Time-Resolved Multi-Spectral X-ray Computed Tomography of Cryoprotectant Diffusion Into Biomimetic Material

TL;DR

This paper presents a novel multi-spectral X-ray computed tomography method that noninvasively measures the distribution of cryoprotectants in tissue mimics, enabling better cryopreservation protocols.

Contribution

Introduction of MSCT with a photon-energy bin selection algorithm for accurate, in situ cryoprotectant quantification without contrast agents.

Findings

Resolved cryoprotectant volume fractions within 5% accuracy

Observed heterogeneous diffusion not detectable by conventional methods

Validated technique with DMSO, glycerol, and water solutions

Abstract

Cryopreservation via vitrification requires loading cryoprotective cocktails. Insufficient loading may lead to freezing, precluding successful recovery; overloading is toxic. Yet, existing in situ measurements of cryoprotectant permeation remain largely unvalidated and do not resolve individual cryoprotectant concentrations. We introduce multi-spectral X-ray computed tomography (MSCT) to noninvasively quantify the spatiotemporal distribution of cryoprotectants diffusing into a tissue mimicking phantom. A developed photon-energy bin selection algorithm achieves sensitivity to low contrast cryoprotectants without contrast agents or fluorescence edges. The technique is validated with a dimethyl sulfoxide, glycerol, and water solution, resolving cryoprotectant volume fractions to within 5% accuracy. We observe heterogeneous diffusion of the cryoprotectants into the tissue mimicking hydrogel, a phenomenon not observable with conventional techniques. MSCT improves upon existing X ray CT methods because it is not underdetermined for multicomponent solutions and does not implicitly assume homogeneous diffusion. These advancements enable the systematic development of cryoprotectant loading protocols and provide diagnostics to assess vitrifiability before cryopreservation.