Name: Establishing the 3D Stromal-Endothelial Cell Co-Culture
Uploaded: 2023-05-19T14:50:00
Duration: 1 min 48 s
Description: Watch this Scientific Journal Video about Establishing the 3D Stromal-Endothelial Cell Co-Culture at JoVE.com

establishing the 3d stromal-endothelial cell co-culture

Ensaio de co-cultura 3D: nichos osteogênicos vascularizados para triagem de drogas contra o câncer

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 em destaque: Estabelecimento simples de um nicho osteogênico vascularizado da medula óssea usando hidrogéis de poli (etilenoglicol) (PEG) pré-moldados em uma microplaca de imagem  

Estabelecimento simples de um nicho osteogênico vascularizado da medula óssea usando hidrogéis pré-moldados de poli(etilenoglicol) (PEG) em uma microplaca de imagem

Most in vitro vascularization studies use naturally-derived matrices. While typically very conductive to biological processes, their inherent biological activity complicates the study of influences of single parameters. Here, we can selectively add our players of interest and assess how the system changes.The sequential seeding of cells on our blank slate synthetic hydrogels enables the formation of very defined niches. Here, hBMMSs colonize the hydrogels and deposit their inherent extracellular matrix. This provides the substrate for the subsequently seeded endothelial cells.This feature is not commonly found in other 3D in vitro models. The possibility to add different cell types sequentially allows for the creation of high-throughput compatible in vitro models with great complexity. These models can be adapted to generate more specific osteogenic vascularized niches and to investigate the functions of cellular and molecular players.

Watch this Scientific Journal Video about Establishing the 3D Stromal-Endothelial Cell Co-Culture at JoVE.com

Establishing the 3D Stromal-Endothelial Cell Co-Culture  

Estabelecimento da Co-Cultura 3D de Células Endoteliais-Estromas

Para começar, pegue o pellet das GFP-HUVECs cultivadas a um nível de confluência de 80 a 100% e ressuspenda as células em um volume apropriado de meio basal para atingir uma concentração de uma vez 10 a sétima células por mililitro de solução estoque. Preparar tubos de centrífuga cônica de 50 mililitros contendo o volume necessário de meio basal suplementado com os respectivos fatores de crescimento. Adicionar as GFP-HUVECs da solução-mãe numa diluição de um a 66,67 para atingir uma concentração de 1,5 vezes 10 à quinta células por microlitro.Em seguida, aspirar o meio da placa contendo a monocultura estromal e adicionar 200 microlitros por poço da suspensão GFP-HUVECs preparada. Incubar a 37 graus Celsius em 5% de dióxido de carbono em uma atmosfera umedecida, trocando o meio a cada três ou quatro dias. Monitorar o desenvolvimento da cultura por microscopia de campo claro e fluorescência usando uma objetiva de cinco vezes.Diferenças entre as culturas puderam ser observadas a partir das imagens de campo claro e fluorescência mostrando apenas as GFP-HUVECs. As culturas mostraram-se menos desenvolvidas, sem qualquer fator de crescimento, porém, observou-se desenvolvimento mais rápido na presença do FGF-2. Em contraste, as imagens de campo claro mostraram as culturas mais densas na presença de BMP-2.No entanto, redes vasculares semelhantes formaram-se em ambas as condições contendo fator de crescimento e as redes mais extensas e interconectadas foram formadas com FGF-2.

Research

JoVE Journal

Bioengineering

Establishing the 3D Stromal-Endothelial Cell Co-Culture

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 ...

Bioengenharia

Establishing the 3D Stromal Monoculture

Estabelecendo a Monocultura Estromal 3D

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

Quantificando a Formação da Rede Celular Endotelial Após a Co-Cultura 3D de Células Estromais-Endoteliais

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

Avaliação da diferenciação osteogênica de células estromais-endoteliais 3D através do monitoramento da atividade da fosfatase alcalina