Accelerating cryoprotectant delivery using vacuum infiltration

The ability to cryopreserve bone marrow within the vertebral body (VB) would offer significant clinical and research benefits. However, cryopreservation of large structures, such as VBs, is challenging due to mass transport limitations that prevent the effective delivery of cryoprotectants into the tissue. To overcome this challenge, we examined the potential of vacuum infiltration, along with carbonation, to increase the penetration of cryoprotectants. In particular, we hypothesized that initial exposure to high-pressure carbon dioxide gas would introduce bubbles into the tissue and that subsequent vacuum cycling would cause expansion and contraction of the bubbles, thus enhancing the transport of cryoprotectant into the tissue. Experiments were carried out using colored dye and agarose gel as a model revealing that carbonation and vacuum cycling result in a 14% increase in dye penetration compared to the atmospheric controls. Experiments were also carried out by exposing VBs isolated from human vertebrae to 40% (v/v) DMSO solution. CT imaging showed the presence of gas bubbles within the tissue pores for carbonated VBs as well as control VBs. Vacuum cycling reduced the bubble volume by more than 50%, most likely resulting in replacement of this volume with DMSO solution. However, we were unable to detect a statistically significant increase in DMSO concentration within the VBs using CT imaging. This research suggests that there may be a modest benefit to carbonation and vacuum cycling for introduction of cryoprotectants into larger structures, like VBs.