Use of Cryopreserved Human Hepatocytes in Sandwich Culture to Measure Hepatobiliary Transport
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 x 10(6) cells/well in 500 microl, 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 microl/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 microl/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.
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The sandwich culture method has long been demonstrated to produce hepatocyte cell cultures where morphology and function more accurately reproduce in vivo behavior.
“The sandwich culture method, e.g., placing cells between two layers comprised of ECM, polyacrylamide, or collagen, has long been demonstrated to produce hepatocyte cell cultures, where morphology and function more accurately reproduce in vivo behavior (9,29,34,65,73).”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [9]
The effect of cell polarization in 2D culture can be mitigated using a sandwich culture method that adds a layer of ECM atop the cells to eliminate apical-basal polarity and mimic 3D ECM.
“The effect of cell polarization in 2D cell culture can be mitigated using a sandwich culture method that adds a layer of ECM atop the cells (9,29,34,65,73) to eliminate apical-basal polarity and provide a mimic of 3D ECM.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [9]
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References 21 · Cited by 216
- Authors
- Bi, Y.-A., Kazolias, D., & Duignan, D. B.
- Affiliations
- Pfizer (United States) (US)
- Venue
- Drug Metabolism and Disposition
- Publisher
- Elsevier BV
- Published
- 2006-09
- Volume
- 34
- Issue
- 9
- Pages
- 1658-1665
- ISSN
- 0090-9556 (ISSN-L)
- OpenAlex
- W2157772258
- Cited by
- 216
- References
- 21
- Citations by year
- 2026: 4 · 2025: 4 · 2024: 3 · 2023: 4 · 2022: 9 · 2021: 7 · 2020: 6 · 2019: 10 · 2018: 7 · 2017: 16 · 2016: 14 · 2015: 16 · 2014: 14 · 2013: 19 · 2012: 18
- FWCI
- 6.43
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Drug Metabolism and Disposition (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Keywords
- cryopreserved human hepatocytes, sandwich culture, bile canaliculi, hepatobiliary transport, biliary excretion, taurocholate, organic anion transporting polypeptides, bile salt export pump, MDR1, ABCG2, MRP2, multidrug efflux transporter
- MeSH terms
- Adolescent, Aged, Bile Canaliculi, Biological Transport, Cells, Cultured, Child, Preschool, Digoxin, Drug Evaluation, Preclinical, Estradiol, Female, Humans, Kinetics, Male, Middle Aged, Taurocholic Acid, Cryopreservation, Cell Culture Techniques, Hepatocytes, Membrane Transport Proteins
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