Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

Eur J Pharm Sci. 2011 Jul 17;43(4):297-307. doi: 10.1016/j.ejps.2011.05.002. Epub 2011 May 14.


While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β-D-glucuronide (E(2)-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly (p<0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant (p<0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2)=0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Methyl-4-phenylpyridinium / metabolism
  • Adolescent
  • Adult
  • Biological Transport
  • Corticosterone / pharmacology
  • Cryopreservation*
  • Digoxin / metabolism
  • Estradiol / analogs & derivatives
  • Estradiol / metabolism
  • Estrone / analogs & derivatives
  • Estrone / metabolism
  • Hepatocytes / metabolism*
  • Humans
  • Male
  • Membrane Transport Proteins / metabolism
  • Middle Aged
  • Organic Anion Transporters / metabolism*
  • Organic Anion Transporters, Sodium-Dependent / metabolism*
  • Organic Anion Transporters, Sodium-Independent / genetics
  • Organic Anion Transporters, Sodium-Independent / metabolism
  • Organic Cation Transport Proteins / antagonists & inhibitors
  • Organic Cation Transport Proteins / metabolism
  • Organic Cation Transporter 1 / antagonists & inhibitors
  • Organic Cation Transporter 1 / metabolism*
  • Prazosin / pharmacology
  • RNA, Messenger / genetics
  • Rifampin / pharmacology
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Symporters / metabolism*
  • Taurocholic Acid / metabolism


  • Membrane Transport Proteins
  • Organic Anion Transporters
  • Organic Anion Transporters, Sodium-Dependent
  • Organic Anion Transporters, Sodium-Independent
  • Organic Cation Transport Proteins
  • Organic Cation Transporter 1
  • RNA, Messenger
  • SLCO1B3 protein, human
  • Solute Carrier Organic Anion Transporter Family Member 1B3
  • Symporters
  • solute carrier family 22 (organic cation transporter), member 3
  • sodium-bile acid cotransporter
  • estradiol-17 beta-glucuronide
  • Estrone
  • Estradiol
  • Taurocholic Acid
  • Digoxin
  • estrone sulfate
  • 1-Methyl-4-phenylpyridinium
  • Rifampin
  • Corticosterone
  • Prazosin