Macroscopic Diffusion of Cryoprotectant in Biological Materials and Its Microstructure

移植需要生物组织和人工工程组织的低温保存和冷冻保存。冷冻保护剂是一种提高组织活力的化合物，在冷冻前需要引入组织中，解冻后需要从组织中移除。由于高浓度的冷冻保护剂和长期暴露在即使是低浓度的冷冻保护剂中，渗透压的巨大阶跃变化可能会对组织造成损害。因此，冷冻保护剂方案的优化对冷冻保存的成功至关重要，而低温保存得益于组织中冷冻保护剂浓度的测量。本研究将磁共振成像(MRI)这一适用于生物材料的非侵入性方法应用于溶液和假生物组织(琼脂)中扩散过程中低温保护剂浓度的测量。初步确定了质子-核磁共振(~1H-…)谱在核磁共振实验中测得了更多的频移频率和弛豫时间。测量两种介质的MRI强度与冷冻保护剂浓度之间的校准曲线。通过考虑溶液体积分数和驰豫时间引起的MRI强度衰减，得到假生物组织的校准曲线。利用化学位移成像技术，对常见的低温保护剂二甲基亚砜(DMSO)和甘油的一维渗透过程进行了成像。根据校准曲线对图像进行处理，以定量计算冷冻保护剂的浓度。根据低温保护剂浓度的时间序列一维分布，通过反问题分析确定了每种低温保护剂的表面扩散系数。在溶液中低温保护剂浓度为6.0M的范围内，随着低温保护剂浓度的增加，扩散系数略有下降。DMSO的扩散系数是甘油的1.5~2.0倍。冷冻保护剂在假生物组织中的扩散系数小于在溶液中的扩散系数，且随琼脂浓度的增加而减小。琼脂中DMSO的含量比甘油中的含量高50%到80%。这种MRI方法也同样适用于新鲜的鸡肝作为组织。测定了~1H-核磁共振谱、化学位移频率、弛豫时间和校正曲线。组织的校准曲线由溶液的校准曲线和假生物组织的校准曲线估算出来。反问题分析表明，DMSO在肝脏内的表面扩散系数约为溶液中扩散系数的5%。这是因为肝脏显微镜下由细胞和结缔组织组成，抵抗传质。文中还提出了由低温保护剂的松弛时间引起的MRI强度衰减与由水的松弛引起的MRI强度衰减的估算方法。较少

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