Sandwich cultures

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. 

R.Z. Turncliff, P.J. Meier, and K.L. Brouwer. Effect of dexamethasone treatment on the expression and function of transport proteins in sandwich-cultured rat hepatocytes. Drug Metab Dispos. 32 834—839 (2004). 

Hepatocytes were isolated from male Wistar rats, two dogs (age, breed and sex not stated) and two human subjects (69-71 years of age, sex not stated) (Hildebrand et al., 1999). In collagen sandwich cultures, the rat hepatocytes responded to di(2-ethylhexyl) phthalate in the culture medium with slightly increased carnitine acetyltransferase activity, while dog and human hepatocytes did not respond. 

Heindel, J.J. Powell, C.J. (1992) Phthalate ester effects on rat Sertoli cell function in vitro effects of phthalate side chain and age of animal. Toxicol appl. Pharmacol, 115, 116-123 Hellwig, J., Freudenberger, H. Jackh, R. (1997) Differential prenatal toxicity of branched phthalate esters in rats. Food Cosmel Toxicol, 35, 501-512 Hildebrand, H., Schmidt, U., Kempka, G, Jacob, R., Ahr, H.J., Ebener, C., Goretzki, RE. Bader, A. (1999) An in-vitro model for peroxisome proliferation utilizing primary hepatocytes in sandwich culture. Toxicol. In Vitro, 13, 265-273... 

The success of cellular therapies ultimately depends on the stability of the hepatocyte in the architecture in which it must exist. Primary hepatocytes are anchorage dependent. Isolated cells rapidly lose viability when cultured in monolayers or suspensions. Investigators have developed culture models based on features of liver architecture to recapitulate the complex hepatocyte microenvironment. Sandwich culture mimics the environment of hepatocytes in vivo by entrapping cells between two layers of collagen gel. However, such methods introduce additional transport barriers and are difficult to scale up to therapeutic levels. ... 

Liu X, Chism JP, LeCluyse EL, et al. Correlation of biliary excretion in sandwich-cultured rat hepatocytes and in vivo in rats. Drug Metab Dispos 1999 27 637-644. 

Hoffmaster KA, Tumcliff RZ, LeCluyse EL, et al. P-glycoprotein expression, localization, and function in sandwich-cultured primary rat and human hepatocytes relevance to the hepatobiliary disposition of a model opioid peptide. Pharm Res 2004 21 1294-1302. 

To achieve longer incubation times, cultivation of freshly isolated hepatocytes in culture on monolayer (Maslansky 1982 Wang 2002), sandwich culture on variable matrixes (e.g. Maurel 1996 Kem 1997 Wang... 

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. 

Li N, Bi YA, Duignan DB, Lai Y. Quantitative expression profile of hepatobiliary transporters in sandwich cultured rat and human hepatocytes. Mol Pharm. 2009 6(4) 1180-1189. 

Dong JQ, Smith PC. Glucuronidation and covalent protein binding of benoxapro-fen and flunoxaprofen in sandwich-cultured rat and human hepatocytes. Drug Metab Dispos. 2009 37(12) 2314-2322. 

Abe K, Bridges AS, Brouwer KL. Use of sandwich-cultured human hepatocytes to predict biliary clearance of angiotensin II receptor blockers and HMG-CoA reductase inhibitors. Drug Metab Dispos. 2009 37(3) 447-452. 

The third cell-based approach concerns sandwich-cultured rat or human hepatocytes (SCH) [80], which closely mimic the hepatic environment in terms of expression of transporters and metabolizing enzymes. In the SCH model, hepatocytes are cultured in a sandwich configuration between two layers of gelled matrix to form intact bile canaliculi [81]. The advantage of this model is that both hepatic uptake and biliary excretion can be studied. 

Bi YA, Kazolias D, Duignan DB (2006) Use of cryopreserved human hepatocytes in sandwich culture to measure hepatobiliary transport. Drug Metab Dispos Biol Fate Chem 34 1658-1665... 

Several experimental systems to check the inhibition potency of bile add transport have been characterized. Using sandwich-cultured human hepatocytes, bosentan, cyclosporin A, CI-1034 (endothelin-A receptor antagonist), glyburide, erythromycin estolate, and troleandomycin could inhibit the taurocholate efflux to the bile pocket [234]. Moreover, Mita et al. [235] construded NTCP/BSEP double-transfeded cells and some cholestasis-induced compounds inhibited both the NTCP-mediated uptake and the BSEP-mediated efflux of taurocholate. Then, they have found fluorescent bile acids whose transcellular transport was dearly observed, which may be used for the rapid identification of inhibitors of NTCP and BSEP in drug screening process [235]. 

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)...

Again, the interspecies difference should be taken into account. Because rodent hepatocytes tend to dedifferentiate more rapidly they profit much more from sandwich culture conditions than human hepatocytes which have a lower propensity to show EMT-like features. 

Tuschl G, Hrach J, Walter Y, Hewitt PG, Mueller SO (2009) Serum-free collagen sandwich cultures of adult rat hepatocytes maintain liver-like properties long term a valuable model for in vitro toxicity and drug-drug interaction studies. Chem Biol Interact 181(1) 124-137... 

Treijtel N, Barendregt A, Freidig AP, Blaauboer BJ, Van Eijkeren JCH (2004) Modeling the in vitro intrinsic clearance of the slowly metabolized compound tolbutamide determined in sandwich-cultured rat hepatocytes. Drug Metab Dispos 32(8) 884-891... 

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... 

Annaert, P. P., Turncliff, R. Z., Booth, C. L., Thakker, D. R., and Brouwer, K. L. (2001) P-glycoprotein-mediated in vitro biliary excretion in sandwich-cultured rat hepatocytes. Drug Metab. Dispos. 29, 1277-1283. 

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