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Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs.

ABSTRACT: Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs such as blood vessels, liver, diaphragm, and a conduit for nerves by using this method. These constructs have the potential to be applied in patients. However, the process of fabricating tissue constructs (harvesting cells, expanding cells, making spheroids using cultured cells, printing constructs, and maturing constructs) is time-consuming. Therefore, cryopreservation methods for spheroids or constructs should be developed to increase the efficiency of this method for clinical use. Here, we developed a method for cryopreserving spheroids, which were then used to fabricate constructs. Fibroblast cell-based spheroids were cryopreserved in phosphate-buffered saline or cryopreservation solution at -80°C for 1 week. After thawing, spheroids in cryopreservation solution began to fuse on day 1. Cryopreserved spheroids were printed onto a needle array to fabricate a scaffold-free tubular construct using a bio-3D printer. After 7 days, the printed spheroids fused and formed scaffold-free constructs. We confirmed the viability of cells in the cryopreserved spheroids and fabricated tubular constructs. Our results indicate that spheroids can be cryopreserved and used to prepare scaffold-free constructs for clinical use.

SUBMITTER: Arai K

PROVIDER: S-EPMC7117714 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs.

Arai Kenichi K Murata Daiki D Takao Shoko S Verissiomo Ana Raquel AR Nakayama Koichi K

PloS one 20200402 4

Cryopreservation is a method used for preserving living cells by cooling them to very low temperatures. Although cryopreservation methods for oocytes and embryos have been developed for use in reproductive medicine, there are no established methods yet for preserving cell aggregates (spheroids) in regenerative medicine. We have developed a bio-three-dimensional (3D) printer that can fabricate scaffold-free 3D constructs by loading spheroids onto a needle array. We fabricated several constructs s ...[more]

PMID: 32240195

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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.

Dataset Information

Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs.

Publications

Cryopreservation method for spheroids and fabrication of scaffold-free tubular constructs.

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