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

3D Co-Culture Assay: 암 약물 스크리닝을 위한 혈관화된 골형성 틈새

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.

저자 스포트라이트: 이미징 마이크로플레이트에서 프리캐스트 폴리(에틸렌 글리콜)(PEG) 하이드로겔을 사용하여 혈관화된 골형성 골수 틈새의 간단한 확립  

이미징 마이크로플레이트에서 프리캐스트 폴리(에틸렌 글리콜)(PEG) 하이드로겔을 사용하여 혈관화된 골수 틈새의 간단한 설정

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  

3D Stromal-Endothelial Cell Co-Culture 확립

시작하려면 80-100%의 합류 수준으로 성장한 GFP-HUVEC의 펠릿을 취하여 적절한 부피의 기본 배지에 세포를 재현탁하여 원액 밀리리터당 10-7번째 세포의 1배 농도를 달성합니다. 각 성장 인자로 보충된 필요한 양의 기본 배지가 들어 있는 50밀리리터 원추형 원심분리기 튜브를 준비합니다. 원액으로부터 GFP-HUVECs를 1 내지 66.67의 희석액으로 첨가하여 마이크로리터당 1.5 내지 5번째 세포에 1.5배의 농도를 달성한다.이어서, 기질 단일배양물을 함유하는 플레이트로부터 배지를 흡인하고, 제조된 GFP-HUVECs 현탁액을 웰 당 200 마이크로리터 첨가한다. 섭씨 37도에서 5 % 이산화탄소로 가습 된 대기에서 배양하고 3-4 일마다 배지를 교체합니다. 5배 대물렌즈를 사용하여 명시야 및 형광 현미경으로 배양물의 발달을 모니터링합니다.배양 간의 차이는 GFP-HUVEC만 보여주는 명시야 및 형광 이미지에서 관찰할 수 있었습니다. 배양물은 어떠한 성장 인자도 없이 덜 발달된 것으로 나타났지만, FGF-2의 존재 하에서 더 빠른 발달이 관찰되었다. 대조적으로, 명시야 이미지는 BMP-2의 존재 하에서 가장 밀도가 높은 배양을 보여주었습니다.그러나, 성장 인자-함유 조건 및 가장 광범위하고 상호연결된 네트워크 둘 다에서 형성된 혈관 유사 네트워크는 FGF-2로 형성되었다.

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 ...

연구

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3D Stromal 단일 문화 구축

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알칼리성 포스파타제 활성을 모니터링하여 3D 기질-내피 세포의 골형성 분화 평가