Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

Abstract

3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CL int) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CL int within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1e3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.

Primary Human Hepatocyte Spheroids as an In Vitro Tool for Investigating Drug Compounds with Low Hepatic Clearance

“…Similarly, another recent study by Kanebratt et al (2021) applying empirical scaling factors. Furthermore, the study by Kanebratt et al (2021) indicated a trend towards more pronounced deviation between predicted and observed clearance data for drug compounds with higher clearance, which was not evident in our study. Interestingly, the authors pooled three spheroids per incubation in order to increase the metabolic capacity and observed comparable CL int data using pooled and individual hepatocyte spheroids.…”

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro

“…Additionally, cellular polarization was clearly present in pHH spheroids as shown by MRP2 [ 51 , 53 ], P-glycoprotein (Pgp), [ 54 ], and BSEP expression [ 53 , 54 ]. Drug-metabolizing enzyme expression and activity of CYP1A2 [ 53 , 55 , 56 , 57 ], CYP2B6 [ 55 ], CYP2C9 [ 55 ], CYP2C19 [ 58 ], CYP2D6 [ 53 , 55 , 57 ], CYP3A4 [ 50 , 53 , 55 , 56 , 57 , 58 , 59 ], and UGTs [ 56 , 59 ] was stable over several weeks of pHH spheroid culture. CYP2C9 activity even increased with culture time [ 53 , 55 , 56 , 58 ], while CYP2C8 [ 53 , 56 ], and in some cases also CYP1A2, CYP2B6, CYP2D6 and CYP3A4 [ 55 , 58 ] activities decreased with time.…”

Experimental liver models: From cell culture techniques to microfluidic organs‐on‐chip

“…18 In a 3D cell configuration, hepatocytes are inclined to interact in a three-dimensional space, improving their polarity and preventing PHHs dedifferentiation; hence, this environment more closely resembles the in vivo hepatocyte architecture. 61,62 Fueled by the possibility to develop hepatic culture methods characterised by prolonged maintenance of the metabolic phenotype, intense efforts have been made to deeply characterise 3D hepatocyte spheroids. They are generated with two main techniques: Formation of tubular-structure after 20 days in culture; expression of GM130 and secretin receptor (cell polarisation); primary cilia extended in the lumen [81] Cholangiocytes derived from hiPSC Expression of biliary markers (eg CK7, CK19); primary cilia; MDR1 functional activity assessed by the use of rhodamine 123; functional secretory cholangiocytes activity assessed by the exposure to somatostatin and secretin [83] Cholangiocytes derived from hiPSC Expression of biliary markers (eg CK7, CK19); MDR1 functional activity assessed by the use of rhodamine 123; CFTR functional activity assessed by the use of forskolin (i) by self-aggregation, plating a well-defined cell number in an ultralow attachment (ULA) plate, generating size-controlled spheroids, and (ii) by embedding hepatocytes in a hydrogel mimicking ECM protein (Matrigel™ or type I collagen).…”

“…In standard 2D cultures, hepatocyte polarity is not retained and its loss is implicated as one of the main causes related to the dedifferentiation process 18 . In a 3D cell configuration, hepatocytes are inclined to interact in a three‐dimensional space, improving their polarity and preventing PHHs dedifferentiation; hence, this environment more closely resembles the in vivo hepatocyte architecture 61,62 …”

“…PHHsHigh viability up to 21 days in culture; high expression and activity of drug-metabolic enzymes; high expression of key hepatic genes (eg HNF4); high sensitivity and drug metabolism; expression of MRP-2 (functional bile canaliculi)[61,62,66,70] HepaRG High expression and activity of drug-metabolic enzymes; high albumin secretion; expression of MRP-2 (functional bile canaliculi); high response to CYP-inducing compounds[67] PHHs High expression of ADMET proteins; high sensitivity to long-term repeated exposure to toxic compounds (eg APAP, diclofenac)[68,69] Human cells from liver biopsies High viability up to 3 months in culture; glycogen accumulation; albumin secretion; high CYP3A4 activity[71] PHHs and NPCs High viability up to 5 weeks; high albumin release; glycogen storage; expression of MRP-2 (hepatocyte polarisation); high CYPs expression and activity assessed by exposure to CYPinducing compounds; presence of KCs assessed by IHC for CD68 and release of IL-6 upon LPS stimulation[69,76] PHHs and NPCs High viability up to 2 weeks; stable integration of LSEC,KCs and HSCs in 3D spheroids; improved hepatic functionality[77] PHHs and NPCs High viability, albumin release and CYPs activity up to 3 months; presence of KCs and HSCs assed by the release of TNFα, IL-1β and IL-6 upon LPS stimulation; endothelial cells presence assessed by the expression of CD31[78] PHHs and NPCs High viability up to 5 weeks; stable integration of LSEC; improved hepatic functionality, as CYPs expression and albumin release; high sensitivity to toxic compounds (eg APAP…”