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


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.


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



Breast cancer resistance protein


Biliary excretion index


Bile salt export pump


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


Intrinsic unbound biliary clearance of telmisartan-glucuronide


Intrinsic unbound biliary clearance of telmisartan


Intrinsic unbound efflux clearance of telmisartan-glucuronide


Intrinsic unbound efflux clearance of telmisartan


Intrinsic unbound metabolic clearance


Intrinsic unbound uptake clearance of telmisartan-glucuronide


Intrinsic unbound uptake clearance of telmisartan


Uptake clearance


Dichotomous covariate


Coefficient of variation


Dulbecco’s modified Eagle’s medium


Fetal bovine serum


First-order conditional estimation with interaction


Unbound fraction of telmisartan-glucuronide in the buffer


Unbound fraction of telmisartan in the buffer


Intracellular unbound fraction of telmisartan-glucuronide


Intracellular unbound fraction of telmisartan


Hanks’ balanced salt solution


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


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


Sodium fluorescein


Organic anion transporter


Organic anion transporting polypeptide


Organic cation transporter


Objective function value


Phosphate buffered saline


Sandwich-cultured hepatocytes


Suspended hepatocytes


Typical value parameter of clearance


UDP glucuronosyltransferase


Volume of the buffer


Total cellular volume


William’s E medium


Amount of telmisartan-glucuronide in bile


Amount of telmisartan in bile


Amount of telmisartan-glucuronide in the buffer


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



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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