Abstract
We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 ± 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 ± 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at –20 °C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (–85 °C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically – (4 °C for 2 days) or cryogenically – (–85 °C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD50values were obtained, LD10 values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.
Similar content being viewed by others
References
Aden DP, Fogel A, Plotkin S, Damjanov I & Knowles BB (1979) Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature 282: 615–616.
Allenspach AL & Kraemer TG (1989) Ice crystal patterns in arti-ficial gels of extracellular matrix macromolecules after quickfreezing and freeze-substitution. Cryobiology 26: 170–179.
Albalak A, Ziedel ML, Zucker SD, Jackson AA & Donovan JM (1996) Effects of submicellar bile salt concentration on biological membrane permeability to low molecular weight non-ionic solutes. Biochemistry 35: 7936–7945.
Clemedson C et al. (1996b) MEIC evaluation of acute systemic toxicity: Part II. In vitro Results from 68 toxicity assays used to test the first 30 reference chemicals and a comparative cytotoxicity analysis. ATLA: 273–311.
Connolly DT, Knight MB, Harakas NK, Wittwer AJ & Feder J (1986) Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal Biochem 152: 136–140.
Delescluse C, Ledirac N, Sousa G, Paralavorio M, Lesca P & Rahmani R (1998) Cytotoxic effects and induction of cytochromes P450 1A1/2 by insecticides, in hepatic or epidermal cells: binding capability to the Ah receptor, Toxicol Letts 96–97: 33–39.
Gordon DA, Luis MS, Pereyra T & Mackenzie GH (1982) Preservation for transplantation; a review of techniques of islet cell culture and storage. J Surg Res. 32: 182–193.
Hubel A, Toner M, Cravalho EG, Yarmush ML & Tompkins RG (1991) Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel. Biotechnol Prog 7: 554–559.
Knight MJ, Scharp DW & Kemp CB (1973) Effects of cold storage noindent on the function of isolated pancreatic islets. Cryobiology 10: 89–94.
Koebe HG, Dunn JCY, Toner M, Sterling LM, Hubel A, Cravalho EG, Yarmush ML & Tompkins RG (1990) A new approach to the cryopreservation of hepatocytes in a sandwich culture configuration. Cryobiology 27: 576–584.
Martin A & Clynes M (1993) Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays. Cytotechnology 11: 49–58.
Meredith JE, Fazeli B & Schwartz MA (1993) The extracellular matrix as a cell survival factor. Mol Biol Cell 4: 953–961.
Ohno T, Saijo-Kurita K, Miyamoto-Eimori N, Kurose T, Aoki Y & Yoshimura S (1991) A simple method for in situ freezing of anchorage-dependent cell including rat liver parenchymal cells. Cytotechnology 5: 273–277.
Pang JM, Zaleski J & Kauffman FC (1996) Actions of selected hepatotoxicants on freshly isolated and cryopreserved rat hepatocytes, Cryobiology 33: 226–235.
Pang JM, Zaleski J & Kauffman FC (1997) Toxicity of allyl alcohol in primary cultures of freshly isolated and cryopreserved hepatocytes maintained on hydrated collagen gels. Toxicol Appl Pharmacol 142: 87–94.
Schulak JA, Stuart FP & Rechard CR (1978) Physiologic aspects of intrasplenic autotransplantation of pancreatic fragments in the dog after 24 hr of cold storage. J Surg Res 24: 125–128.
Seibert H, Kolossa M & Wassermann O (1989) Bovine spermatozoa as an in vitro model for studies on the cytotoxicity of chemicals: Effects of chlorophenols. Cell Biol Toxicol 5: 315–330.
Senseble L, Charbord P, Berthou C, Agbrall JF, Autrand C & Briere J (1992) Effects of cryopreservation on the adherent layer of human long-term bone marrow cultures: Study of cell phenotypes. Leukemia 6: 459–462.
Smith KO (1981) Low-temperature storage of surface-attached living cell cultures. Cryobiology 18: 251–257.
Song YC, Hunt CJ & Pegg DE (1995) Cryopreservation of the common carotid artery of the rabbit: Optimization of dimethyl sulfoxide concentration and cooling rate. Cryobiology 32: 405–421.
Varani J, Inman DR, Fligiel SEG & Hillagas WJ (1993) Use of recombinant and synthetic peptides as attachment factors for cells on microcarriers. Cytotechnology 13: 89–98.
Watts P & Grant MH (1996) Cryopreservation of rat hepatocyte monolayer cultures. Hum Exp Toxicol 15: 30–37.
Wendland M & Subramani S (1993) Cytosol-dependent peroxisomal protein import in a permeabilized cell system. J Cell Biol 120: 675–685.
Wright G, Reichenbecher V, Green T, Wright GL & Wang S (1997) Paraquat inhibits the processing of human manganese-dependent superoxide dismutase by SF-9 insect cell mitochondria. Exp Cell Res 234: 78–84.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shoji, R., Sakai, Y., Sakoda, A. et al. Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests. Cytotechnology 32, 147–155 (2000). https://doi.org/10.1023/A:1008124228377
Issue Date:
DOI: https://doi.org/10.1023/A:1008124228377