Biological freezing of human articular chondrocytes

Aim: To preserve viable, metabolically active chondrocytes cultured in algi nate beads at -196 degreesC for further use in in vitro and in vivo studies . Methods: Human articular chondrocytes were isolated from femoral condyles w ithin 24 h post mortem. To optimize the biological freezing procedure, the chondrocytes were control-rate frozen in different concentrations of dimeth yl sulfoxide (DMSO) in Dulbecco's MEM supplemented with 10% FCS before bein g thawed and the cell viability was determined by Trypan Blue exclusion tes t. To investigate the effect of control-rate freezing on chondrocyte metabo lism, control-rate frozen chondrocytes in 5% DMSO were thawed and cultured in gelled agarose for 2 weeks. Non-frozen chondrocytes cultured in agarose served as controls. Furthermore, human articular chondrocytes were cultured in 2% alginate beads for 2 weeks after which the beads were incubated with 5% DMSO for 0 h, 2.5 h, 5 h and 10 h and frozen at -196 degreesC. Non-froz en alginate beads containing chondrocytes and incubated with 5% DMSO served as a control. After 2 weeks in culture, chondrocytes in agarose or in algi nate were sulfated with 10 mu Ci (SO4)-S-35/ml for 48 h. The total producti on of aggrecans, and the aggrecan subtypes, were subsequently determined. Results: Five percent DMSO in the culture medium was the optimal condition to control-rate freeze and recover viable and functional isolated chondrocy tes. Total aggrecan synthesis of control-rate frozen chondrocytes cultured in gelled agarose was not significantly reduced when compared with control cells. The proportion of aggrecan in the aggregate form of control-rate fro zen chondrocytes kept in agarose remained unaltered. Chondrocytes, control- rate frozen in the alginate matrix, showed a 0-30% decrease in total aggrec an synthesis rates in culture when compared with the non-frozen chondrocyte s. The optimal pre-incubation time of the alginate beads with 5% DMSO was 5 h, without any change in aggrecan synthesis rates when compared with the c ontrol situation. Shorter pre-incubation times resulted in an insufficient diffusion of DMSO into the beads and in cell death. There was no difference in the synthesis of the different aggrecan subtypes between frozen and non -frozen chondrocytes in alginate. Conclusion: Human articular chondrocytes can be stored at -196 degreesC for 24 h without important decreases in their aggrecan synthesis rates when co ntrol-rate frozen as a cell suspension in 5% DMSO. Proportions of the aggre can subtypes (monomers, aggregates) synthesized by chondrocytes cultured in agarose remained unchanged. The control-rate freezing procedure in the alg inate beads pre-incubated with 5% DMSO for 5 h produced no decrease in tota l aggrecan synthesis rates and no change in the synthesized aggrecan subtyp es. Further experiments have to confirm the suitability of this freezing me thod for long-term storage of chondrocytes allowing us to set up a 'chondro cyte' bank far further use in in vitro and in vivo manipulations. (C) 2001 OsteoArthritis Research Society International.