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Engineering multi-layered tissue constructs using acoustic levitation.

ABSTRACT: Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and fibroblast cell layers. The collective behaviour of the epithelium is dependent on the formation of cell-cell junctions during levitation and contrasts with the behaviour of mono-dispersed epithelial cells where cell-matrix interactions dominate and hinder formation of discrete cell layers. The multilayered tissue model is shown to form a polarised epithelial barrier and respond to apical challenge. The method is useful for engineering a wide range of layered tissue types and mechanistic studies on collective cell migration.

SUBMITTER: Tait A

PROVIDER: S-EPMC6611909 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Engineering multi-layered tissue constructs using acoustic levitation.

Tait Angela A Glynne-Jones Peter P Hill Alison R AR Smart David E DE Blume Cornelia C Hammarstrom Bjorn B Fisher Adam L AL Grossel Martin C MC Swindle Emily J EJ Hill Martyn M Davies Donna E DE

Scientific reports 20190705 1

Engineering tissue structures that mimic those found in vivo remains a challenge for modern biology. We demonstrate a new technique for engineering composite structures of cells comprising layers of heterogeneous cell types. An acoustofluidic bioreactor is used to assemble epithelial cells into a sheet-like structure. On transferring these cell sheets to a confluent layer of fibroblasts, the epithelial cells cover the fibroblast surface by collective migration maintaining distinct epithelial and ...[more]

PMID: 31278312

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Project description:Vascularization remains a substantial limitation to the viability of engineered tissue. By comparing in vivo vascularization dynamics of a self-assembled prevascular endothelial-fibroblast model to avascular grafts, we explore the vascularization rate limitations in implants at early time intervals, during which tissue hypoxia begins to affect cell viability. Scaffold-free prevascular endothelial-fibroblast constructs (SPECs) may serve as a modular and reshapable vascular bed in replacement tissues. SPECs, fibroblast-only spheroids (FOS), and silicone implants were implanted in 54 Sprague Dawley rats and harvested at 6, 12, and 24 h (n = 5 per time point and implant type). We hypothesized that the primary endothelial networks of the SPECs allow earlier anastomosis and increased vessel formation in the interior of the implant compared to FOS and silicone implants within a 24 h window. All constructs were encapsulated by an endothelial lining at 6 h postimplantation and SPEC internal cords inosculated with the host vascular network by this time point. SPECs had a significantly higher microvascular area fraction and branch/junction density of penetrating cords at 6-12 h compared with other constructs. In addition, SPECs demonstrated perivascular cell recruitment, lumen formation, and network remodeling consistent with vessel maturation at 12-24 h; however, these implants were poorly perfused within our observation window, suggesting poor lumen patency. FOS vascular characteristics (microvessel area and penetrating cord density) increased within the 12-24 h period to represent those of the SPEC implants, suggesting a 12 h latency in host response to avascular grafts compared to prevascular grafts. Knowledge of this temporal advantage in in vitro prevascular network self-assembly as well as an understanding of the current limitations of SPEC engraftment builds on our theoretical temporal model of tissue graft vascularization and suggests a crucial time window, during which technological improvements and vascular therapy can improve engineered tissue survival.

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

Engineering multi-layered tissue constructs using acoustic levitation.

Publications

Engineering multi-layered tissue constructs using acoustic levitation.

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