Sandwich-cultured hepatocytes

Lau, Y.Y. et al. 2002. Development of a novel in vitro model to predict hepatic clearance using fresh, cryopreserved, and sandwich-cultured hepatocytes. Drug Met. Disp. 30 1446. 

II.I.4.2 Liver Specific Drug Transport in Sandwich-cultured Hepatocytes. . 540... 

Liver Specific Drug Transport in Sandwich-cultured Hepatocytes... 

To prepare sandwich-cultured hepatocytes, neutralized collagen solution (0.1 ml, 1.5 mg/ml, pH 7.4) is added to the monolayers 24 h after the cells are seeded. Cultures with collagen overlay are incubated for 45 min at 37 °C in a humidified incubator to allow the collagen to gel before addition of DMEM. Medium is changed on a daily basis until the fifth day after the cells are seeded. These hepatocytes are referred to as day 5 or long-termed cultured hepatocytes. 

An exclusive sandwich-cultured hepatocyte system for hepatobiliary disposition is also commercially available by Qualyst, Inc (www.qualyst.com), named B-CLEAR. 

LeeJK, Marion T, AbeK, LimC, PollackGM, Brouwer KL. Hepatobiliary disposition of troglitazone and metabolites in rat and human sandwich-cultured hepatocytes Use of Monte Carlo simulations to assess the impact of changes in biliary excretion on troglitazone sulfate accumulation. f Pharmacol Exp Ther. 2010 332(l) 26-34. 

Hepatocytes cultivated between two layers of soft gel collagen represent the most frequently used hepatocyte in vitro system. They establish an apical pole between the cells which contains bile canaliculi (Fig. 3). The hepatocyte membrane facing the collagen gel corresponds to the basolateral side. Therefore, hepatocyte sandwich cultures represent the easiest to handle 3D culture system, although only one sheet of hepatocytes is represented. The hepatocyte phenotype in sandwich culture is characterized by (1) maintenance of susceptibility to apoptosis, (2) a delayed decrease of drug-metabolizing activities compared to monolayer cultures, (3) establishment and maintenance of bile canaliculi, and (4) a resting cell state where stimulation by HGF and EGF induces almost no proliferation events. As previously mentioned, this cultivation system effectively prevents the spontaneous activation of ERK and Akt which occurs in 2D systems [13]. Consistent with the effects of small chemical inhibitors in 2D cultures, expression of a constitutively active form of Ras in sandwich-cultured hepatocytes induces features of EMT and stress fibers. In contrast to Ras, expression of constitutive active Akt in hepatocytes induces an antiapoptotic phenotype and does not cause EMT [13]. 

Fig. 3 Hepatocyte polarity in different culture conditions, (a) Confocal microscopy reveals formation of bile cana-liculi (white arrows) in primary mouse hepatocytes. These structures are formed within 24 h when hepatocytes are cultivated between two layers of soft gel collagen (i.e., sandwich culture S) but not in monolayer confluent (Mc) or monolayer subconfluent (Ms) cultures. Green fluorescence corresponds to DPPIV staining (a marker for bile canaliculi). Nuclei appear blue (DAPI staining), (b) Bile canaliculi lumen is further revealed in z-stack confocal imaging in sandwich-cultured hepatocytes. Red corresponds to F-actin and green to DPPIV. Co-localization of the two markers is seen in yellow and corresponds to bile canaliculi. Nuclei appear blue (DAPI staining)...

De Bruyn T, Chatterjee S, Fattah S, Keemink J, Nicolai J, Augustijns P, Annaert P (2013) Sandwich-cultured hepatocytes utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 9 589-616. doi 10. 1517/17425255.2013.773973... 

Susukida T, Sekine S, Ogimura E, Aoki S, Oizumi K, Horie T, Ito K (2015) Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes. Toxicol In Vitro 29 1454—63. 

Swift B, Pfeifer ND, Brouwer KL (2010) Sandwich-cultured hepatocytes an in vitro model to evaluate hepatobiliary transporter-based drug interactions and hepatotoxicity. Drug Metab Rev 42, 446-71. 

NAD+ oxidized nicotinamide adenine dinucleotide SCH sandwich cultured hepatocytes... 

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