Photon counting multi-spectral x-ray computed tomography of multi-component cryoprotectant diffusion into tissue...

The ability to cyropreserve biological samples is essential for facilitating biomedical research, but current methods do not scale beyond cells. Cryopreservation by vitrification holds promise for extending this practice to tissues and potentially whole organs, but a key step -loading cryoprotectant chemicals -is not well understood in-situ, leading to inconsistent and poor recovery. Insufficient cryoprotectant loading may lead to freezing, precluding successful recovery while over-loading results in chemical toxicity. Despite the importance of this step, existing in-situ measurements of cryoprotectant permeation remain largely unvalidated and do not resolve individual cryoprotectant concentrations. We demonstrate photon counting multi-spectral X-ray computed tomography for non-invasively quantifying the spatiotemporal distribution of cryoprotectants diffusing into a hydrogel tissue-surrogate phantom and develop a photon-energy bin selection algorithm that achieves sensitivity to low-contrast cryoprotectants without contrast agents or fluorescence edges. Validating the technique with a dimethyl sulfoxide-glycerol-water solution, we resolve cryoprotectant volume fractions to within 5% accuracy and observe heterogeneous diffusion of the cryoprotectants, a phenomenon not observable with energy-integrating X-ray CT techniques currently standard in cryoprotectant perfusion experiments. These advancements provide the quantitative information needed for rational design of cryoprotectant loading protocols and validation of multi-component permeation models. Additionally, multi-spectral X-ray CT (MSCT) can be integrated into cryopreservation workflows to assess cryoprotectant loading success before vitrification with the goal of improving recovery outcomes.