VIABILITY AND FUNCTION IN PRIMARY CULTURE OF ADULT HEPATOCYTES FROM VARIOUS ANIMAL SPECIES AND HUMAN-BEINGS AFTER CRYOPRESERVATION

被引:56
作者
CHESNE, C
GUYOMARD, C
FAUTREL, A
POULLAIN, MG
FREMOND, B
DEJONG, H
GUILLOUZO, A
机构
[1] HOP PONTCHAILLOU, INSERM, U49, UNITE RECH HEPATOL, F-35033 RENNES, FRANCE
[2] INST RECH INT SERVIER, F-92415 COURBEVOIE, FRANCE
[3] BIOPREDIC, RENNES ATALANTE VILLEJEAN, F-35000 RENNES, FRANCE
关键词
D O I
10.1002/hep.1840180227
中图分类号
R57 [消化系及腹部疾病];
学科分类号
摘要
Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4-degrees-C cell suspensions were transferred to - 20-degrees-C and then to - 70-degrees-C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells. These results show that a simple and reproducible freeze/thaw protocol can be used to cryopreserve hepatocytes from various species including human beings and that after thawing a large fraction of the cells is still viable and capable of expressing various functions in vitro.
引用
收藏
页码:406 / 414
页数:9
相关论文
共 34 条
  • [1] INSIGHTS INTO THE CRYOPROTECTIVE MECHANISM OF DIMETHYL-SULFOXIDE FOR PHOSPHOLIPID-BILAYERS
    ANCHORDOGUY, TJ
    CECCHINI, CA
    CROWE, JH
    CROWE, LM
    [J]. CRYOBIOLOGY, 1991, 28 (05) : 467 - 473
  • [2] THE BASIS FOR TOXICITY OF CERTAIN CRYOPROTECTANTS - A HYPOTHESIS
    ARAKAWA, T
    CARPENTER, JF
    KITA, YA
    CROWE, JH
    [J]. CRYOBIOLOGY, 1990, 27 (04) : 401 - 415
  • [3] BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
  • [4] CRYOPRESERVATION OF ISOLATED RAT HEPATOCYTES - A CRITICAL-EVALUATION OF FREEZING AND THAWING CONDITIONS
    CHESNE, C
    GUILLOUZO, A
    [J]. CRYOBIOLOGY, 1988, 25 (04) : 323 - 330
  • [5] USE OF CRYOPRESERVED ANIMAL AND HUMAN HEPATOCYTES FOR CYTOTOXICITY STUDIES
    CHESNE, C
    GUYOMARD, C
    GRISLAIN, L
    CLERC, C
    FAUTREL, A
    GUILLOUZO, A
    [J]. TOXICOLOGY IN VITRO, 1991, 5 (5-6) : 479 - 482
  • [6] DRUG-METABOLISM AND VIABILITY STUDIES IN CRYOPRESERVED RAT HEPATOCYTES
    COUNDOURIS, JA
    GRANT, MH
    SIMPSON, JG
    HAWKSWORTH, GM
    [J]. CRYOBIOLOGY, 1990, 27 (03) : 288 - 300
  • [7] THE EFFECTS OF CRYOPRESERVATION ON MEMBRANE INTEGRITY, MEMBRANE-TRANSPORT, AND PROTEIN-SYNTHESIS IN RAT HEPATOCYTES
    DELOECKER, R
    FULLER, BJ
    GRUWEZ, J
    DELOECKER, W
    [J]. CRYOBIOLOGY, 1990, 27 (02) : 143 - 152
  • [8] FARRANT J, 1980, LOW TEMPERATURE PRES, P287
  • [9] FULLER BJ, 1980, CRYOLETTERS, V1, P139
  • [10] FULLER BJ, 1985, CRYO-LETT, V6, P49