Many attempts were made to use pHHs in 3D culture with the aim to maintain and to prolong their viability and to prevent dedifferentiation. PHH spheroids were viable for at least 2 and up to 7 weeks with stable albumin production ^{[2][3][4][5][6]}[50,51,52,53,54], urea synthesis ^[3][4][51,52] and glycogen storage ^[6][54]. Additionally, cellular polarization was clearly present in pHH spheroids as shown by MRP2 ^[3][5][51,53], P-glycoprotein (Pgp), ^[6][54], and BSEP expression ^[5][6][53,54]. Drug-metabolizing enzyme expression and activity of CYP1A2 ^[5][7][8][9][53,55,56,57], CYP2B6 ^[7][55], CYP2C9 ^[7][55], CYP2C19 ^[10][58], CYP2D6 ^[5][7][9][53,55,57], CYP3A4 ^{[2][5][7][8][9][10][11]}[50,53,55,56,57,58,59], and UGTs ^[8][11][56,59] was stable over several weeks of pHH spheroid culture. CYP2C9 activity even increased with culture time ^{[5][7][8][10]}[53,55,56,58], while CYP2C8 ^[5][8][53,56], and in some cases also CYP1A2, CYP2B6, CYP2D6 and CYP3A4 ^[7][10][55,58] activities decreased with time. In general, maintenance of their initial hepatic transcriptomic and metabolomic profiles was possible for at least 2–5 weeks ^{[3][5][11][12]}[51,53,59,60], which allowed long-term and repeated-dose toxicity testing. EC50 values for acetaminophen, bosentan, diclofenac, troglitazone, and other hepatotoxic compounds significantly decreased upon long-term treatment ^[5][12][53,60] and were closer to the corresponding in vivo C_max than acute toxicity EC50 values ^[5][11][53,59]. Importantly, liver spheroids cultured in chemically-defined medium reached 100% specificity and 69% sensitivity when 123 hepatotoxic compounds were tested. The model system could reliably distinguish between hepatotoxic substances and their nontoxic structural analogues ^[13][61]. The authors used a repeated-dose approach for 14 days and an easy-to-handle ATP assay as readout. Such comprehensive studies corroborate the utility of pHH spheroids in drug development, toxicity testing and DILI prediction. Further, those studies used panels of drugs with different toxicity mechanisms, including mitochondrial dysfunction, steatosis, cholestasis, and fibrosis. This implies that spheroid cultures of pHHs can be used to study different mechanisms of drug induced hepatotoxicity ^{[5][11][12][13][14][15]}[53,59,60,61,62,63].

Spheroids can also be formed by using pHHs together with non-parenchymal cells (NPCs). This approach intends to further improve hepatic characteristics. Those spheroids, often called microtissues, displayed pronounced glycogen storage and albumin synthesis capabilities as well as expression of CYP1A2, CYP2C9 and CYP3A4. Maintenance of cell polarity could be shown by MRP2 and BSEP expression ^[16][17][18][64,65,66]. Viability of the spheroids could be maintained for up to 7 weeks in one of the studies, but albumin secretion declined from day 28 on. Interestingly, CYP1A1/2, CYP2C9 and CYP3A4/5 transcript levels were higher in those microtissues than in clinical liver specimens or pHHs upon long-term culture ^[17][18][65,66]. However, on protein level, only CYP2B6 and UGT1A1 levels remained stable for 7 weeks of culture. Other CYP and phase II enzyme protein levels dropped already at day 7 of culture. Additionally, a decline of CYP1A2, CYP2B6, CYP2C9 and CYP2C19 activities could be observed after 4 weeks in culture, which is in accordance with the reduction in albumin production at this time-point ^[18][66]. Others directly compared hepatic spheroids in monoculture and in co-culture with NPCs over a culture time of 14 days. Spheroid functionality was increased in the co-culture setup as shown by expression of albumin (ALB) and CYP3A4, as well as by albumin synthesis ^[19][67]. A contradictory study, however, showed that ALB and CYP1A2 and CYP3A4 expression was higher in hepatocyte-only spheroids than in NPC-containing spheroids ^[20][68]. Thus, it remains to be shown whether co-culture with NPCs really adds a benefit to pHH spheroid culture.

Other studies used such 3D human liver microtissues for comprehensive toxicology analyses and tested a panel of 100 and 110 hepatotoxic compounds, respectively. Results were directly compared with 2D pHH cultures. It must be noted that spheroids were treated for 7 and 14 days, while monolayer cultures were treated for 24 and 72 h, respectively. Overall, specificity of spheroids for detection of hepatotoxic substances was high (79–85%), and sensitivity was moderate (19–61%) indicating that also NPC containing spheroid cultures of pHHs are suitable for hepatotoxicity risk assessment in drug development processes and for the study of toxicity mechanisms ^[21][22][69,70].

Further improvement of spheroid co-cultures might be achieved by using bioprinting techniques. Here, spheroids are formed by bioprinting pHHs together with non-parenchymal cells such as endothelial and stellate cells on transwell culture inserts of multi-well plates ^[23][71]. Upon this culture system, albumin synthesis and glycogen storage capacity could be sustained, and albumin secretion even increased over the culture period of 4 weeks. Expression levels of CYP1A2, CYP2B6, CYP2C9, CYP2D6 and CYP3A4 also increased significantly over time, with a peak at day 14 followed by a slight decrease at day 28. In addition, EC50 values for trovafloxacin were lower in those so-called 3D liver tissues than in corresponding 2D pHH cultures ^[23][71]. Bioprinting methods are emerging technologies, are often expensive and difficult in handling. Further studies are required to provide easy-to-use and reproducible protocols and to prove the utility of the method for toxicology analyses.

Primary liver characteristics could also be maintained when spheroids were generated in a stirred tank bioreactor. The authors described stable urea and albumin synthesis as well as stable expression of phase I enzymes (CYP1A2, CYP2C9, CYP3A4), the phase II enzymes GSTA1 and UGT2B7 and the polarization marker F-actin ^[24][72]. Gene expression of CYP1A2, CYP2C9 and CYP3A4 could be further improved in stirred tank bioreactors by adding an outer layer of bone marrow-derived mesenchymal stem cells to the liver spheroids, while albumin and urea production remained unaffected with this strategy ^[25][73]. The greatest disadvantage of spheroids in stirred tank bioreactors is the single reaction vessel. Spheroids generated by this method a) vary greatly in size and b) need to be separated and transferred to other culture vessels for toxicology testing. Therefore, this method is not suitable for high-throughput analyses and spheroids generated by the hanging drops or ULA method should be preferred.

Many groups use hepatoma cell lines as HepG2 for spheroid culture due to their ease of use and low cost. It could be shown that culture in this 3D format indeed improves hepatic properties at several levels.

Albumin synthesis ^{[26][27][28][29][30]}[74,75,76,77,78] and urea production ^[28][76] were elevated when HepG2 cells were cultured as spheroids and compared to their 2D cultured counterparts. However, one study showed a contradictory result: albumin secretion levels were significantly higher in 2D than in 3D spheroid cultures ^[31][79]. Gene expression of drug-metabolizing enzymes such as CYP1A1/2 ^{[26][27][30][32]}[74,75,78,80], CYP3A4 ^[26][27][32][74,75,80] and UGT1A1 ^[26][27][74,75] could be increased upon 3D spheroid culture. Importantly, in addition, cellular polarization could be restored. This was shown by positive MRP2 and Pgp staining as well as by functional transporter assays indicating presence of canalicular-like structures ^[28][76].

Co-culture of HepG2 cells with endothelial cells and application of a cell coating technique resulted in elevated albumin synthesis, MRP2 expression and CYP enzyme activities ^[33][81]. Similarly, HepG2 cells cultured together with endothelial and mesenchymal cells as tubular 3D liver microtissues had higher expression levels of drug-metabolizing enzymes than 2D cultured counterparts ^[34][82].

Additionally, bioprinted 3D HepG2 spheroids showed stable albumin and urea synthesis for at least 7 days ^[35][36][83,84]. Such bioprinted HepG2 spheroids displayed higher CYP1A2 gene expression and higher sensitivity towards acetaminophen than 2D cultured HepG2 cells ^[36][84]. Bioprinting HepG2 cells together with fibroblasts resulted in elevated albumin production ^[37][85].

Cell lines as HepG2 are by nature very well suited for high-throughput screens, which is desirable for any toxicological test system. Indeed, HepG2 cells cultured in 3D spheroids were more susceptible to hepatotoxic compounds and showed considerably lower EC50 values for many different drugs than 2D cultured HepG2 cells ^[28][76]. However, other studies have shown that HepG2 spheroids were more resistant to hepatotoxins than their 2D counterparts, both in acute toxicity and repeated-dose toxicity experiments ^[38][86]. Furthermore, they were still considerably less sensitive to hepatotoxic compounds than 3D cultured pHHs. This might be explained by the low expression of CYP and other drug-metabolizing enzymes. As their basal expression is very low to absent in HepG2 cells, also an increase in expression levels upon spheroid culture might not lead to sufficiently elevated enzyme activities. Therefore, the value of such cells for drug development and DILI prediction is questionable.

HepaRG cells also benefit from spheroid culture conditions. They displayed improved albumin synthesis capacity upon culture as spheroids in hanging droplets and when compared to HepaRG monolayer culture. Those HepaRG spheroids needed several days for maturation. Albumin synthesis was elevated at day 6 of spheroid culture, and mRNA levels of CYP1A2, CYP2B6 and CYP3A4 first dropped, then slightly increased at day 4 and were elevated 1.2–3 -fold at day 7 when compared to day 0 ^[29][77]. Additionally, others have shown that HepaRG spheroids (self-aggregated or bioprinted) could be cultured over several weeks and that those cultures maintained several hepatic properties: HepaRG spheroids were capable of (a) albumin ^{[39][40][41][42][43]}[87,88,89,90,91] and urea ^[39][42][87,90] production, (b) displayed expression and activity of CYP1A2 ^[40][41][88,89], CYP2B6 ^[41][89] and CYP3A4 ^{[38][40][41][42][44]}[86,88,89,90,92], (c) showed phase II enzyme activity ^[41][43][89,91], and (d) displayed cellular polarization shown by MRP2 ^[39][40][42][87,88,90] and Pgp ^[43][91] expression, and by F-actin bands, indicative of bile canalicular structures ^[41][43][44][89,91,92]. Regarding expression and activity of phase I enzymes, however, several studies have shown that basal levels in spheroids were unaffected, in part even slightly lower than in monolayer cultures. This might be explained by the relatively high basal expression of several CYP enzymes in HepaRG cells. However, the authors of those studies have deemed it necessary to elevate expression and activity of CYPs by induction using β-naphthoflavone (for CYP1A2), phenobarbital (for CYP2B6) or rifampicin (for CYP2C9 and CYP3A4) to reach more relevant levels ^{[39][41][43][45]}[87,89,91,93].

Importantly, and in accordance with the presence of hepatic characteristics, HepaRG spheroids were also more sensitive to hepatotoxic compounds than monolayer cultures. This was shown by smaller EC50 values in a range of substances including acetaminophen, tamoxifen, and aflatoxin B1 ^{[38][40][42][43][46]}[86,88,90,91,94]. Interestingly, a recent study showed in a high-throughput 384-well format that HepG2 spheroids were more sensitive to hepatotoxic compounds than HepaRG spheroids. When 150 compounds were tested, HepG2 spheroids displayed a sensitivity of 58% and a specificity of 83%, while HepaRG spheroids showed sensitivity of 47% and specificity of 86% ^[47][95]. Static HepaRG spheroid cultures could also be combined with lactate and oxygen microsensors. This allowed live, long-term, and fast measurement of the cellular metabolic activity. This test system was validated using antimycin A and bosentan as test substances, and lactate production increased or decreased accordingly ^[48][96]. Further, it was shown that HepaRG spheroids were able to predict steatosis and mitochondrial dysfunction, while cholestasis could only be predicted in 2D culture models in this work ^[40][88]. Another study presented spheroid models to study cholestatic liability of compounds and showed that HepaRG spheroids as well as pHH spheroids were both able to reliably predict cholestasis ^[14][62]. A model to detect liver fibrosis could be generated by co-culturing HepaRG cells and hepatic stellate cells in spheroid cultures ^[49][97]. Those studies imply that HepaRG cells are well suited to study different types of hepatotoxicity.

Culture of HepaRG as 3D spheroids indeed seems a promising system to maintain primary hepatic properties. However, some studies found that expression of drug-metabolizing enzymes needed to be elevated by prototypic CYP inducers. This, together with the required time-consuming differentiation procedure, renders the system somewhat complicated for toxicity testing. In summary, however, HepaRG cells constitute a more reliable test system than HepG2 cells, especially regarding their drug-metabolizing enzyme expression, and more closely resemble pHHs than other cell lines of cancerous origin.

2.4. Spheroids from iPSC-Derived Hepatocytes

Studies on spheroid cultures of iPSC-heps for toxicity testing are still rare. It was shown that hepatic properties improved upon 3D culture of those liver cells, but they still maintained a more fetal-like phenotype, and adult marker genes (including CYP1A2, CYP2C9, CYP3A4 and ALB) were only expressed at low levels ^[50][51][52][98,99,100]. For example, albumin production was not significantly increased when iPSC-heps were cultured as spheroids instead of 2D monolayers. However, an increase in urea production could be observed at least at day 12 of spheroid culture ^[51][99]. In contrast, others have shown that spheroids of iPSC-heps were capable of albumin production, urea synthesis, glycogen storage and gene expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, UGT2B7). However, again, levels remained far below those of pHHs ^[53][101]. A very recent study compared 2D and 3D cultured iPSC-heps with pHHs cultured in 2D and 3D spheroids. Basal activities of CYP1A2, CYP2B6 and CYP3A4, as well as canaliculi formation, were similar in all culture systems, but CYPs could be induced successfully only in pHH cultures, which is an important feature of functional human hepatocytes ^[54][102].

Bioprinted 3D spheroids of iPSC-heps displayed higher albumin and urea synthesis as well as CYP1A2 and CYP3A4 expression than their 2D cultured counterparts ^[55][103]. Co-culturing of iPSC-hep spheroids with endothelial cells seems to improve maturity of the cells as shown by albumin and urea secretion and CYP enzyme activities ^[56][104].

Interestingly, iPSC-hep spheroids were more sensitive to a set of 24 hepatotoxic compounds than HepG2 spheroids in one study ^[52][100], while another study showed opposite results for 10 out of 23 tested compounds ^[57][105]. When compared to pHH spheroids, IC50 values of iPSC-hep spheroids were similar in 12/15 tested hepatotoxic compounds ^[53][101]. In another study, the authors tested a set of seven hepatotoxic compounds and found that pHHs generally displayed higher sensitivity ^[54][102].

Hepatic differentiation of iPSCs is a promising approach for modelling the human liver in vivo. To date, however, those hepatocytes have a more fetal-like than mature phenotype and efforts must be made to optimize the differentiation procedure towards adult characteristics. Three-dimensional cultures seem to add only little towards this goal, and contradictory data on utility for toxicological analyses render this system, still, an immature technology for such applications.

2.5. Spheroids from Upcyte and Primary-Like Hepatocytes

Data for spheroid culture of upcyte or other primary-like hepatocytes are even scarcer than those for iPSC-heps. Despite their characteristics seem to make them a promising tool for in vitro toxicology testing, they might be still rather unknown in the community. One study compared gene expression of drug-metabolizing enzymes in 2D and 3D spheroid cultures of upcyte hepatocytes. The results indicate that expression levels of CYP2C19, CYP3A4, MRP2 and OATP-C in spheroids were slightly higher than in 2D cultures, while CYP2C8 and BSEP expression was reduced in 3D cultures ^[58][41]. A second study used spheroids of primary-like hepatocytes generated from proliferation-competent liver progenitor cells. Those spheroids showed elevated ALB and CYP1A2, CYP2D6, CYP3A4, UGT1A1 and MRP2 expression, albumin and urea synthesis as well as glycogen storage. However, these levels did not reach those of pHHs. Gene-expression profiling showed that spheroids of primary-like hepatocytes clustered with pHHs and differentiated HepaRG cells, but not with HepG2 cells, indeed indicating a mature phenotype ^[59][106]. And a third study showed that spheroids of so called ProliHHs were comparable to pHHs regarding albumin expression and secretion, but expression and activity levels of CYP1A2, CYP2B6 and CYP3A4, as well as BSEP and MRP2 expression levels were considerably lower than in pHHs ^[60][48].