Cryopreserved, transiently transfected HepG2 cells were compared to freshly transfected HepG2 cells for use in a pregnane X receptor (PXR) transactivation assay. Assay performance was similar for both cell preparations; however, cryopreserved cells demonstrated less interassay variation. Validation with drugs of different PXR activation potencies and efficacies demonstrated an excellent correlation (r2 > 0.95) between cryopreserved and fresh cells. Cryopreservation did not change the effect of known CYP3A4 inducers that have poor cell permeability, indicating that cryopreservation had little effect on membrane permeability. In addition, cryopreserved HepG2 cells did not exhibit enhanced susceptibility to cytotoxic compounds compared to transiently transfected control cells. The use of cryopreserved cells enables this assay to run with enhanced efficiency.
Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, et al: Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci U S A1998;95:12208-12213.
2.
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA: The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest1998;102:1016-1023.
3.
Zhu Z, Kim S, Chen T, Lin J-H, Bell A, Bryson J, et al: Correlation of high-throughput pregnane X receptor (PXR) transactivation and binding assays. J Biomol Screen2004;9:533-540.
4.
Finnigan D, Surey D, King A, McLaren-Douglas A, Middleton B, Coote J, et al: Cellular HTS using frozen cells: uncoupling the link between cell supply and HTS. Abstract presented at the annual conference of SBS, Orlando, FL, September 2004.
5.
Cong M, Fursov N, Federici M, Tacke R, Livelli T, Zhong Z: Improving consistency of cell-based assays by using division-arrest cell reagents. Abstract presented at the annual conference of SBS, Orlando, FL, September 2004.
6.
Kunapuli P, Zheng W, Weber M, Solly K, Mull R, Platchek M, et al: Application of division arrest technology to cell-based HTS: comparison with frozen and fresh cells. Assay Drug Dev Technol2005;3:17-26.
7.
Kafert-Kasting S, Alexandrova K, Barthold M, Laube B, Friedrich G, Arseniev L, et al: Enzyme induction in cryopreserved human hepatocyte cultures. Toxicology2006;220:117-125.
8.
Reinach B, de Sousa G, Dostert P, Ings R, Gugenheim J, Rahmani R: Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes. Chem Biol Interact1999;121:37-48.
9.
Hengstler JG, Ringel M, Biefang K, Hammel S, Milbert U, Gerl M, et al: Cultures with cryopreserved hepatocytes: applicability for studies of enzyme induction. Chem Biol Interact2000;125:51-73.
10.
Hengstler JG, Utesch D, Steinberg P, Platt KL, Diener B, Ringel M, et al: Cryopreserved primary hepatocytes as a constantly available in vitro model for the evaluation of human and animal drug metabolism and enzyme induction. Drug Metab Rev2000;32:81-118.
11.
Roymans D, Annaert P, Van Houdt J, Weygers A, Noukens J, Sensenhauser C, et al: Expression and induction potential of cytochromes P450 in human cryopreserved hepatocytes. Drug Metab Dispos2005;33:1004-1016.
12.
Roymans D, Van Looveren C, Leone A, Parker JB, McMillian M, Johnson MD, et al: Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes. Biochem Pharmacol2004;67:427-437.
13.
Gebhardt R, Hengstler JG, Muller D, Glockner R, Buenning P, Laube B, et al: New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab Rev2003;35:145-213.
14.
Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen1999;4:67-73.
15.
Guyatt G, Jaeschke R, Heddle N, Cook D, Shannon H, Walter S: Basic statistics for clinicians: 1. Hypothesis testing. CMAJ1995;152:27-32.
16.
Desbiens NA: A novel use for the word “trend” in the clinical trial literature. Am J Med Sci2003;326:61-65.
17.
Cleophas TJ: Clinical trials: renewed attention to the interpretation of the P values—review. Am J Ther2004;11:317-322.
18.
Balimane PV, Han YH, Chong S: Current industrial practices of assessing permeability and P-glycoprotein interaction. AAPS J2006;8:E1-E13.
19.
Sinz M, Kim S, Zhu Z, Chen T, Anthony M, Dickinson K, et al: Evaluation of 170 xenobiotics as transactivators of human pregnane X receptor (hPXR) and correlation to known CYP3A4 drug interactions. Curr Drug Metab2006;7:375-388.
20.
Yee S: In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man—fact or myth. Pharm Res1997;14: 763-766.
21.
Roymans D, Van Looveren C, Leone A, Parker JB, McMillian M, Johnson MD: Determination of cytochrome P450 1A2 and cytochrome P450 3A4 induction in cryopreserved human hepatocytes. Biochem Pharmacol2004; 67:427-437.
22.
Griffiths JB, Cox CS, Beadle DJ, Hunt CJ, Reid DS: Changes in cell size during the cooling, warming and post-thawing periods of the freeze-thaw cycle. Cryobiology1979;16:141-151.
23.
Fujikawa S: Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice. Cryobiology1980;17:351-362.
24.
Hidayat AA, LaPiana FG, Kramer KK, Whitmore PV, Wertz FD, Rao NA: The effect of rapid freezing on uveal melanomas. Am J Ophthalmol1987;103:66-80.
25.
Levin RL: Osmotic behavior of cells during freezing and thawing. In McGrath JJ, Diller KR (eds): Low-Temperature Biotechnology: Emerging Applications and Engineering Contributions. New York: ASME, 1988: 177-188.
26.
Koch AL: Shrinkage of growing Escherichia coli cells by osmotic challenge. J Bacteriol1984;159:919-924.
27.
Mazur P, Rall WF, Rigopoulos N: Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophys J1981;36:653-675.
28.
Taylor MJ: Physicochemical principles in low-temperature biology. In Grout BW, Morris GJ (eds): The Effects of Low Temperatures on Biological Systems. London: Arnold, 1987:3-71.
29.
Farrant J, Walter CA, Lee H, McGann LE: Use of two-step cooling procedures to examine factors influencing cell survival following freezing and thawing. Cryobiology1977;14:273-286.
30.
Leibo SP, Mazur P: The role of cooling rates in low-temperature preservation. Cryobiology1971;8:447-452.
31.
Phelan MC: Techniques for mammalian cell tissue culture. In Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds): Current Protocols in Molecular Biology. New York: John Wiley & Sons; 1998:A.3F1-A.3F12.
32.
McGann LE: Optimal temperature ranges for control of cooling rate. Cryobiology1979;16:211-216.
33.
Paul J: Cell and Tissue Culture. New York: Churchill Livingstone; 1975.
34.
Porterfield JS, Ashwood-Smith MJ: Preservation of cells in tissue culture by glycerol and dimethyl sulphoxide. Nature1962;193:548-550.
35.
Dawson M: Cryopreservation. In Butler M, Dawson M (eds): Cell Culture Labfax. Oxford, UK: BIOS; 1992:141-145.
