title: Vitreous cryopreservation of nanofibrous tissue-engineered constructs generated using mesenchymal stromal cells.
abstract: Development of an effective preservation strategy to fulfill off-the-shelf availability of tissue-engineered constructs (TECs) is demanded for realizing their clinical potential. In this study, the feasibility of vitrification, ice-free cryopreservation, for precultured ready-to-use TECs was evaluated. To prepare the TECs, bone marrow-derived porcine mesenchymal stromal cells (MSCs) were seeded in polycaprolactone-gelatin nanofibrous scaffolds and cultured for 3 weeks before vitrification treatment. The vitrification strategy developed, which involved exposure of the TECs to low concentrations of cryoprotectants followed by a vitrification solution and sterile packaging in a pouch with its subsequent immersion directly into liquid nitrogen, was accomplished within 11min. Stepwise removal of cryoprotectants, after warming in a 38 degrees C water bath, enabled rapid restoration of the TECs. Vitrification did not impair microstructure of the scaffold or cell viability. No significant differences were found between the vitrified and control TECs in cellular metabolic activity and proliferation on matched days and in the trends during 5 weeks of continuous culture postvitrification. Osteogenic differentiation ability in vitrified and control groups was similar. In conclusion, we have developed a time- and cost-efficient cryopreservation method that maintains integrity of the TECs while preserving MSCs viability and metabolic activity, and their ability to differentiate.
authors.affiliation: Low Temperature Preservation Unit, National University Medical Institutes, National University of Singapore, Singapore, Singapore.
authors.forename: Feng
authors.initials: F
authors.lastname: Wen
authors.forename: Raquel
authors.initials: R
authors.lastname: Magalhães
authors.forename: Sok Siam
authors.initials: SS
authors.lastname: Gouk
authors.forename: Gajadhar
authors.initials: G
authors.lastname: Bhakta
authors.forename: Bhakta
authors.initials: B
authors.lastname: Gajadhar
authors.forename: Kong Heng
authors.initials: KH
authors.lastname: Lee
authors.forename: Dietmar W
authors.initials: DW
authors.lastname: Hutmacher
authors.forename: Lilia L
authors.initials: LL
authors.lastname: Kuleshova
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000880:Anthraquinones; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation; D048430:Cell Shape; D002470:Cell Survival; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D015925:Cryopreservation / Q000379:methods*; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology; D049329:Nanostructures / Q000737:chemistry* / Q000648:ultrastructure; D010012:Osteogenesis; D017154:Stromal Cells / Q000166:cytology* / Q000201:enzymology; D013499:Surface Properties; D034421:Sus scrofa; D023822:Tissue Engineering; D054457:Tissue Scaffolds
chemical_list: D000880:Anthraquinones; D024042:Collagen Type I; C010078:alizarin; D000469:Alkaline Phosphatase; D002118:Calcium