Macroscopic Diffusion of Cryoprotectant in Biological Materials and Its Microstructure

点击翻译按钮获取中文摘要

Cryopreservation, preservation by freezing, of biological tissues and artificial engineered tissues are required for transplantation. Cryoprotectants, chemical compounds to improve viability of the tissues, need to be introduced into the tissues before freezing and to be removed from the tissues after thawing. Large step change in osmolarity due to high concentration of cryoprotectants and long-term exposure to even low concentration of cryoprotectants can cause damage to the tissues. Therefore, optimization of cryoprotectant protocols is important for success of cryopreservation, and cryopreservation benefits from the measurement of cryoprotectant concentration in the tissues.In this study, magnetic resonance imaging (MRI), a noninvasive method applicable to biological materials, was applied to the measurement of cryoprotectant concentration during the diffusion in solutions and pseudobiological tissues (agar). Preliminarily, proton-nuclear magnetic resonance (^1H-NMR) spectrum, chemi … More cal shift frequency, and relaxation times were measured in NMR experiments. Calibration curves between MRI intensity and cryoprotectant concentration for both the media were measured. The calibration curves for the pseudobiological tissues was found to be estimated from that for the solutions by considering the attenuation of MRI intensity due to solution volumetric fraction and relaxation time.Transient one-dimensional permeation process of dimethyl sulfoxide (DMSO) and glycerol, common cryoprotectants, penetrating cells, were imaged by means of a chemical shift imaging technique. The image was processed to quantitate the cryoprotectant concentration based on the calibration curve. Superficial diffusivity of each cryoprotectant was determined by means of the inverse problem analysis based on the time-series one-dimensional distribution of cryoprotectant concentration. In the range of cryoprotectant concentration up to 6.0M in the solution, the diffusivity decreased moderately with an increase in cryoprotectant concentration. The diffusivity of DMSO was found to be 1.5 to 2.0 times that of glycerol. The diffusivity of cryoprotectant in pseudobiological tissues was smaller than that in the solution and decreased with an increase in agar concentration. The values for DMSO in the agar was found to be by 50% to 80% larger than those for glycerol.This MRI method was also applied similarly to fresh liver of chicken as tissues. ^1H-NMR spectrum, chemical shift frequency, relaxation times, and calibration curve were measured. The calibration curve for the tissues was found to be estimated from that for the solutions as well as that for the pseudobiological tissues. The inverse problem analysis indicated that superficial diffusivity of DMSO within the liver was about 5% of the value in the solution. This is because the liver microscopically consists of cells and connective tissues, resistance of mass transfer. Also, the way to estimate the attenuation of MRI intensity due to the relaxation time of cryoprotectant from that due to the relaxation of water was proposed. Less