Swales N J, Johnson T, Caldwell J
Imperial College School of Medicine at St. Mary's, London, UK.
Drug Metab Dispos. 1996 Nov;24(11):1224-30.
Cryopreservation of hepatocytes is widely used, but to validate the use of cryopreserved (CP) hepatocytes in metabolic studies, CP cells must compare favorably with fresh cell activities. We have assessed the metabolic capacity of fresh and CP rat and mouse hepatocytes in primary culture. Total cytochrome P450 (P450) contents and metabolism of testosterone were measured up to 72 hr in culture. At 0 hr, total P approximately 450 in CP rat hepatocytes was 102.5 +/- 32.8 pmol/10(6) cells, compared with fresh rat hepatocytes that had 148.2 +/- 75.7 pmol/10(6) cells. The P450 contents of mouse hepatocytes were also unaltered by cryopreservation (176.7 +/- 56.0 pmol/ 10(6) fresh cells; 196.4 +/- 59.9 pmol/10(6) CP cells). There were no significant differences in the total P450 contents of fresh and CP rat and mouse cell cultures with time over 72 hr in culture. The overall metabolism of testosterone was lower in CP suspensions than in freshly isolated hepatocytes. When CP hepatocyte suspensions were permeabilized (with digitonin) and incubated with NADPH and ATP, testosterone metabolism was significantly increased. Testosterone hydroxylase activities (16 alpha-, 6 beta-, 2 alpha-, and 7 alpha-hydroxylase) were equivalent in fresh and CP rat hepatocytes over 72 hr in culture. There was a marked and sustained loss of 6 beta-hydroxylase activity in CP mouse hepatocyte cultures, compared with fresh hepatocytes throughout 72 hr in culture (436.9 +/- 118.0 pmol/min/10(6) cells and 37.3 +/- 41.0 pmol/min/10(6) cells at 72 hr in fresh and CP mouse hepatocytes, respectively). The total metabolism of testosterone was, however, unaffected because 16 alpha-hydroxylase activity increased in CP mouse hepatocytes (475.4 +/- 80.8 pmol/min/10(6) CP cells, compared with 148.7 +/- 39.4 pmol/min/10(6) fresh cells).
肝细胞的冷冻保存被广泛应用，但为了验证冷冻保存（CP）肝细胞在代谢研究中的应用，CP细胞必须在活性方面与新鲜细胞相媲美。我们评估了原代培养的新鲜和CP大鼠及小鼠肝细胞的代谢能力。在培养长达72小时的过程中，测定了总细胞色素P450（P450）含量和睾酮的代谢情况。在0小时时，CP大鼠肝细胞中的总P450约为102.5±32.8 pmol/10⁶细胞，而新鲜大鼠肝细胞的该值为148.2±75.7 pmol/10⁶细胞。冷冻保存对小鼠肝细胞的P450含量也没有影响（新鲜细胞为176.7±56.0 pmol/10⁶细胞；CP细胞为196.4±59.9 pmol/10⁶细胞）。在培养72小时的过程中，新鲜和CP大鼠及小鼠细胞培养物的总P450含量随时间没有显著差异。CP悬浮液中睾酮的总体代谢低于新鲜分离的肝细胞。当CP肝细胞悬浮液用洋地黄皂苷通透化并与NADPH和ATP一起孵育时，睾酮代谢显著增加。在培养72小时的过程中，新鲜和CP大鼠肝细胞的睾酮羟化酶活性（16α-、6β-、2α-和7α-羟化酶）相当。与整个培养72小时的新鲜肝细胞相比，CP小鼠肝细胞培养物中6β-羟化酶活性有明显且持续的损失（新鲜和CP小鼠肝细胞在72小时时分别为436.9±118.0 pmol/min/10⁶细胞和37.3±41.0 pmol/min/10⁶细胞）。然而，睾酮的总代谢不受影响，因为CP小鼠肝细胞中16α-羟化酶活性增加（CP细胞为475.4±80.8 pmol/min/10⁶细胞，而新鲜细胞为148.7±39.4 pmol/min/10⁶细胞）。
