Nikon Optiphot-2 microscopio. Secciones ópticas de los canales verde y rojo fueron recogidos y una estación de trabajo de Silicon Graphix Carmesí VGX equipado con Vital Images Voxel Ultra View software de imagen se utiliza para asignar colores falsos para ambos canales y para fusionar las imágenes, revelando las configuraciones anatómicas generado. (A) sección óptica confocal a través de un envolvimiento total que demuestra de las células del corazón (aquí, en un pseudo-color amarillo) por las células hepáticas en pseudocolor azul (de 8). (B) Sección óptica confocal a través de un envolvimiento total de demostración de alta de N-cadherina células que expresan (verde) por los que expresan niveles más bajos de N-cadherina (rojo) (de 6).

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Los estudios han demostrado que el cultivo de células en un contexto de tres dimensiones (3D) produce la morfología celular y la señalización distinta en comparación con un rígido de dos dimensiones del sistema de cultivo (2D) 9. Por ejemplo, poblada de fibroblastos-geles de colágeno demostrar que la morfología de fibroblastos en 3D es muy distinta de la observada en 2D 10,11. Del mismo modo, la cultura 3D puede inducir la diferenciación de tejidos específicos de las células epiteliales ...

<ul><li> contador de células automático (BioRad TC10) </li><li> sacudiendo baño de agua con CO 2 Modelo trata de dos aguas 3540 (Lab-line) </li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

Los estudios de la cohesión célula-célula y la adhesión célula-sustrato históricamente han llevado a cabo en cultivos de monocapa adherida a sustratos rígidos. Las células en un tejido, sin embargo, suelen ser encerrado dentro de una masa de tejido muy juntos en el cual las células establecer conexiones íntimas con muchos casi vecinos y con componentes de la matriz extracelular. En consecuencia, el entorno químico y físico experimentado por las fuerzas de las células en un tejido 3D son fundamentalmente diferentes de las experimentadas por las células cultivadas en cultivo en monocapa. Esto se ha demostrado que el impacto notablemente la morfología celular y la señalización. Varios métodos han sido diseñados para generar culturas de células en 3D incluyendo encapsulación de células en geles de colágeno o un biomaterial en andamios 2. Estos métodos, aunque útiles, no recapitulan la íntima directa célula-célula arquitectura de adhesión se encuentra en los tejidos normales. Por el contrario, más de cerca los sistemas de aproximación de la cultura en la que las células individuales están más o menos dispersas dentro de una malla 3D de productos de ECM. Aquí se describe un método simple en el cual las células se colocan en la cultura colgantes caída y se incubaron en condiciones fisiológicas, hasta que la verdadera forma de esferoides 3D en el que las células están en contacto directo con ellos y con componentes de la matriz extracelular. El método no requiere equipo especializado y puede ser adaptado para incluir la adición de cualquier agente biológico en cantidades muy pequeñas que pueden ser de interés en el esclarecimiento de los efectos en la célula-célula o la interacción célula-ECM. El método también puede utilizarse para el co-cultivo de dos (o más) de diferentes poblaciones de células con el fin de dilucidar el papel de las interacciones célula-célula o célula-ECM en la especificación de las relaciones espaciales entre las células. Célula-célula la cohesión y la adhesión célula-ECM son los pilares de los estudios de desarrollo embrionario, la interacción, las células tumorales del estroma en la invasión maligna, la cicatrización de heridas, y para aplicaciones de ingeniería de tejidos. Este sencillo método proporcionará un medio de generar tejido como agregados celulares para la medición de las propiedades biomecánicas o para el análisis molecular y bioquímico en un modelo fisiológicamente relevantes.

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Se describe un método simple, rápido de generación de 3D, como los tejidos esferoides y su potencial aplicación para cuantificar las diferencias en las interacciones célula-célula.

Una simple gota colgante celular Cultura Protocolo para la generación de esferoides 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

a-simple-hanging-drop-cell-culture-protocol-for-generation-3d

Research

JoVE Journal

Bioengineering

56.6K vistas.  UMDNJ-Robert Wood Johnson Medical School. Se describe un método simple, rápido de generación de 3D, como los tejidos esferoides y su potencial aplicación para cuantificar las diferencias en las interacciones célula-célula.

Video: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparación de fibrina Andamios 3D para Aplicaciones madre para cultivo celular

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Investigación

Bioingeniería

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Generación y Recuperación de β-células esferoides de PEG-péptido de crecimiento Paso hidrogeles

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D hidrogel andamios para articular condrocitos cultura y cartílago Generación

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Los hidrogeles sintonizable de la matriz extracelular pulmonar por la 3D de Cultivos Celulares

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Un protocolo para el seguimiento en tiempo real 3D sola partícula

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Construcción de una hoja de varias capas células madre mesenquimales con un sistema de cultivo dinámico 3D

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

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.

Un modelo de cultivo de matriz-adipocitos extracelulares 3D humano para el estudio de la conversación cruzada metabólica Matrix-Cell

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

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Producción automatizada a gran escala de esferoides de células madre derivadas de tejido adiposo para bioimpresión 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Generación de microambientes celulares multicue mediante fotopatronaje UV de sustratos de cultivo celular tridimensional

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

Generación, detección de alto rendimiento y biobancos de esferoides cardíacos derivados de células madre pluripotentes inducidos por humanos

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...