Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo
Autor: | Emily R. Nelson, Benjamin M. Wu, Min Lee, Benjamin Levi, Aaron W. James, Michelle Peng, George W. Commons, Michael T. Longaker |
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Rok vydání: | 2011 |
Předmět: |
Male
Bone Regeneration Stromal cell Cell Survival Cellular differentiation Transplantation Heterologous Gene Expression Mice Nude Biology Cell morphology Osteocytes Mice chemistry.chemical_compound Original Research Reports Freezing Cell Adhesion Animals Humans Oil Red O Cells Cultured Cell Proliferation Cryopreservation Multipotent Stem Cells Skull Cell Differentiation Cell Biology Hematology Middle Aged Alkaline Phosphatase Molecular biology Adipose Tissue chemistry Adipogenesis Multipotent Stem Cell Female Trypan blue Fetal bovine serum Developmental Biology |
Zdroj: | Stem Cells and Development. 20:427-439 |
ISSN: | 1557-8534 1547-3287 |
Popis: | Human adipose-derived stromal cells (hASCs) represent a multipotent stromal cell type with a proven capacity to undergo osteogenic differentiation. Many hurdles exist, however, between current knowledge of hASC osteogenesis and their potential future use in skeletal tissue regeneration. The impact of frozen storage on hASC osteogenic differentiation, for example, has not been studied in detail. To examine the effects of frozen storage, hASCs were harvested from lipoaspirate and either maintained in standard culture conditions or frozen for 2 weeks under standard conditions (90% fetal bovine serum, 10% dimethyl sulfoxide). Next, in vitro parameters of cell morphology (surface electron microscopy [EM]), cell viability and growth (trypan blue; bromodeoxyuridine incorporation), osteogenic differentiation (alkaline phosphatase, alizarin red, and quantitative real-time (RT)–polymerase chain reaction), and adipogenic differentiation (Oil red O staining and quantitative RT–polymerase chain reaction) were performed. Finally, in vivo bone formation was assessed using a critical-sized cranial defect in athymic mice, utilizing a hydroxyapatite (HA)-poly(lactic-co-glycolic acid) scaffold for ASC delivery. Healing was assessed by serial microcomputed tomography scans and histology. Freshly derived ASCs differed significantly from freeze–thaw ASCs in all markers examined. Surface EM showed distinct differences in cellular morphology. Proliferation, and osteogenic and adipogenic differentiation were all significantly hampered by the freeze–thaw process in vitro (*P |
Databáze: | OpenAIRE |
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