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

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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.

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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

F u,buffer,glu :

Unbound fraction of telmisartan-glucuronide in the buffer

F u,buffer,tel :

Unbound fraction of telmisartan in the buffer

F u,cell,glu :

Intracellular unbound fraction of telmisartan-glucuronide

F u,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

V buffer :

Volume of the buffer

V cell :

Total cellular volume

WEM:

William’s E medium

X bile,glu :

Amount of telmisartan-glucuronide in bile

X bile,tel :

Amount of telmisartan in bile

X buffer,glu :

Amount of telmisartan-glucuronide in the buffer

X buffer,tel :

Amount of telmisartan in the buffer

X cells + bile,glu :

Amount of telmisartan-glucuronide with standard buffer in cell lysate

X cells + bile,tel :

Amount of telmisartan with standard buffer in cell lysate

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Acknowledgements

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

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Author notes

  1. Janneke Keemink

    Present address: Drug Delivery, Department of Pharmacy, Uppsala University, Uppsala, Sweden

  2. Tom De Bruyn

    Present address: Genentech, Inc, South San Francisco, California, USA

  3. Janneke Keemink and Neel Deferm contributed equally to this work.

Authors and Affiliations

  1. Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, Gasthuisberg O&N2 Herestraat 49-box 921, 3000, Leuven, Belgium

    Janneke Keemink, Neel Deferm, Tom De Bruyn, Patrick Augustijns, Thomas Bouillon & Pieter Annaert

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Correspondence to Pieter Annaert.

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Keemink, J., Deferm, N., De Bruyn, T. et al. Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes. AAPS J 20, 33 (2018). https://doi.org/10.1208/s12248-018-0188-7

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