VIII. HIGHER-THROUGHPUT SCREENING (HrTS) WITH HUMAN HEPATOCYTES
Higher-throughput screening (HrTs) and human hepatocytes are not compatible, due to the difficulty most laboratories have in procuring human livers for research. The success in cryopreservation, however, has dramatically enhanced the availability of human hepatocytes for research. Currently, cryopreserved human hepatocytes are commercially available. Furthermore, as cryopreserved hepatocytes can be used at the convenience of the investigator, HrTS with human hepatocytes is now a reality. The typical protocols for HrTS assays are given below .
As the HrTS assays all require the use of cryopreserved human hepatocytes, the procedures for thawing of the cells are described here. This thawing procedure needs to be followed carefully, as deviations may result in lowered viability. On the day of assay, the vials containing the cryopreserved hepatocytes are removed from liquid nitrogen storage. The cells are thawed by gently shaking the vials in a 37°C water bath. As soon as the contents are thawed (approx. 75-90 sec), the vials are transferred to an ice bucket. The hepatocyte suspensions (approximately 1 mL) are then transferred into a 50 mL centrifuge tube on ice. Cold medium (15 mL) is then added to the cell suspension slowly. After dilution, the cells are centrifuged at 50 X g for 3 min. The resulting pellet is resuspended in 2 mL of medium. Viability in general can be determined based on trypan blue exclusion [13,15].
The cryopreserved human hepatocytes are resuspended in an isotonic buffer at physiological (e.g., Krebs-Hensleit buffer) pH of 7.2-7.5 in a cell density of 2 X 106 cells/mL and seeded onto 96-well filter plates (Millipore, MultiScreen) in a volume of 50 |L (0.1 X 106 cells). Aliquots of 50 |L of buffer containing 2X concentrations of the chemical to be tested are added to the wells preseeded with the hepatocytes to achieve a final 1X concentration. A pragmatic endpoint is the quantification of the disappearance of the parent chemical (a final concentration between 1 and 10 |M). The plates are incubated at 37°C for 4-6 hr. At the end of the incubation period, 100 |L of ice-cold organic solvent (methanol or acetonitrile) is added to each well to stop the reaction. The resulting samples are filtered by centrifugation into a recipient 96-well plate for later analysis. At present, LC/MS analysis is the most efficient. Incubation of test chemical with killed hepatocytes (freeze-thaw cycle at -20°C or heat-inactivation) under identical conditions is used as control. Metabolic stability is expressed as a percentage of parent disappearance using the equation
Cryopreserved human hepatocytes are thawed and resuspended in incubation medium at a density of 1.25 X 106 cells/mL. A volume of 40 |L/well of the cell suspension is loaded into 96-well plates, followed by a 40 |L/well addition of
% Disappearance peak area (live hepatocytes) peak area (dead hepatocytes)
peak area (live hepatocytes) peak area (dead hepatocytes)
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