Glöckner R, Steinmetzer P, Drobner C, Müller D
Institute of Pharmacology and Toxicology, Friedrich Schiller University Jena, Germany.
Exp Toxicol Pathol. 1998 Sep;50(4-6):440-9. doi: 10.1016/S0940-2993(98)80031-6.
Principle steps necessary for cryopreservation of precision-cut liver slices as currently applied by different groups are summarized including own results concerning mode of freezing. Now we use rapid freezing by immersion in liquid nitrogen after exposure to 10% DMSO as the cryoprotectant for rat liver slices. The results indicate well-maintained cytochrome P450 (CYP)-dependent deethylation rates in slice homogenate after short-term incubation. ECOD rate in intact thawed slices was even higher than in fresh ones after 2 h incubation. In contrast to fresh slices all parameters except protein content decreased to marginal levels during long-term incubation of thawed slices for 24 h. The first preliminary experiments on albumin secretion by thawed rat liver slices, measured between the 2nd and the 4th hour of incubation, showed partial maintenance of this liver specific differentiated function. Trials to induce CYP1A1 in thawed rat liver slices in vitro by beta-naphthoflavone (BNF) resulted in increased expression of CYP1A1-mRNA within 6 h as shown by RT-PCR and quantified by competitive RT-PCR. The decline of deethylation rates, determined in slice homogenates, and of viability within 24 h incubation was not prevented by exposure to BNF or DMSO. The results derived from one sample of cryopreserved human liver slices indicate a quite acceptable maintenance of function up to 6 h, if the same protocol as developed for rat liver slices was used.
总结了目前不同研究小组在精密肝切片冷冻保存方面所必需的主要步骤，包括我们自己关于冷冻方式的研究结果。目前我们将大鼠肝切片暴露于10%二甲基亚砜（DMSO）作为冷冻保护剂后，通过浸入液氮进行快速冷冻。结果表明，短期孵育后切片匀浆中细胞色素P450（CYP）依赖性脱乙基化率保持良好。解冻后的完整切片在孵育2小时后的乙氧基香豆素-O-脱乙基酶（ECOD）活性甚至高于新鲜切片。与新鲜切片相比，解冻后的切片在24小时的长期孵育过程中，除蛋白质含量外，所有参数均降至极低水平。在孵育的第2至4小时之间对解冻后的大鼠肝切片白蛋白分泌进行的首次初步实验表明，这种肝脏特异性分化功能部分得以维持。用β-萘黄酮（BNF）在体外诱导解冻后的大鼠肝切片中CYP1A1，结果显示，通过逆转录聚合酶链反应（RT-PCR）检测，并通过竞争性RT-PCR定量，CYP1A1-mRNA在6小时内表达增加。在切片匀浆中测定的脱乙基化率下降以及在24小时孵育内的活力下降并未因暴露于BNF或DMSO而得到阻止。从一份冷冻保存的人肝切片样本得出的结果表明，如果使用与大鼠肝切片相同的方案，其功能在长达6小时内的维持情况相当可观。
