Generation of high-density human induced pluripotent stem cell derived-liver organoid by enabling growth factors accumulation in a simple dialysis medium refinement culture platform

doi:10.21203/rs.3.rs-1814052/v1

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Generation of high-density human induced pluripotent stem cell derived-liver organoid by enabling growth factors accumulation in a simple dialysis medium refinement culture platform

https://doi.org/10.21203/rs.3.rs-1814052/v1

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The human pluripotent stem cells-derived liver organoid (HLOs) has recently become a promising alternative source for liver regenerative therapy. To realize this application, a large number of the hiPSCs derived-liver cells are required for partial liver replacement in transplantation. This method requires a stepwise induction by using costly growth factors to direct the hiPSCs into the hepatic lineage. Therefore, we developed a simple dialysis culture system to fully utilize the growth factors accumulation with continuous medium refinement. The results showed that the dialysis culture system was able to accumulate the four essential growth factors required in each differentiation stage, such as Activin A, BMP4, HGF, and OSM. As a result, this culture system allowed a very high-density bipotential hepatic differentiation in approximately 4.5×10⁷ cells/mL of human liver organoids (HLOs), mainly consisting of hiPSCs derived-hepatocytes (HLCs) and cholangiocytes (CLCs). Moreover, the differentiated liver organoids showed a better or comparable hepatic marker and physiological activity with the organoids differentiated in suspension culture with conventional daily medium replacement in a lower cell density. This simple miniaturized dialysis culture system demonstrated the feasibility of cost-effective hepatic differentiation in high density.

hiPSCs

HLOs

high-density hepatic differentiation

growth factor

dialysis culture

Liver is an important organ that plays a role in various metabolic functions and hundreds of other vital functions. A partial- or total liver dysfunction may cause a systemic life-threatening physiological disturbance. Currently, orthotopic liver transplantation is the most effective and preferable therapy to cure liver failure; however, the shortage of liver donors has become a great limitation of their therapeutic modalities. The human liver organoids (HLOs) consist of hepatocyte like-cells (HLCs) and cholangiocyte like-cells (CLCs) which are differentiated from human-induced pluripotent stem cells (hiPSCs) may become a good source for personalized regenerative therapy for liver disease^1–3. In most cases, 2 x 10⁸ liver cells per kg body weight is required to regenerate and restore the physiological function for liver failure or liver cirrhosis are necessary to ensure the therapeutic efficacy⁴. To realize this translational application, a large-scale and cost-effective biomanufacturing process essentially needed to be employed to improve the production of hiPSCs derived-liver cells⁵. Nutrition supply, waste metabolic byproduct removal, and mechanical-related stress becoming the main challenges that must be overcome in the current hiPSCs culture. Additionally, the use of growth factors caused by daily medium removal can lead to high production costs and inefficient differentiation processes^6–8.

The dialysis culture system is one of the potential methods that can be utilized for improving differentiation efficiency. This culture system was firstly utilized to produce the monoclonal antibody from hybridoma cell lines in a high cell density^9,10. Our previous study has shown their potential in the highly efficient production of undifferentiated hiPSCs by the accumulation of important growth factors required for maintaining pluripotency and proliferation; while simultaneously maintaining the medium refinement in a very high cell density^7,11. Since the differentiation process required a larger amount of these expensive factors, the cost reduction may be significantly enhanced by using this dialysis device.

Here, we assessed the feasibility of hepatic differentiation of hiPSCs in a simple dialysis culture system. This system has successfully enabled the accumulation of the main growth factor in each step of differentiation that is necessary to direct the differentiation into a hepatic lineage. Since the high-density culture can increase the hydrodynamic shear stress and aggregates collision, we added a gellan gum-based-biopolymer FP001 to increase the medium viscoelasticity that reduces the stress caused by dynamic suspension culture. The results showed that the hepatic differentiation of hiPSCs in a high density using simple dialysis culture was supported by a growth factors accumulation and low mechanical stress environment. These enhancements of culture conditions not only reduce the cost generated by growth factor usage but also improved the hepatic function of resulted HLOs. Although this study was limited to hepatic differentiation, a similar method can be broadly applied for the differentiation of pluripotent stem cells into various lineages.

Simple dialysis culture system and conventional medium replacement system. To assess the differentiation efficiency, we performed a stepwise hepatic differentiation in three different configurations (Fig. 1a). In this study, we improve the utilization of the important growth factors by selectively accumulating them while maintaining the exchange of low molecular weight nutrition and waste metabolic byproduct, enabling the hepatic differentiation in a high cell density using a simple dialysis culture device (D/HD) (Fig. 1a and 1b). To evaluate the performance of this system, we also include the hepatic differentiation in a standard routine daily complete medium replacement, both using a low density (C/LD) and high density (C/HD) (Fig. 1c).

Protection of hiPSCs aggregates from mechanical stress during hepatic differentiation in a high density. A physical condition, such as excessive mechanical stress may significantly affect the differentiation efficiency into the hepatic lineage. Although a gentle dynamic culture system has been known to improve the hepatic differentiation^12,13, the mechanical stress coming from hydrodynamic mixing and aggregates collision in rotational culture may negatively interfere with the differentiation process⁷. The previous study by Vosough et al was shown that excessive shear stress can reduce the hepatic differentiation efficiency during culture in stirred tank bioreactor¹⁴. Additionally, the high-density culture may increase the risk of aggregates agglomeration, resulting in a large fused aggregate which is prone to form a necrotic core formation^15,16. To address this problem, we include the gellan gum-based-biopolymer FP001 to reduce the mechanical stress and excess agglomeration while enabling efficient high-density culture at the same time^17,18. Based on our previous study⁷, the application of this biopolymer has shown its potential in protecting the high-density hiPSCs aggregates from mechanical stress induced-apoptosis and necrosis, which may negatively affect cell proliferation and cell quality. In this study, we confirmed that the inclusion of this biopolymer has shown its potential to improved the HLCs production (Supplementary Fig. 1a) and hepatic differentiation (Supplementary Fig. 1b-d).

Dense morphology and improved cellular proliferation. The HLOs from D/HD and C/LD represent similar morphological features that consisted of a dense opaque core surrounded by small cystic structures in the periphery area. Meanwhile, the hiPSCs aggregates differentiated in C/HD showing an enlarged cystic structure (Figs. 2a and 2b). The enhancement of cellular proliferation was observed in D/HD compared to C/LD and C/HD groups. The cell was proliferating until the foregut endoderm stage up to day 8 in a very high density (the high-density culture criteria was determined by ≥ 10⁷ cells/ml, according to Griffith, et al.¹⁹) and started to decrease the proliferation afterward (Fig. 2c). The reduction of this proliferation may be correlated with the loss of self-renewal capability during further cell fate specification²⁰. This evidence suggests that the early stage of differentiation is an essential step to producing a large number of cells, facilitated by continuous medium refinement and growth factors accumulation in the dialysis culture system. On the contrary, the cellular proliferation in both C/LD and C/HD was limited by the volume constraint of culture media, resulting in a limited cell number, up to approximately 5–6 x 10⁶ cells/ml (Fig. 2d).

Medium refinement enhanced the culture environment during hepatic differentiation. To evaluate the proper culture environment during the high-density hepatic differentiation, the glucose and lactate concentration in the culture compartment was measured daily. Although the glucose consumption was increased due to the proliferating cells, the dialysis support was able to accommodate the continuous supply of low molecular weight nutrition, such as glucose from the dialysate compartment (Fig. 3a). In the last stage of differentiation, the glucose in C/LD and C/HD showed a lower concentration due to a low glucose formulation in the HBM (hepatocyte basal medium), which is necessary to adapt the HLOs toward a specific way of hepatic metabolism^21,22. The dialysis culture system also shows a decent continuous lactate removal so it can be maintained under the critical lactate concentration that may damage the hiPSCs through lactate acidification which was previously described by Ouyang et al²³. On the contrary, the C/LD group showed an increased lactate accumulation, reaching above its critical concentration during the third stage of differentiation (Fig. 3b). This high lactate concentration may negatively impact proliferation, as well as hepatic differentiation. The condition was even more severe in C/HD which experiences a high lactate environment all time during the hepatic differentiation, resulting in morphological alteration into low proliferated-cystic like-structure. Based on our previous study in dialysis culture, the decreasing proliferation rate is more affected by a high lactate concentration, rather than glucose starvation⁷.

Accumulation of the four essential key factors in hepatic differentiation. One of the main advantages of the dialysis culture system is its capability to selectively accumulate the large molecular weight growth factors that are essentially required in directed differentiation. To represent the growth factors accumulation, we select four representative growth factors that play a central role in hepatic differentiation, such as Activin A, BMP-4, HGF, and OSM. These essential growth factors were successfully accumulated in D/HD, while their remaining concentration was completely replenished during complete medium replacement in C/LD and C/HD. Since the Activin A and BMP-4 are also endogenously secreted by the hiPSCs^24,25, their concentration still can be maintained even better hiPSCs during the definitive- and foregut endodermal differentiation step (Fig. 4a and 4b). HGF and OSM are important factors that direct the hiPSCs differentiation and maturation towards hepatic lineage. Normally, the dose of these two components may not be sufficient to be administrated for high-density differentiation because they were not natively secreted by hepatocytes and cholangiocytes. In addition, the HGF was very sensitive to temperature-induced degradation over time^26,27. Therefore, we investigate the potential component that may improve the stabilization of these growth factors. The results showed that the inclusion of fetal calf serum in the medium formulation significantly improved the stabilization of HGF and OSM in culture medium by protecting them from degradation (Supplementary Fig. 2a). Next, we performed the optimization to obtain the optimum concentration of the HGF and OSM for the high-density HLOs production (Supplementary Fig. 2b). Based on these optimizations, we increase the HGF and OSM concentration in the culture compartment up to 200 ng ml^− 1 and 120 ng ml^− 1, respectively. Then, by using these concentrations, the HGF and OSM can be sufficiently maintained and accumulated in the culture compartment (Figs. 4c and 4d).

Continuous medium refinement and growth factors accumulation supported hepatic differentiation. We further analyzed the comprehensive gene expression profiles of fetal and adult liver-specific markers (Fig. 5). The results indicate that the HLOs generated from the D/HD system showed a relatively higher expression of mature hepatic markers and their nuclear receptors. Their physiological functions related-gene expression such as CYP450 enzymes, hepatic transporters, conjugating enzymes, mitochondrial genes, and urea cycle were improved as well, compared to other culture systems in this study. However, all resulting HLOs from hiPSCs still showed a relatively higher hepatic progenitor marker such as AFP, and lower hepatic marker compared to the primary human liver (PHL), which indicated that the maturation step is still needed to be optimized.

The liver progenitor often exhibited bipotential differentiation towards hepatic lineage and cholangiocyte lineage^28–34. The differentiation tendency of these lineages can be potentially affected by the condition of the culture environment. Interestingly, the high level of cholangiocyte markers in cystic-HLOs generated from C/HD has revealed that exposure to a high lactate culture environment may increase higher differentiation tendency into cholangiocyte lineage. Its tendency was confirmed by the upregulation of cholangiocyte marker gene expression (Fig. 5) as well as the localized expression of KRT19 and KRT7 among ALB positive cells which localized in the cystic-like structure area, mainly in C/HD (Figs. 6a and 6b). In accordance with the other study conducted by Ramli et al.³⁰ and Wu, et al³⁵, this evidence was shown that the forming of this peripheral cystic structure was related to the development of cholangiocyte lineage during hepatic differentiation. In our study, we found that this cystic-like structure was increased and persistently grow during the exposure to a high lactate culture environment.

The dialysis culture produced a better hepatic physiological function. Although the higher albumin secretion was detected in the HLCs produced in the D/HD system (Fig. 7a), it still showed a decent amount of AFP. Its secretion level was comparable to AFP secretion with the C/LD system, and relatively produce less AFP than the C/HD at the end of differentiation (Fig. 7b). These increasing AFP in the last stage of differentiation may be indicating an immature liver development.

To assess the HLCs drug metabolic activity, we select and evaluate three of the CYP450 enzyme family. The HLCs produced in the D/HD system showed comparable CYP3A4 and CYP1A2 activity with the one that was produced in C/LD, while the C/HD showed the lowest activity of these two enzymes. There is no difference in the activity of CYP2B6 among all culture systems (Fig. 7c). Additionally, the resulted HLCs aggregates that differentiated from all the culture groups showed a similar capability of glycogen conversion and storage (Fig. 7d).

Interestingly, the presence of functional bile canaliculi was highly observed in cystic HLOs produced from C/HD. The 5-carboxy-2,7-dichlorofluorescein diacetate (CDFDA) is accumulated in the cystic space of all HLOs, indicating the activity of transporter protein of bile canaliculi which mainly consisted of cholangiocytes (Fig. 7e).

High-cost production cost is the main problem in the translational application of hiPSCs based-regenerative therapy for the liver organ. Most of the differentiation process involved a stepwise induction by the combination of multiple exogenous growth factors, which became the most expensive components of the differentiation medium^33,36. A simple dialysis culture system is a potential method to fully utilize the growth factors accumulation while maintaining a decent exchange of small molecule nutrition and waste metabolic product, supporting proliferation and differentiation which enable a high-density volumetric yield⁷. This strategy can be potentially useful in low-cost production of functional HLOs.

A decent culture environment was achieved in the production of HLOs from hiPSCs in a high-density manner through a continuous supply of micromolecule nutrition such as glucose and waste metabolic products such as lactate. Although the glucose concentration sufficiently remained in all experimental groups, the accumulation of secreted lactate might become a problem by potentially disturbing the proliferation and cellular viability through the decrease of the pH of the culture medium over time. The usage of dialysis culture systems was not only maintaining the proliferation in an early stage but also improved the functional maturation of hepatic lineage. Interestingly, the high exposure of lactate was shown to induce the morphological alteration into a complete cystic-like structure with lineage specification tendency into cholangiocyte. These findings provide important information that the exposure of lactate may affect the differentiation results of three-dimensional hiPSCs aggregates.

Our current study showed a successful accumulation of four essential growth factors during high-density hepatic differentiation in dialysis culture. Activin A and BMP4 are the most important and costly growth factors in definitive endodermal and foregut endoderm differentiation which is required in a relatively high concentration (100ng/ml and 20 ng/ml)³⁷. The inefficient utilization of these growth factors may cause failure in the early lineage specification, resulting in non-endodermal cells which could not be developed into hepatic lineage¹⁴. Besides supplemented exogenously, these growth factors are also endogenously produced by the hiPSCs as a paracrine factor to support their homeostasis, which may reinforce their accumulation in culture compartment. On the contrary, the HGF and OSM that are required for the hepatic specification and maturation were not natively produced by the hiPSCs. These growth factors are produced by different counterparts of the main liver organ. The HGF is produced by mesenchymal cells³⁸ and relatively exhibits a short half-life in vivo^39–41, and OSM is produced by activated T cells and monocytes/macrophages during inflammatory response⁴². Therefore, the increasing amount is necessary to sufficiently maintain their availability and optimal usage by recycling the remaining daily dose of HGF and OSM, which are also preserved from the degradation in FCS supplemented differentiation medium (Supplementary Fig. 2a).

Generally, the growth factor accumulation was able to improve the HLOs differentiation with mixed cells containing hepatocytes and cholangiocytes-like cells, which normally developed from the bipotential hepatic progenitor cells (hepatoblast)^43,44. The HLOs differentiated in dialysis culture also resulted in a higher composition of HLCs, indicated by the higher mature hepatocytes marker and their physiological functions such as albumin secretion, drug metabolism, and glycogen storage, (Fig. 7b, 7c and 7d); while the CLCs may be existed in a smaller amount to provide a functional bile canaliculi ductular structure (Fig. 7e). This condition may correlate with an accumulation of the endogenous and exogenous signals which are known to contribute to promoting HLOs maturation⁴⁵.

Our study on HLO generation showed that the utilization of dialysis culture system potentially contributed to a significant cost saving per unit of produced cells (Supplementary Fig. 3), compared to the routine culture method with low density and manual daily medium replacement. This simple culture platform also can be utilized to optimize various culture conditions to optimize various parameters, such as cell metabolism, endogenous and exogenous growth factor kinetics, and differentiation efficiency with less complexity compared to other types of dialysis culture systems. Although we were successfully improved the HLOs production from the hiPSCs, several problems still becoming a challenge that needs to be addressed in the future to improve their efficacy for translational application. First, our resulted liver organoid still showed a lower expression marker of mature hepatocytes counterparts compared to the primary human liver, which was also indicated by a high expression fetal liver AFP. Although these profiles correspond with most of the hepatic differentiation studies from PSCs⁴⁶, the fully matured organoid is essentially required, mainly for transplantation purposes. Several alternatives, such as a prolonged culture period with a maturation medium may be effective, but the physiological function was often decreased over time during extended in vitro culture. A further fundamental study needs to be performed to elucidate the mechanism behind this phenomenon. Secondly, for liver therapeutic purposes, further scalable production is necessary. For example, to regenerate one-tenth of adult liver mass, approximately 10⁹ cells need to be transplanted into the patients⁴⁷. Although multiple units of this simple dialysis platform may be employed in parallel, a larger dialysis culture system needs to be designed. Several studies showed the possibility to perform the hepatic differentiation PSCs inside a hollow fiber-based-dialysis^48,49, but the complexity and difficulties in harvesting were also becoming a significant technical challenge that needs to be addressed. Therefore, we are currently trying a scaling-up method by a larger volume of culture compartment in a rotational culture with perfused medium into a separate hollow fiber module.

In conclusion, the simple dialysis culture system was able to enhance the HLOs differentiation at a high density by facilitating the essential growth factors accumulation. Our study provides an insight into a feasible minimum growth factors utilization which promotes a significant cost reduction during liver organoid differentiation.

Design of the simple dialysis culture system. The simple dialysis-culture system consisted of the upper culture compartment and lower dialysate compartment. A mesh bottom-cell strainer (PluriSelect, Leipzig, Germany) was modified by cutting and replacing the bottom mesh layer with a 12-kDa MWCO Spectra/Por 4 dialysis membrane (Spectrum Chemical, New Brunswick, NJ, USA). The dialysis membrane was affixed to the bottom side of the strainer using alkyl-α-cyanoacrylate-based surgical-grade tissue adhesive (Aron Alpha A; Daiichi Sankyo, Japan). The upper dialysis culture compartment inserts were then placed in 6-deep well plates (Corning, NY, USA). To represent the control condition, the cell strainer was directly affixed to the bottom surface of six-well plates (Iwaki, Tokyo, Japan) using Aron Alpha A tissue adhesive (Daiichi Sankyo). All devices were sterilized using an ethylene oxide gas sterilizer before use.

Monolayer hiPSCs culture. The TkDN-4M hiPSC line was provided by the Stem Cell Bank, Centre for Stem Cell Biology and Regenerative Medicine, University of Tokyo (Tokyo, Japan)⁵⁵. This cell was maintained in vitronectin-coated tissue-culture dishes using Essential 8 (E8) culture medium (Thermo Fisher Scientific, Waltham, MA, USA).

Hepatic differentiation of hiPSCs in Suspension culture. The hiPSCs were harvested from monolayer culture and dissociated into single-cell suspension by passing through a 40µm strainer (Corning, USA). hiPSC aggregates were formed by inoculation of a 2 × 10⁶ single-cell suspension/well in 6-well plates for 24-h culture in mTeSR1 medium using a 90-rpm rotary shaker. The differentiation protocols were adapted from the previous study³⁷ with several modifications. To generate the HLOs in high density using a dialysis culture, the differentiation components were divided into two groups (Table 1). The low molecular weight components (LMW component) consisted of basal medium with antioxidants and antibiotics. The high molecular weight components (HMW component) consisted of growth factors differentiation cocktails, fetal calf serum, and a gellan gum-based-biopolymer that prevent excess agglomeration and reduced shear stress. On day 0, 2ml of LMW component was placed in culture compartments, and 15 ml of the same component was placed in the dialysate compartment. hiPSCs aggregates were collected from 4 wells of 6 well plates containing a total of 8 × 10⁶ cells collected using a 10 ml tube. After the aggregates were sedimented, the mTeSR1 medium was removed and replaced by 2ml of LMW and HMW components, so the total cell density will be 4 × 10⁶ cells/ml. This cell suspension was then placed on the upper culture compartment. To continuously support the high-density differentiation, 15 ml of the LMW component was placed in the lower dialysate compartment. The dialysis culture was performed under 120 rpm of rotational culture.

The daily medium change was only performed by replacing the LMW component in the dialysate compartment; while a daily dose of growth factors was administrated in the culture compartment, without removing the LMW component. The exception was applied at the end of each differentiation stage, all component was completely replaced with the next differentiation induction components, and the daily medium change can be performed afterward using the same method.

Table 1. The composition of low- and high-molecular-weight components in this study.

Differentiation stage	Low molecular weight component	High molecular weight components
Differentiation stage	Low molecular weight component	Growth factors	Another supplement
Definitive endoderm	º IMDM/F12 (1:1) º 55µM Monothioglycerol º 1% Penicillin-streptomycin	º 5% fetal calf serum (FCS) º 1% Insulin-Transferrin-Selenium (ITS) º 10 ng ml^-1 FGF2 º 100 ng ml^-1 Activin A º 10 ng ml^-1 TGFβ-1	º 2% FP001
Foregut endoderm	º IMDM/F12 (1:1) º 55µM Monothioglycerol º 1% Penicillin-streptomycin	º 5% fetal calf serum (FCS) º 1% Insulin-Transferrin-Selenium (ITS) º 10 ng ml^-1 FGF4 º 20 ng ml^-1 BMP4	º 2% FP001
Pre-hepatic spesification	º IMDM/F12 (1:1) º 55µM Monothioglycerol º 1% Penicillin-streptomycin	º 5% fetal calf serum (FCS) º 1% Insulin-Transferrin-Selenium (ITS) º 1 µg ml^-1 WIF-1 º 0.1 µg ml^-1 DKK-1	º 2% FP001
Hepatic lineage	º HBM º 55µM Monothioglycerol º 1% Penicillin-streptomycin	º 5% fetal calf serum (FCS) º 1% Insulin-Transferrin-Selenium (ITS) º 200 ng ml^-1 HGF º 120 ng ml^-1 OSM	º 2% FP001

Morphological observation. The aggregate groups were moved to 60 mm culture dish (Iwaki, Tokyo, Japan), and the morphological images were obtained by Olympus IX83 light microscope (Olympus, Tokyo, Japan)

Cell counting. The aggregates was isolated by centrifugation at 1000 rpm for 3 min, and the upper supernatant was carefully removed from the tubes. The aggregates were dissociated using 1 mL of Accutase (Thermo Fisher Scientific) and incubated for 10 min to 25 min at 37°C, followed by gentle homogenization using the pipette to obtain the single cells. The cells solution was diluted in Trypan blue (Thermo Fisher Scientific) and counted using a hemocytometer (SLGC, Yokohama, Japan).

Hematoxylin-eosin of cross-sectioned aggregates. Aggregates were fixated using 4% paraformaldehyde (Wako Pure Chemical) overnight followed by incubation with 30% PBS-sucrose solution (Wako Pure Chemical) for 24 h, at 4°C. The sucrose solution was removed, and the aggregates were embedded with Tissue-Tek OCT compound (Sakura, Alphen aan den Rijn, The Netherlands) in cryo mold at −20°C until hardened. 10 µm thin section slices were obtained using a cryostat and mounted onto glass slides for hematoxylin-eosin staining. The sample was observed using Olympus IX83 light microscope (Olympus).

Measurement of glucose and lactate concentrations. Glucose and lactate concentrations during hepatic differentiation were performed by collecting 50-µL of medium samples every 24 hours and measured by OSI BF-48AS Bioanalyzer (Oji Scientific Instrument, Hyogo, Japan).

Protein level measurement by enzyme linked-immunoabsorbent assays (ELISA). For the measurements of growth factors accumulation during differentiation, such as Activin A, BMP4, HGF, and OSM, 100 µL of the culture medium was directly isolated from the culture compartment every 24 hours.

To measure the secretion level of AFP and ALB, approximately 50 aggregates were isolated from the high-density culture, washed with PBS 3 times, and moved into 6 well plates. Aggregates were cultured for 24 hours using the differentiation medium. To measure the secretion level of ALB and AFP, the concentration was normalized with the cell number that dissociated from the aggregates.

The Activin A, BMP4, OSM, AFP, and ALB concentration was measured by Duo Set Elisa Kit (R&D Biosystems, Minnesota, USA), and the HGF was measured by HGF Quantikine ELISA kit (R&D Biosystems, Minnesota, USA), according to a similar manufacturer’s instruction. A 100-µL solution of diluted capture antibody per well of 96-well enzyme-linked immunosorbent assay (ELISA) plates was immobilized in each well and incubated overnight at 37°C. Afterward, the plate was then washed using 200 µL of wash buffer, followed by the incubation with 300 µL of blocking buffer (reagent diluent) for 1 h at 25^oC.

100 µL of standard protein solution or each medium sample was diluted in reagent diluent and plated in each well, followed by incubation for 2 hours at room temperature. The plates were washed using wash buffer and incubated with 100 µL/well of the diluted detection antibody (R&D Biosystems) for 2 h at 25°C. After washing by wash buffer, 100 µL of streptavidin-horseradish peroxidase (HRP) solution (R&D Biosystems) was added and incubated at room temperature for 20-39 min at 25°C. The color reaction was generated by washing the plates with wash buffer and adding 100 µL of a substrate solution to each well. The sample was incubated for 20 min at 25°C in dark places. 50 µL/well stop solution was added to stop the color reaction. Fluorescence intensity measurement was detected using a Wallace Arvo SX 1420 multilabel counter (PerkinElmer).

Quantitative real-time PCR (qRT-PCR). The RNA was isolated using Trizol reagent (Life Technologies) and cDNA was synthesized using ReverTra Ace master mix (Toyobo, Osaka, Japan). Afterward, the qPCR analysis was carried out using Thunderbird SYBR qPCR mix (Toyobo) according to the manufacturer`s instruction. Gene amplification was performed using a StepOnePlus kit qPCR (Thermo Fisher Scientific). The primer sequences used in this analysis are listed in supplementary table 1.

Comprehensive gene expression analysis. The total RNA was isolated using Trizol reagent (Life Technologies) and reverse-transcribed using ReverTra Ace master mix (Toyobo, Osaka, Japan), followed by comprehensive qPCR analysis using Primer Array Hepatic Differentiation Kit (Takara Bio, Shiga, Japan) and KOD qPCR master mix (Toyobo) according to manufacturer`s instructions. Gene amplification was performed using a StepOnePlus kit qPCR (Thermo Fisher Scientific, Massachusetts, USA) and the comprehensive gene expression analysis was analyzed using PrimerArray® Analysis Tool for Hepatic Differentiation (Takara Bio, Japan).

Immunohistochemistry. An approximately 30 HLOs were collected using microtubes and fixed with 4% paraformaldehyde for 12 hours at 4 °C. The aggregates were then permeabilized with 1% Triton X (Thermo Fisher Scientific, Massachusetts, USA) in PBS for 1 hour, followed by additional incubation using gelatin blocking buffer solution containing 1% PBS-Tween (Nacalai Tesque, Kyoto, Japan) for 1 hour at room temperature by laying the tube horizontally in an orbital shaker. The protein targets were detected by incubation with 1 µg/mL primary antibodies for 3-4 days, followed by washing with PBS followed by incubation with 1 µg/mL secondary antibodies for 12-24 hours at 4 °C in a rotary shaker. The aggregates were incubated with 1:1000 nuclear staining 4', 6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan) for 30–40 min before observation. All antibodies used in this study are listed in supplementary table 2. Fluorescence imaging of the protein target was performed using an FV1200 confocal microscope (Olympus, Tokyo, Japan).

CYP450 Assays. Following the manufacturer's instruction, the cytochrome P450 enzyme activity was performed using P450-Glo Assay Kit (Promega, Madison, USA). Approximately 100 aggregates were isolated from each group of culture systems at the end of the hepatic differentiation. For the CYP3A4 assay, the aggregates were incubated by HCM Hepatocyte Culture Medium with BulletKit supplementation containing 20μM Rifampicin solution (Sigma-Aldrich, Missouri, USA) for 48 hours. The CYP1A2 activity was evaluated by incubating the aggregates in HCM Hepatocyte Culture Medium BulletKit containing 50 μM Omeprazole solution (Sigma-Aldrich) for 48 hours. For the CYP2B6 activity assay, the aggregates were incubated with HCM Hepatocyte Culture Medium BulletKit containing 1000μM Phenobarbital solution (Sigma-Aldrich, USA) for 48 hours. The CYP activity value was normalized by the tested cell number. The activity of CYP2B6 using P450-Glo CYP2B6 (Promega, Madison, USA), CYP3A4 using P450-Glo CYP3A4 (Promega, Madison, USA), and the CYP1A2 using P450-Glo CYP1A2 (Promega, Madison, USA).

Periodic acid Schiff staining. The periodic acid Schiff staining was performed for the thin sectioned aggregates by periodic acid Schiff staining (PAS) staining system kit (Sigma-Aldrich, USA) according to the manufacturer`s instruction. The slide was immersed in the periodic acid solution for 5 minutes at room temperature followed by 3 times rinsing with distilled water. Afterward, the slides were incubated with Schiff`s reagent for 15 minutes at room temperature followed by washing in tap water for 5 minutes. The counterstain was performed by 90-second incubation with Hematoxylin solution, Gill no. 3 followed by rinsing in running tap water. Xylene-based mounting solutions were applied to the slides and the slides were observed by the light microscope Olympus IX83 light microscope (Olympus).

Visualization of bile canaliculi formation. An approximately 30 resulted HLOs were used for bile canaliculi visualization. The organoids were incubated in HCM consisting 1 µmol/L CDFDA at 37^OC for 20-30 minutes. Afterward, the HLOs were washed with HCM without CDFDA 2 times and observed using FV1200 laser confocal microscope (Olympus, Tokyo, Japan).

Statistical analysis. Statistical analysis was calculated using GraphPad Prism software (v.9.1.1; GraphPad Software, San Diego, CA, USA). Statistical significances were determined using a one-way analysis of variance followed by Tukey's multiple comparison test. A p < 0.05 was considered significant.

Data availability

The datasets generated and/or analysed during the current study are available in the Open Science Network (OSF) public repository, DOI:10.17605/OSF.IO/JBDNT

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Generation of high-density human induced pluripotent stem cell derived-liver organoid by enabling growth factors accumulation in a simple dialysis medium refinement culture platform

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