OBJECTIVE: To generate in vitro hyaline cartilage from cryogenically preserved human septal chondrocytes in a simulated microgravity environment on a 3-dimensional biodegradable scaffolding material. METHODS: In this experiment, cryogenically frozen chondrocytes were thawed and cultured in a monolayer in serum-based chondrocyte media. They were seeded onto 3-dimensional biopolymer scaffolds in a spinner flask. The seeded constructs were then transferred to a bioreactor (an environment of solid-body rotation) for 6 weeks. Chondrocyte growth and extracellular matrix production in the constructs were confirmed by cell count, cell viability, and histologic analysis and by electron microscopy. RESULTS: Histologic sections stained with hematoxylin-eosin and Alcian blue (for acidic proteoglycans) confirmed the presence of hyaline cartilage in the cartilage constructs. Ultrastructural examination using transmission electron microscopy demonstrated matrix formation and chondrocyte viability. CONCLUSIONS: This study proves that chondrocytes that are cryogenically stored for extended periods can be used to grow cartilage in vitro. Cryogenically preserved chondrocytes retain their ability to grow in tissue culture, redifferentiate, and produce extracellular matrix.
OBJECTIVE: To generate in vitro hyaline cartilage from cryogenically preserved human septal chondrocytes in a simulated microgravity environment on a 3-dimensional biodegradable scaffolding material. METHODS: In this experiment, cryogenically frozen chondrocytes were thawed and cultured in a monolayer in serum-based chondrocyte media. They were seeded onto 3-dimensional biopolymer scaffolds in a spinner flask. The seeded constructs were then transferred to a bioreactor (an environment of solid-body rotation) for 6 weeks. Chondrocyte growth and extracellular matrix production in the constructs were confirmed by cell count, cell viability, and histologic analysis and by electron microscopy. RESULTS: Histologic sections stained with hematoxylin-eosin and Alcian blue (for acidic proteoglycans) confirmed the presence of hyaline cartilage in the cartilage constructs. Ultrastructural examination using transmission electron microscopy demonstrated matrix formation and chondrocyte viability. CONCLUSIONS: This study proves that chondrocytes that are cryogenically stored for extended periods can be used to grow cartilage in vitro. Cryogenically preserved chondrocytes retain their ability to grow in tissue culture, redifferentiate, and produce extracellular matrix.
Authors: Marsha S Reuther; Van W Wong; Kristen K Briggs; Angela A Chang; Quynhhoa T Nguyen; Barbara L Schumacher; Koichi Masuda; Robert L Sah; Deborah Watson Journal: Otolaryngol Head Neck Surg Date: 2012-05-17 Impact factor: 3.497