Cryopreserving the intact intervertebral disc without compromising viability

Abstract

Background

Tissue cryopreservation requires saturation of the structure with cryoprotectants (CPAs) that are also toxic to cells within a short timeframe unless frozen. The race between CPA delivery and cell death is the main barrier to realizing transplantation banks that can indefinitely preserve tissues and organs. Unrealistic cost and urgency leaves less life-threatening ailments unable to capitalize on traditional organ transplantation systems that immediately match and transport unfrozen organs. For instance, human intervertebral discs (IVD) could be transplanted to treat back pain or used as ex vivo models for studying regenerative therapies, but both face logistical hurdles in organ acquisition and transport. Here we aimed to overcome those challenges by cryopreserving intact IVDs using compressive loading and swelling to accelerate CPA delivery.

Methods

CPAs were tested on bovine nucleus pulposus cells to determine the least cytotoxic solution. Capitalizing on our CPAs Computed Tomography (CT) contrast enhancement, we imaged and quantified saturation time in intact bovine IVDs under different conditions in a bioreactor. Finally, the entire protocol was tested, including 1 week of frozen storage, to confirm tissue viability in multiple IVD regions after thawing.

Results

Results showed cryopreserving medium containing dimethyl sulfoxide and ethylene glycol gave over 7.5 h before cytotoxicity. While non-loaded IVDs required over 3 days to fully saturate, a dynamic loading protocol followed by CPA addition and free-swelling decreased saturation time to <5 h. After cryopreserving IVDs for 1 week with the optimized CPA and permeation method, all IVD regions had 85% cell viability, not significantly different from fresh unfrozen controls.

Conclusions

This study created a novel solution to a roadblock in IVD research and development. Using post-compression swelling CPA can be delivered to an intact IVD over 20× more quickly than previous methods, enabling cryopreservation of the IVD with no detectable loss in cell viability.