title: Expansion of mouse hematopoietic progenitor cells in three-dimensional cocultures on frozen-thawed stromal cell layers formed within porous scaffolds.
abstract: To establish a highly efficient method of ex vivo expansion of hematopoietic cells (HCs), three-dimensional (3D) cocultures of HCs and stromal cell lines were performed using porous polymer scaffolds. Hematopoietic cells derived from mouse fetal livers were expanded by two successive cultures without the use of exogenous cytokines, namely, 3D cultures of stromal cells (DAS 104-8 cell line) to form stromal layers within the scaffolds, and, subsequently, by cocultures of the HCs on the stromal cell layers for 2 weeks. To expand the HCs more conveniently, in some experiments the stromal layers formed within the scaffolds were frozen (3D freezing) before the cocultures, then stored and applied to the cocultures after thawing. When the HCs were cocultured on the stromal layers of the DAS 104-8 cells, primitive HCs (c-kit(+) and CD34(+) cells) were expanded several fold during the cocultures. In contrast, the expansion of these primitive HCs was remarkably enhanced in the cocultures using the 3D frozen-thawed DAS 104-8 stromal layers (c-kit(+) cells > fifteenfold and CD34(+) cells > thirtyfold), and these expansions were significantly higher than those without the 3D freezing. The expansions enhanced by cocultures on the 3D frozen-thawed stromal layers were also observed in the cocultures with another stromal cell line (DAS 104-4). Because 3D frozen-thawed stromal cell lines are easy to handle, 3D coculture of HCs on frozen-thawed stromal cell lines may be an effective and convenient method for expanding primitive HCs.
authors.affiliation: Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan. Electronic address: hmiyoshi@md.tsukuba.ac.jp.
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Miyoshi
authors.affiliation: Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
authors.forename: Misa
authors.initials: M
authors.lastname: Morita
authors.affiliation: Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Ohshima
authors.affiliation: Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Sato
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism / Q000648:ultrastructure*; D049109:Cell Proliferation; D018920:Coculture Techniques / Q000379:methods*; D004622:Embryo, Mammalian; D005333:Fetus; D005615:Freezing; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000648:ultrastructure*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D011145:Polyvinyls / Q000737:chemistry; D016062:Porosity; D061251:Primary Cell Culture; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism / Q000648:ultrastructure*; D054457:Tissue Scaffolds
chemical_list: D018952:Antigens, CD34; D015415:Biomarkers; D011145:Polyvinyls; D019009:Proto-Oncogene Proteins c-kit