Use of Cryopreserved Human Hepatocytes in Sandwich Culture to Measure Hepatobiliary Transport

Abstract

ABSTRACT:Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 ؋ 10 6 cells/well in 500 l, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na ؉ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 l/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 l/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.

Characterization of Digoxin Uptake in Sandwich-Cultured Human Hepatocytes

“…We have previously shown that SCHH possess significant OATP activity (Bi et al, 2006), and a previous study showed that OATP1B1 and OATP1B3 protein levels are well maintained on day 6 in SCHH (Hoffmaster et al, 2004). Our data show that the uptake clearance of digoxin in sandwich culture is similar to that observed in noncultured hepatocyte suspension, and OATP1B3-specific activity (as measured by CCK-8 uptake clearance) is appreciable in sandwich culture.…”

“…We previously reported the ability to use cryopreserved human hepatocytes in sandwich culture (Bi et al, 2006) and have shown that uptake and efflux transporter activities are similar to those in fresh hepatocytes. The purpose of this study was to investigate the hepatic uptake of digoxin using cryopreserved human hepatocytes in sandwich culture.…”

Use of Sandwich-Cultured Human Hepatocytes to Predict Biliary Clearance of Angiotensin II Receptor Blockers and HMG-CoA Reductase Inhibitors

“…Approximately 5-fold variability was also observed for the uptake of estradiol-17␤-D-glucuronide in suspended human hepatocytes and sandwich-cultured human hepatocytes prepared from cryopreserved human hepatocytes (Shitara et al, 2003;Bi et al, 2006). Multiple OATPs are involved in the hepatic uptake of these ARBs and statins (Nakagomi-Hagihara et al, 2006;Yamashiro et al, 2006).…”

“…at ASPET Journals on May 12, 2018 dmd.aspetjournals.org Downloaded from Bi et al (2006) reported that sandwich-cultured human hepatocytes prepared from cryopreserved human hepatocytes form intact bile canalicular networks and exhibit functional uptake and transportermediated excretion. Use of cryopreserved human hepatocytes would be more convenient than isolating and preparing sandwich-cultured human hepatocytes from fresh human liver.…”

“…Many drugs, including cardiovascular, anticancer, and anti-infective agents (Bi et al, 2006;, undergo extensive biliary excretion in humans. Biliary excretion often plays an important role in the pharmacologic and pharmacokinetic behavior of these compounds.…”