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In vitro Assessment of Transporter-Mediated Uptake in Plated Cryopreserved Hepatocytes

互联网2013-11-13

549

实验设备

37°C water bath

37°C / 5% CO₂ humidified incubator

Orbital shaker placed inside incubator

实验步骤

1. Prepare compound stocks: test articles (TA) and positive control(s) (PC) dissolved in an organic solvent such as methanol or DMSO to desired concentration, such as 1 mM.

2. In separate conical tubes, add the test compounds and positive control(s) to warm Incubation Medium to yield the desired working concentration(s). For example, prepare 1 μM by adding 20 μL of 1 mM test article stock solution to 20 mL of Incubation Medium. Note: if DMSO is used as a solvent, the concentration should not exceed 0.1%, with a maximum of 1% in the final Incubation Medium.

3. Remove the wash medium from the hepatocytes and replace with an appropriate volume of Incubation Medium containing the TA or PC for the appropriate time point. Incubate on an orbital shaker for 0, 1, 2, 4, 6 and 8 hr at 80-120 rpm for a 24-well plate. Some low-turnover compounds typically require up to 24 hr incubation periods.

4. Stop the incubations by addition of the appropriate quenching solvent. Mix briefly and collect samples and eithe freeze at -70°C, or directly extract as per analytical methods. If plate is to be returned to incubator, aspirate remaining quenching solvent.

5. Analyze parent disappearance (and metabolite formation as desired) by analytical method(s) of choice, typically HPLC or LC-MS/MS.

6. Determine the in vitro half-life (t_1/2 ) of the parent compound by regression analysis of the percent parent disappearance vs. time curve.

7. Intrinsic clearance in vitro (Cl_{int in vitro} ) can be calculated according to the equation: Cl_{int in vitro} = kV/N, where k = 0.693/t1/2, V = incubation volume and N = number of hepatocytes per well (Table 1).

8. Clint in vitro may be scaled to in vivo predictions according to Obach and McGinnity.

Table 1. Parameters used in intrinsic clearance in vitro (Clint,in vitro) calculations for human hepatocytes. The assumption for N is that all hepatocytes adhered to the well with respect to seeding density.

Plate format	Seeding density (10⁶ total cells/mL)	Incubation volume V (mL)	Hepatocytes per well N (10⁶ cells)
12-well	Either 0.7 / 0.8 / 0.9	1.0	0.7 / 0.8 / 0.9
24-well	Either 0.7 / 0.8 / 0.9	0.5	0.35 / 0.4 / 0.45
48-well	Either 0.7 / 0.8 / 0.9	0.2	0.14 / 0.16 / 0.18
96-well	Either 0.7 / 0.8 / 0.9	0.1	0.07 / 0.08 / 0.09

注意事项

Review this protocol, as well as the protocol, Thawing and Use of Plateable and Suspension Cryopreserved Hepatocytes, to ensure you have all the necessary reagents and equipment prior to starting the procedure. Once thawed, cryopreserved hepatocytes must be used immediately and will not maintain viability if refrozen.

Use universal safety precautions and appropriate biosafety cabinet when handing primary hepatocytes.

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