In vitro characterization of scaffold-free three-dimensional mesenchymal stem cell aggregates

• Published in: Cell and tissue research Vol. 358; no. 2; pp. 395 - 405
• Main Authors: Rettinger, Christina L, Fourcaudot, Andrea B, Hong, Seok J, Mustoe, Thomas A, Hale, Robert G, Leung, Kai P
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.11.2014; Springer Berlin Heidelberg; Springer; Springer Nature B.V
• Subjects: Adipose Tissue - cytology; angiogenesis; Animals; Biomedical and Life Sciences; Biomedicine; Care and treatment; cell aggregates; Cell Aggregation; Cell Count; Cell culture; Cell differentiation; Cell Movement; Cell Proliferation; Cell Shape; Cell Survival; Chemical properties; chemokines; Cytokines - genetics; Cytokines - metabolism; Enzyme-linked immunosorbent assay; fibroblasts; Gene expression; Gene Expression Regulation; genes; growth factors; Human Genetics; Humans; Keratinocytes - cytology; Mesenchymal Stromal Cells - cytology; Mesenchymal Stromal Cells - ultrastructure; Molecular Medicine; Monomolecular films; Neovascularization, Physiologic; new methods; plastics; protein synthesis; Proteomics; quantitative polymerase chain reaction; Rabbits; Regular Article; RNA, Messenger - genetics; RNA, Messenger - metabolism; Stem cells; tissue culture; Tissue engineering; tissue repair; Tissue Scaffolds - chemistry; Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; viability; wells; Wound Healing; Wounds and injuries; Cell aggregates/spheroids; Wound healing; Three-dimensional culture; Rabbit; Adipose-derived stem cells; Scarring; Mesenchymal stem cells
• ISSN: 0302-766X; 1432-0878
• DOI: 10.1007/s00441-014-1939-0

Mesenchymal stem cells (MSCs) are capable of self-renewal and differentiation along multiple cell lineages and have potential applications in a wide range of therapies. These cells are commonly cultured as monolayers on tissue culture plastic but possibly lose their cell-specific properties with time in vitro. There is growing interest in culturing adherent cells via three-dimensional (3D) techniques in order to recapitulate 3D in vivo conditions. We describe a novel method for generating and culturing rabbit MSCs as scaffold-free 3D cell aggregates by using micropatterned wells via a forced aggregation technique. The viability and proliferative capability of MSC aggregates were assessed via Live/Dead staining and 5-ethynyl-2’-deoxyuridine (EdU) incorporation. Enzyme-linked immunosorbent assay and antibody-based multiplex protein assays were used to quantify released growth factors and chemokines. The gene expression profile of MSCs as 3D aggregates relative to MSCs grown as monolayers was evaluated via quantitative real-time polymerase chain reaction. The rabbit MSCs were able to form compact cell aggregates and remained viable in 3D culture for up to 7 days. We also demonstrated enhanced gene and protein expression related to angiogenesis and wound healing in MSCs cultured under 3D conditions. In vitro tube formation and scratch assay revealed superior neovessel formation and greater cell recovery and migration in response to 3D conditioned media after wounding. Our data further suggest that adipose-derived stem cell aggregates have greater potential than dermal fibroblasts or bone-marrow-derived MSCs in accelerating wound healing and reducing scarring.