Extended-term culture of bone cells in a compartmentalized bioreactor.
A specialized bioreactor is used to grow mineralizing, collagenous tissue up to 150 microm thick from an inoculum of isolated murine (mouse calvaria MC3T3-E1, American Type Culture Collection (ATCC) CRL-2593) or human (hFOB 1.19 ATCC CRL-11372) fetal osteoblasts over uninterrupted culture periods longer than 120 days (4 months). Proliferation and phenotypic progression of an osteogenic-cell monolayer into a tissue consisting of 6 or more cell layers of mature osteoblasts in the bioreactor was compared with cell performance in conventional tissue-culture polystyrene (TCPS) controls. Cells in the bioreactor basically matched results obtained in TCPS over a 15-day culture interval, but loss of insoluble extracellular matrix and an approximate doubling of apoptosis rates in TCPS after 30 days indicated that progressive instability of cultures maintained in TCPS with periodic refeeding but without subculture. In contrast, stable cultures were maintained in the bioreactor for more than 120 days, suggesting that extended-term tissue maintenance is feasible with little or no special technique. Transmission electron microscopy ultramorphology of tissue derived from hFOB 1.19 recovered from the bioreactor after only 15 days of culture showed evidence of osteocytic-like processes and gap junctions between cells like those observed in vivo, in addition to elaboration of the usual osteoblastic markers such as alkaline phosphatase activity and mineralization (alizarin red). Thus, the bioreactor design based on the principle of simultaneous growth and dialysis was shown to create an extraordinarily stable peri-cellular environment that better simulates the in vivo condition than conventional tissue culture. The bioreactor shows promise as a tool for the in vitro study of osteogenesis and osteopathology.
Dhurjati
Ravi
R
Department of Materials Science and Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Liu
Xiaomei
X
Gay
Carol V
CV
Mastro
Andrea M
AM
Vogler
Erwin A
EA
D016475:3T3 Cells; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D019149:Bioreactors; D001842:Bone and Bones / Q000166:cytology* / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D002113:Calcification, Physiologic / Q000502:physiology; D018929:Cell Culture Techniques; D003956:Dialysis; D004577:Electron Probe Microanalysis; D004867:Equipment Design; D005109:Extracellular Matrix / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D005240:Feasibility Studies; D005333:Fetus; D017629:Gap Junctions / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure*; D011137:Polystyrenes / Q000737:chemistry; D013997:Time Factors
D015415:Biomarkers; D011137:Polystyrenes; D000469:Alkaline Phosphatase