Dataset Information

Correlative Imaging of Three-Dimensional Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications.

ABSTRACT: Three-dimensional (3D) tissue engineering (TE) is a prospective treatment that can be used to restore or replace damaged musculoskeletal tissues, such as articular cartilage. However, current challenges in TE include identifying materials that are biocompatible and have properties that closely match the mechanical properties and cellular microenvironment of the target tissue. Visualization and analysis of potential 3D porous scaffolds as well as the associated cell growth and proliferation characteristics present additional problems. This is particularly challenging for opaque scaffolds using standard optical imaging techniques. Here, we use graphene foam (GF) as a 3D porous biocompatible substrate, which is scalable, reproducible, and a suitable environment for ATDC5 cell growth and chondrogenic differentiation. ATDC5 cells are cultured, maintained, and stained with a combination of fluorophores and gold nanoparticles to enable correlative microscopic characterization techniques, which elucidate the effect of GF properties on cell behavior in a 3D environment. Most importantly, the staining protocol allows for direct imaging of cell growth and proliferation on opaque scaffolds using X-ray MicroCT, including imaging growth of cells within the hollow GF branches, which is not possible with standard fluorescence and electron microscopy techniques.

SUBMITTER: Sawyer M

PROVIDER: S-EPMC10521016 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Correlative Imaging of Three-Dimensional Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications.

Sawyer Mone't M Eixenberger Josh J Nielson Olivia O Manzi Jacob J Francis Cadré C Montenegro-Brown Raquel R Subbaraman Harish H Estrada David D

ACS applied bio materials 20230901 9

Three-dimensional (3D) tissue engineering (TE) is a prospective treatment that can be used to restore or replace damaged musculoskeletal tissues, such as articular cartilage. However, current challenges in TE include identifying materials that are biocompatible and have properties that closely match the mechanical properties and cellular microenvironment of the target tissue. Visualization and analysis of potential 3D porous scaffolds as well as the associated cell growth and proliferation chara ...[more]

PMID: 37655758

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Project description:White adipose tissue (WAT) is becoming widely used in regenerative medicine/cell therapy applications, and its physiological and pathological importance is increasingly appreciated. WAT is a complex organ composed of differentiated adipocytes, stromal mesenchymal progenitors known as adipose stromal cells (ASC), as well as endothelial vascular cells and infiltrating leukocytes. Two-dimensional (2D) culture that has been typically used for studying adipose cells does not adequately recapitulate WAT complexity. Improved methods for reconstruction of functional WAT ex vivo are instrumental for understanding of physiological interactions between the composing cell populations. Here, we used a three-dimensional (3D) levitation tissue culture system based on magnetic nanoparticle assembly to model WAT development and growth in organoids termed adipospheres. We show that 3T3-L1 preadipocytes remain viable in spheroids for a long period of time, while in 2D culture, they lose adherence and die after reaching confluence. Upon adipogenesis induction in 3T3-L1 adipospheres, cells efficiently formed large lipid droplets typical of white adipocytes in vivo, while only smaller lipid droplet formation is achievable in 2D. Adiposphere-based coculture of 3T3-L1 preadipocytes with murine endothelial bEND.3 cells led to a vascular-like network assembly concomitantly with lipogenesis in perivascular cells. Adipocyte-depleted stromal vascular fraction (SVF) of mouse WAT cultured in 3D underwent assembly into organoids with vascular-like structures containing luminal endothelial and perivascular stromal cell layers. Adipospheres made from primary WAT cells displayed robust proliferation and complex hierarchical organization reflected by a matricellular gradient incorporating ASC, endothelial cells, and leukocytes, while ASC quickly outgrew other cell types in adherent culture. Upon adipogenesis induction, adipospheres derived from the SVF displayed more efficient lipid droplet accumulation than 2D cultures. This indicates that 3D intercellular signaling better recapitulates WAT organogenesis. Combined, our studies show that adipospheres are appropriate for WAT modeling ex vivo and provide a new platform for functional screens to identify molecules bioactive toward individual adipose cell populations. This 3D methodology could be adopted for WAT transplantation applications and aid approaches to WAT-based cell therapy.

Dataset Information

Correlative Imaging of Three-Dimensional Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications.

Publications

Correlative Imaging of Three-Dimensional Cell Culture on Opaque Bioscaffolds for Tissue Engineering Applications.

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