Intervertebral disc cryopreservation

Abstract

Cryopreserved allogeneic intervertebral disc (IVD) transplantation has been performed clinically for treating disc degeneration. However, the transplanted allograft has undergone progressive degeneration 1. Conventional disc cryopreservation method has been shown to greatly decrease the disc cell viability and hence may be a cause of the degeneration. In this study, we aim to search for a freeze-thaw protocol that is able to retain a maximal cell viability and metabolic activity in the cryopreserved IVD. Porcine lumbar IVDs with endplates were harvested and cryopreserved in various freezing medium 1) 45% Cryo-protective agents (CPAs) (DMSO (dimethylsulfoxide) + propylene glycol), 2) 20% CPAs (DMSO + glycerol), 3) 10% DMSO with synthetic ice-blocker , 4) 10% DMSO, or 5) without CPA. IVDs were incubated at 4°C for 1 hour, freezed at -80°C overnight, and then stored in liquid nitrogen. The frozen IVDs were thawed quickly at 37°C for analysis. Cell prol iferation and proteoglycan production rate were evaluated using Alarma blue and 35S-sulphate incorporation respectively. The distribution of live and dead cells was determined microscopically using a LIVD/DEAD staining. IVD cryopreserved in freezing medium composed of 45% CPAs showed higher disc cell viability compared with that in 10% DMSO. Cell viability was 50% of the fresh IVD tissue when cryopreserved in 45% CPAs but merely 20% when cryopreserved with 10% DMSO. Combination of DMSO and propylene glycol as CPAs for IVD cryopreservation has an advantage over conventional cryopreservation protocol in maintaining disc cell viability. Reference: 1. Ruan D, et al. Lancet 2007; 369:993-9.