Macroscopic Diffusion of Cryoprotectant in Biological Materials and Its Microstructure

移植需要对生物组织和人工工程组织进行冷冻保存。冷冻保护剂，即提高组织活力的化合物，需要在冷冻前引入组织中，并在解冻后从组织中除去。由于高浓度的冷冻保护剂和长期暴露于甚至低浓度的冷冻保护剂导致的渗透压的大的阶跃变化可对组织造成损伤。本研究应用磁共振成像技术（MRI）对生物材料在溶液和假生物组织（琼脂）中扩散过程中的保护剂浓度进行了测量。首先，质子-核磁共振（^1H-NMR）谱、化学组成和分子结构。 ...更多信息 在NMR实验中测量了校准位移频率和弛豫时间。测量两种培养基的MRI强度和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弛豫时间对磁共振强度的衰减，得到了伪生物组织的校正曲线，并利用化学位移成像技术对常用冷冻保护剂二甲基亚砜（DMSO）和甘油的瞬态一维渗透过程进行了成像。处理图像以基于校准曲线定量冷冻保护剂浓度。每种冷冻保护剂的表面扩散系数是通过基于冷冻保护剂浓度的时间序列一维分布的反问题分析来确定的。在溶液中的冷冻保护剂浓度高达6.0M的范围内，扩散系数随着冷冻保护剂浓度的增加而适度降低。发现DMSO的扩散系数是甘油的1.5至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，并随琼脂浓度的增加而减小。琼脂中DMSO的值比甘油的值大50%~ 80%，该方法同样适用于新鲜鸡肝组织。测量了1H-NMR谱、化学位移频率、弛豫时间和校准曲线。发现组织的校准曲线是根据溶液和假生物组织的校准曲线估计的。反问题分析表明，DMSO在肝脏内的表面扩散系数约为溶液中的值的5%。这是因为肝脏在显微镜下由细胞和结缔组织组成，抵抗质量转移。提出了由水的弛豫时间估算冷冻保护剂弛豫时间引起的MRI强度衰减的方法。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