establishing the 3d stromal monoculture

Ensayo de cocultivo 3D: nichos osteogénicos vascularizados para el cribado de fármacos oncológicos

The bone and bone marrow are structurally and functionally complex organs central to human health. This is reflected by the existence of distinct niches that regulate hematopoiesis and bone maintenance1. It is now widely accepted that in healthy bone marrow, the maintenance and expansion of hematopoietic and skeletal stem cells, as well as their progeny, are controlled by distinct niches. These niches comprise various cell types, including osteolineage cells, mesenchymal stem cells, endothelial and perivascular cells, neuronal and glial cells, adipocytes, osteoclasts, macrophages, and neutrophils2. Not surprisingly, these mostly vasculature-associated niches are also involved in the development of various types of leukemia3 and are the site of metastasis for different cancers4. Owing to its specific roles in bone formation, remodeling, and bone (marrow) maintenance, the bone-associated vasculature has distinct specialized structures different from the vasculature found elsewhere in the body5,6,7. Thus, anti-angiogenic or vasculature-modulating drugs applied systemically may have different effects within these specialized environments8. Therefore, models to investigate the molecular mechanisms involved in maintaining the bone and bone marrow physiological properties, bone and bone marrow regeneration, and responses to therapeutic treatments are highly desirable.
Classical two-dimensional (2D) tissue cultures and in vivo investigations using animal models have provided invaluable insight into the roles of different cells and molecular players involved in the development of bone and bone marrow9,10. Models that allow for high-throughput experiments with relevant human cells could improve our understanding of how to modulate selected parameters in these highly complex systems.
In the past decade, principles derived from tissue engineering have been employed to generate 3D tissue models11,12. These have mostly relied on the encapsulation of tissue-relevant cells into biomaterials to establish 3D mono- or co-cultures13. Among the most used biomaterials are fibrin14, collagen15, and Matrigel16,17, all of which are highly biocompatible and provide appropriate conditions for the growth of many cell types. These biomaterials have the ability to generate in vitro models that recapitulate key aspects of the different vascular niches found in vivo18. Moreover, the use of microfluidic devices to generate perfused vascular bone and bone marrow models has contributed to the generation of in vitro models of higher complexity19,20,21,22.
The difficulty in controlling the composition and engineering the properties of naturally occurring biomaterials has inspired the development of synthetic analogs that can be rationally designed with predictable physical, chemical, and biological properties23,24. We have developed fully synthetic factor XIII (FXIII) cross-linked poly(ethylene glycol) (PEG)-based hydrogels, which are functionalized with RGD peptides and matrix metalloprotease (MMP) cleavage sites to facilitate cell attachment and remodeling25,26. The modular design of these biomaterials has been successfully used to optimize conditions for the formation of 3D vascularized bone and bone marrow models27,28.
For the testing of larger numbers of different culture conditions and new therapeutics, models with a higher throughput capability are required. We have recently shown that the FXIII cross-linking of our PEG hydrogel can be controlled through an electrochemical process such that an in-depth hydrogel stiffness gradient is formed29. When cells are added on top of such hydrogels, they migrate toward the interior and gradually develop into highly interconnected 3D cellular networks30. The elimination of the need to encapsulate cells into the hydrogel, which is usually present with other 3D scaffolds, not only simplifies the experimental design but also allows the sequential addition of different cell types at different time points to generate complex co-culture systems. These hydrogels are available pre-cast onto glass-bottom 96-well imaging plates, thus making the establishment of 3D cultures achievable by manual as well as automated cell seeding protocols. The optical transparency of the PEG hydrogels renders the platform compatible with microscopy.
Here, we present a straightforward method for the generation and characterization of vascularized osteogenic niches within this&#160;ready-to-use, synthetic plug-and-play platform. We show that the development of vascular networks can be stimulated with a growth factor commonly used for inducing in vitro osteogenesis, bone morphogenetic protein-2 (BMP-2), while osteogenic differentiation can be prevented by supplementation of fibroblast growth factor 2 (FGF-2)27,31. The networks formed are different when compared to FGF-2-stimulated networks in terms of the overall appearance, as well as the cell and ECM distribution. Moreover, we monitored the osteogenic induction using alkaline phosphatase as a marker. We demonstrate the increased expression of this marker over time and compare the expression to that in FGF-2 stimulated networks using qualitative and quantitative methods. Finally, we demonstrate the suitability of the generated niches of this model for two potential applications. Firstly, we performed a proof-of-concept drug sensitivity assay by adding bevacizumab to pre-formed niches and monitoring the degradation of the vascular networks in its presence. Secondly, we added MDA-MB-231 breast cancer and U2OS osteosarcoma cells to pre-formed osteogenic niches, showing that the niches can be used to study interactions between cancer cells and their environment.

Autor destacado: Establecimiento simple de un nicho de médula ósea osteogénica vascularizada utilizando hidrogeles prefabricados de poli(etilenglicol) (PEG) en una microplaca de imagen  

Establecimiento simple de un nicho de médula ósea osteogénica vascularizada utilizando hidrogeles de poli(etilenglicol) (PEG) prefabricados en una microplaca de imágenes

Watch this Scientific Journal Video about Establishing the 3D Stromal Monoculture at JoVE.com

Establishing the 3D Stromal Monoculture  

Establecimiento del monocultivo del estroma 3D

Para comenzar, tome un tubo de hBM-MSC y granularlos por centrifugación. Desechar el sobrenadante y resuspender el pellet en un volumen apropiado de medio basal. Luego, prepare tubos de centrífuga cónica de 50 mililitros que contengan el volumen requerido de medio basal complementado con los respectivos factores de crecimiento.Finalmente, agregue los hBM-MSC de la solución madre a una dilución de uno a 66.67 para lograr una concentración de 1.5 veces 10 a la quinta celda por mililitro. Retire la película adhesiva de polipropileno que cubre la placa de hidrogel de 96 pocillos para sembrar la placa. Aspire cuidadosamente el tampón de almacenamiento que cubre los hidrogeles colocando la punta del aspirador contra la pared del pozo y moviéndose lentamente hacia el borde del pozo interior.Durante la siembra, mezcle la suspensión celular periódicamente para mantener la mezcla homogénea y agregue 200 microlitros de la suspensión celular a cada pocillo de la placa. Luego, mantenga los cultivos a 37 grados centígrados y 5% de dióxido de carbono en una atmósfera humidificada. Alternativamente, use una lavadora de placas automatizada con las boquillas de aspiración colocadas al menos a 3,8 milímetros del portador de la placa y hacia el borde del pozo y aspire el amortiguador de almacenamiento.Sembrar la placa mezclando las células con una pipeta serológica y dispensando un número igual de células al pocillo de la placa. Monitorear el desarrollo de los cultivos por microscopía de campo claro con un objetivo de cinco veces y adquirir imágenes de referencia aproximadamente 30 minutos después de la siembra para evaluar el número de células agregadas.

Research

JoVE Journal

Bioengineering

Establishing the 3D Stromal Monoculture

283 vistas. Para comenzar, tome un tubo de hBM-MSC y granularlos por centrifugación. Desechar el sobrenadante y resuspender el pellet en un volumen apropiado de medio basal. Luego, prepare tubos de centrífuga cónica de 50 mililitros que contengan el volumen requerido de medio basal complementado con los respectivos factores de crecimiento.Finalmente, agregue los hBM-MSC de la solución madre a una dilución de uno a 66.67 para lograr una concentración de 1.5 veces 10 a la quinta celda por mili...

Video: Establishing the 3D Stromal Monoculture  

Simple Establishment of a Vascularized Osteogenic Bone Marrow Niche Using Pre-Cast Poly(ethylene Glycol) (PEG) Hydrogels in an Imaging Microplate

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models recapitulating bone- and bone marrow-specific functions, including vascularization, that are compatible with drug screening are highly desirable. Such models can bridge the gap between simplistic, structurally irrelevant two-dimensional (2D) in vitro models and the more expensive, ethically challenging in vivo models. This article describes a controllable three-dimensional (3D) co-culture assay based on engineered poly(ethylene glycol) (PEG) matrices for the generation of vascularized, osteogenic bone-marrow niches. The PEG matrix design allows the development of 3D cell cultures through a simple cell seeding step requiring no encapsulation, thus enabling the development of complex co-culture systems. Furthermore, the matrices are transparent and pre-cast onto glass-bottom 96-well imaging plates, rendering the system suitable for microscopy. For the assay described here, human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) are cultured first until a sufficiently developed 3D cell network is formed. Subsequently, GFP-expressing human umbilical vein endothelial cells (HUVECs) are added. The culture development is followed by bright-field and fluorescence microscopy. The presence of the hBM-MSC network supports the formation of vascular-like structures that otherwise&#160;would not form and that remain stable for at least 7 days. The extent of vascular-like network formation can easily be quantified. This model can be tuned toward an osteogenic bone-marrow niche by supplementing the culture medium with bone morphogenetic protein 2 (BMP-2), which promotes the osteogenic differentiation of the hBM-MSCs, as assessed by increased alkaline phosphatase (ALP) activity at day 4 and day 7 of co-culture. This cellular model can be used as a platform for culturing various cancer cells and studying how they interact with bone- and bone marrow-specific vascular niches. Moreover, it is suitable for automation and high-content analyses, meaning it would enable cancer drug screening under highly reproducible culture conditions.

An in vitro model of the bone marrow vascular niche is established by seeding mesenchymal and endothelial cells onto pre-cast 3D PEG hydrogels. The endothelial networks, ECM components, and ALP activity of the niches vary depending on the growth factor used. The platform can be used for advanced cancer models.

The bone and bone marrow are highly vascularized and structurally complex organs, and are sites for cancer and metastasis formation. In vitro models ...

Investigación

Bioingeniería

Establishing the 3D Stromal-Endothelial Cell Co-Culture

Establecimiento del cocultivo 3D de células estromales-endoteliales

Quantifying the Endothelial Cell Network Formation After the 3D Stromal-Endothelial Cell Co-Culture

Cuantificación de la formación de la red celular endotelial después del cocultivo 3D de células estromal-endoteliales

Assessing the Osteogenic Differentiation of 3D Stromal-Endothelial Cells by Monitoring the Alkaline Phosphatase Activity

Evaluación de la diferenciación osteogénica de células estromal-endoteliales 3D mediante el monitoreo de la actividad de la fosfatasa alcalina