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Conditions for maintenance of hepatocyte differentiation and function in 3D cultures

ABSTRACT: Summary Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different conditions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymal transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function - a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research. Graphical abstract Highlights • 3D spheroid cultures were established in 384-well format• Eight different media variants were used to optimize the 3D cultures• Optimized William's medium was as good as expensive commercial medium• The 3D cultures were used to study drug metabolism and toxicity Biological sciences; Proteomics; Methodology in biological sciences

SUBMITTER: Handin N

PROVIDER: S-EPMC8551077 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Conditions for maintenance of hepatocyte differentiation and function in 3D cultures

Project description:3D spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. However, the 3D spheroids are maintained under different conditions, with possible cofounding results. Here we performed an in-depth analysis of how various culture conditions influence 3D spheroids. Our aim was to find optimal conditions for the maintenance of a normal PHH phenotype.

2022-02-17 | PXD024632 | Pride

Comparison of commercially available media for hepatic differentiation and hepatocyte maintenance.

Project description:Human hepatocytes are essential materials in pharmaceutical researches. Not only primary human hepatocytes (PHH) but also human iPS cell-derived hepatocyte-like cells (human iPS-HLCs) are expected to be applied as materials for pharmaceutical researches. To date, several culture media have been developed for culturing human hepatocytes. However, there have been no reports comparing these media to determine which is most suitable for culturing human hepatocytes. In this study, we compared five commercial media (Hepatocyte Culture Medium (HCM), HepatoZYME-SFM, Cellartis Power Primary HEP Medium, DMEM/F12, and William's E Medium (WEM)) to determine which is most suitable for culturing PHH and human iPS-HLCs. In hepatic differentiation of human iPS cells (day 14-25 of differentiation), albumin (ALB) and urea secretion abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM or WEM. During maintenance of human iPS-HLCs, ALB and urea producing abilities and CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM. Importantly, we found that human iPS-HLCs cultured in HCM were maintained for 3 weeks or more without impairment of their hepatic functions. These results suggest that it is necessary to select an optimal medium for hepatic differentiation and maintenance of human iPS-HLCs. In the case of PHH culture, there was little difference in hepatic functions among the five media. However, the CYP2C9, CYP2C19, and CYP3A4 activities were the highest when using HCM and WEM. In conclusion, it is important to select the optimal medium for specific application when carrying out pharmaceutical researches using human hepatocytes.

| S-EPMC7046223 | biostudies-literature

Splicing factor SRSF3 is crucial for hepatocyte differentiation and metabolic function.

Project description:SR family RNA binding proteins regulate splicing of nascent RNAs in vitro but their physiological role in vivo is largely unexplored, as genetic deletion of many SR protein genes results in embryonic lethality. Here we show that SRSF3HKO mice carrying a hepatocyte-specific deletion of Srsf3 (homologous to human SRSF3/SRp20) have a disrupted hepatic architecture and show pre- and postnatal growth retardation. SRSF3HKO mice exhibit impaired hepatocyte maturation with alterations in glucose and lipid homeostasis characterized by reduced glycogen storage, fasting hypoglycemia, increased insulin sensitivity and reduced cholesterol synthesis. We identify various splicing alterations in the SRSF3HKO liver that explain the in vivo phenotype. In particular, loss of SRSF3 causes aberrant splicing of Hnf1?, Ern1, Hmgcs1, Dhcr7 and Scap genes, which are critical regulators of glucose and lipid metabolism. Our study provides the first evidence for a SRSF3-driven genetic programme required for morphological and functional differentiation of hepatocytes that may have relevance for human liver disease and metabolic dysregulation.

| S-EPMC4314219 | biostudies-literature

Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures.

Project description:Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require time-consuming culture (?28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

| S-EPMC4998578 | biostudies-literature

The assembly of integrated rat intestinal-hepatocyte cultures.

Project description:The jejunum is the segment of the small intestine responsible for several metabolism and biotransformation functions. In this report, we have cultured rat jejunum explants in vitro and integrated them with hepatocyte cultures. We have also investigated the changes in jejunum function at different locations since spatial variations in intestinal functions have been reported previously. We divided the length of the rat jejunum into three distinct regions of approximately 9 cm each. We defined the regions as proximal (adjacent to the duodenum), medial, and distal (adjacent to the ileum). Spatiotemporal variations in functions were observed between these regions within the jejunum. Alkaline phosphatase activity (a marker of enterocyte function), decreased twofold between the proximal and distal regions at 4 hr. Lysozyme activity (a marker of Paneth cell function) increased from the proximal to the distal jejunum by 40% at 24 hr. Mucin-covered areas, a marker of goblet cell function, increased by twofold between the proximal and distal segments of the jejunum at 24 hr. When hepatocytes were integrated with proximal jejunum explants, statistically higher urea (~2.4-fold) and mucin (57%) production were observed in the jejunum explants. The integrated intestine-liver cultures can be used as a platform for future investigations.

| S-EPMC6971435 | biostudies-literature

Long-term expansion and differentiation of adult murine epidermal stem cells in 3D organoid cultures.

Project description:Mammalian epidermal stem cells maintain homeostasis of the skin epidermis and contribute to its regeneration throughout adult life. While 2D mouse epidermal stem cell cultures have been established decades ago, a long-term, feeder cell- and serum-free culture system recapitulating murine epidermal architecture has not been available. Here we describe an epidermal organoid culture system that allows long-term, genetically stable expansion of adult epidermal stem cells. Our epidermal expansion media combines atypically high calcium concentrations, activation of cAMP, FGF, and R-spondin signaling with inhibition of bone morphogenetic protein (BMP) signaling. Organoids are established robustly from adult mouse skin and expand over at least 6 mo, while maintaining the basal-apical organization of the mouse interfollicular epidermis. The system represents a powerful tool to study epidermal homeostasis and disease in vitro.

| S-EPMC6642409 | biostudies-literature

3D culture conditions support Kaposi's sarcoma herpesvirus (KSHV) maintenance and viral spread in endothelial cells.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumorigenic virus and the etiological agent of an endothelial tumor (Kaposi's sarcoma) and two B cell proliferative diseases (primary effusion lymphoma and multicentric Castleman's disease). While in patients with late stage of Kaposi's sarcoma the majority of spindle cells are KSHV-infected, viral copies are rapidly lost in vitro, both upon culture of tumor-derived cells or from newly infected endothelial cells. We addressed this discrepancy by investigating a KSHV-infected endothelial cell line in various culture conditions and in tumors of xenografted mice. We show that, in contrast to two-dimensional endothelial cell cultures, KSHV genomes are maintained under 3D cell culture conditions and in vivo. Additionally, an increased rate of newly infected cells was detected in 3D cell culture. Furthermore, we show that the PI3K/Akt/mTOR and ATM/γH2AX pathways are modulated and support an improved KSHV persistence in 3D cell culture. These mechanisms may contribute to the persistence of KSHV in tumor tissue in vivo and provide a novel target for KS specific therapeutic interventions. KEY MESSAGES: In vivo maintenance of episomal KSHV can be mimicked in 3D spheroid cultures 3D maintenance of KSHV is associated with an increased de novo infection frequency PI3K/Akt/mTOR and ATM/ γH2AX pathways contribute to viral maintenance.

| S-EPMC7900040 | biostudies-literature

Hepatocyte Differentiation Metabolism

Project description:In the first group of samples, we are looking into the different metabolites and their concentrations within our human embryonic stem cells over the course of differentiation. Our goal is to study how the metabolism of stem cells are changing as they undergo this conversion to become hepatocyte-like cells. In the second group of samples labeled HyCell, we would like to understand the concentrations of metabolites within our CHO cells in order to update the parameters for our metabolic model. Included are two samples from different days of a suspension batch culture which may have different lactate production qualities.

2017-08-02 | ST000837 | MetabolomicsWorkbench

Discovering networks of perturbed biological processes in hepatocyte cultures.

Project description:The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs) and collagen sandwiches (CS). Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs). CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.

| S-EPMC3016309 | biostudies-literature

Direct Differentiation of Human Embryonic Stem Cells to 3D Functional Hepatocyte-like Cells in Alginate Microencapsulation Sphere.

Project description:BackgroundThe lack of a stable source of hepatocytes is one of major limitations in hepatocyte transplantation and clinical applications of a bioartificial liver. Human embryonic stem cells (hESCs) with a high degree of self-renewal and totipotency are a potentially limitless source of a variety of cell lineages, including hepatocytes. Many techniques have been developed for effective differentiation of hESCs into functional hepatocyte-like cells. However, the application of hESC-derived hepatocyte-like cells (hESC-Heps) in the clinic has been constrained by the low yield of fully differentiated cells, small-scale culture, difficulties in harvesting, and immunologic graft rejection. To resolve these shortcomings, we developed a novel 3D differentiation system involving alginate-microencapsulated spheres to improve current hepatic differentiation, providing ready-to-use hESC-Heps.MethodsIn this study, we used alginate microencapsulation technology to differentiate human embryonic stem cells into hepatocyte-like cells (hESC-Heps). Hepatic markers of hESC-Heps were examined by qPCR and Western blotting, and hepatic functions of hESC-Heps were evaluated by indocyanine-green uptake and release, and ammonia removal.ResultsThe maturity and hepatic functions of the hESC-Heps derived from this 3D system were better than those derived from 2D culture. Hepatocyte-enriched genes, such as HNF4α, AFP, and ALB, were expressed at higher levels in 3D hESC-Heps than in 2D hESC-Heps. 3D hESC-Heps could metabolize indocyanine green and had better capacity to scavenge ammonia. In addition, the 3D sodium alginate hydrogel microspheres could block viral entry into the microspheres, and thus protect hESC-Heps in 3D microspheres from viral infection.ConclusionWe developed a novel 3D differentiation system for differentiating hESCs into hepatocyte-like cells by using alginate microcapsules.

| S-EPMC9562699 | biostudies-literature

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