The AAPS Journal

, 20:33 | Cite as

Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes

  • Janneke Keemink
  • Neel Deferm
  • Tom De Bruyn
  • Patrick Augustijns
  • Thomas Bouillon
  • Pieter Annaert
Research Article
  • 109 Downloads

Abstract

Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.

KEY WORDS

cryopreservation Mrp2 Oatp rat hepatocytes sandwich-culture suspended hepatocytes telmisartan UGT 

Abbreviations

Bcrp

Breast cancer resistance protein

BEI

Biliary excretion index

Bsep

Bile salt export pump

CDF(DA)

5 (and 6)-Carboxy-2′,7′-dichlorofluorescein (diacetate)

CLint,u,bile,glu

Intrinsic unbound biliary clearance of telmisartan-glucuronide

CLint,u,bile,tel

Intrinsic unbound biliary clearance of telmisartan

CLint,u,eff,glu

Intrinsic unbound efflux clearance of telmisartan-glucuronide

CLint,u,eff,tel

Intrinsic unbound efflux clearance of telmisartan

CLint,u,met

Intrinsic unbound metabolic clearance

CLint,u,up,glu

Intrinsic unbound uptake clearance of telmisartan-glucuronide

CLint,u,up,tel

Intrinsic unbound uptake clearance of telmisartan

CLuptake

Uptake clearance

Cov

Dichotomous covariate

CV

Coefficient of variation

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

FOCE+I

First-order conditional estimation with interaction

Fu,buffer,glu

Unbound fraction of telmisartan-glucuronide in the buffer

Fu,buffer,tel

Unbound fraction of telmisartan in the buffer

Fu,cell,glu

Intracellular unbound fraction of telmisartan-glucuronide

Fu,cell,tel

Intracellular unbound fraction of telmisartan

HBSS

Hanks’ balanced salt solution

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

KHB

Krebs Henseleit buffer; Mrp2, multidrug resistance-associated protein 2

NaFluo

Sodium fluorescein

Oat

Organic anion transporter

Oatp

Organic anion transporting polypeptide

Oct

Organic cation transporter

OFV

Objective function value

PBS

Phosphate buffered saline

SCH

Sandwich-cultured hepatocytes

SH

Suspended hepatocytes

TVCL

Typical value parameter of clearance

Ugt

UDP glucuronosyltransferase

Vbuffer

Volume of the buffer

Vcell

Total cellular volume

WEM

William’s E medium

Xbile,glu

Amount of telmisartan-glucuronide in bile

Xbile,tel

Amount of telmisartan in bile

Xbuffer,glu

Amount of telmisartan-glucuronide in the buffer

Xbuffer,tel

Amount of telmisartan in the buffer

Xcells + bile,glu

Amount of telmisartan-glucuronide with standard buffer in cell lysate

Xcells + bile,tel

Amount of telmisartan with standard buffer in cell lysate

Notes

Acknowledgements

We would like to acknowledge Niels Graindor for his contributions to the experimental work.

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Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Janneke Keemink
    • 1
    • 2
  • Neel Deferm
    • 1
  • Tom De Bruyn
    • 1
    • 3
  • Patrick Augustijns
    • 1
  • Thomas Bouillon
    • 1
  • Pieter Annaert
    • 1
  1. 1.Drug Delivery and DispositionKU Leuven Department of Pharmaceutical and Pharmacological SciencesLeuvenBelgium
  2. 2.Drug Delivery, Department of PharmacyUppsala UniversityUppsalaSweden
  3. 3.Genentech, IncSouth San FranciscoUSA

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