Shinya Yamanaka.

Dr. Shinya Yamanaka is recognized for his discovery of the induction of pluripotent stem cells from fibroblasts by a combination of defined factors. In this interview with Cell, he discusses the progress of the field, what's next for clinical applications of iPS cells, and the state of science in Japan and the rest of the world.

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

Limited oxygen in standard cell culture alters metabolism and function of differentiated cells.

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic, but pericellular oxygen levels, which are affected by oxygen diffusivity and consumption, are rarely reported. Here, we provide evidence that several cell types in culture actually experience local hypoxia, with important implications for cell metabolism and function. We focused initially on adipocytes, as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions, cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria, lowered HIF1α activity, and resulted in widespread transcriptional rewiring. Functionally, adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli, recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages, hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types, and that considering pericellular oxygen improves the quality, reproducibility and translatability of culture models.

Generation of complex bone marrow organoids from human induced pluripotent stem cells.

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.

Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designs.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the µm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.

Structurally and mechanically tuned macroporous hydrogels for scalable mesenchymal stem cell-extracellular matrix spheroid production.

Mesenchymal stem cells (MSCs) are essential in regenerative medicine. However, conventional expansion and harvesting methods often fail to maintain the essential extracellular matrix (ECM) components, which are crucial for their functionality and efficacy in therapeutic applications. Here, we introduce a bone marrow-inspired macroporous hydrogel designed for the large-scale production of MSC-ECM spheroids. Through a soft-templating approach leveraging liquid-liquid phase separation, we engineer macroporous hydrogels with customizable features, including pore size, stiffness, bioactive ligand distribution, and enzyme-responsive degradability. These tailored environments are conducive to optimal MSC proliferation and ease of harvesting. We find that soft hydrogels enhance mechanotransduction in MSCs, establishing a standard for hydrogel-based 3D cell culture. Within these hydrogels, MSCs exist as both cohesive spheroids, preserving their innate vitality, and as migrating entities that actively secrete functional ECM proteins. Additionally, we also introduce a gentle, enzymatic harvesting method that breaks down the hydrogels, allowing MSCs and secreted ECM to naturally form MSC-ECM spheroids. These spheroids display heightened stemness and differentiation capacity, mirroring the benefits of a native ECM milieu. Our research underscores the significance of sophisticated materials design in nurturing distinct MSC subpopulations, facilitating the generation of MSC-ECM spheroids with enhanced therapeutic potential.

Origins and functional differences of blood endothelial cells.

The interests in blood endothelial cells arise from their therapeutic potential in vascular repair and regeneration. Our understanding of blood endothelial cells that exist in the circulation has been evolving significantly from the original concept of endothelial progenitor cells. Many studies have uncovered heterogeneities of blood endothelial subtypes where some cells express both endothelial and hematopoietic antigens, and others possess either mature or immature endothelial markers. Due to the lack of definitive cell marker identities, there have been momentums in the field to adopt a technical-oriented labeling system based on the cells' involvement in postnatal neovascularization and cell culture derivatives. Our review streamlines nomenclatures for blood endothelial subtypes and standardizes understanding of their functional differences. Broadly, we will discuss about myeloid angiogenic cells (MACs), endothelial colony-forming cells (ECFCs), blood outgrowth endothelial cells (BOECs) and circulating endothelial cells (CECs). The strategic location of blood endothelial cells confers them essential roles in supporting physiological processes. MACs exert angiogenic effects through paracrine mechanisms, while ECFCs are recruited to sites of vascular injury to participate directly in new vessel formation. BOECs are an in vitro derivative of ECFCs. CECs are shed into the bloodstream from damaged vessels, hence reflective of endothelial dysfunction. With clarity on the functional attributes of blood endothelial subtypes, we present recent advances in their applications in disease modelling, along with serving as biomarkers of vascular tissue homeostasis.

Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle.

ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Thyroid cancer cell metabolism: A glance into cell culture system-based metabolomics approaches.

Thyroid cancer is the most common malignancy of the endocrine system and the seventh most prevalent cancer in women worldwide. It is a complex and diverse disease characterized by heterogeneity, underscoring the importance of understanding the underlying metabolic alterations within tumor cells. Metabolomics technologies offer a powerful toolset to explore and identify endogenous and exogenous biochemical reaction products, providing crucial insights into the intricate metabolic pathways and processes within living cells. Metabolism plays a central role in cell function, making metabolomics a valuable reflection of a cell's phenotype. In the OMICs era, metabolomics analysis of cells brings numerous advantages over existing methods, propelling cell metabolomics as an emerging field with vast potential for investigating metabolic pathways and their perturbation in pathophysiological conditions. This review article aims to look into recent developments in applying metabolomics for characterizing and interpreting the cellular metabolome in thyroid cancer cell lines, exploring their unique metabolic characteristics. Understanding the metabolic alterations in tumor cells can lead to the identification of critical nodes in the metabolic network that could be targeted for therapeutic intervention.

In vitro modeling of skeletal muscle ischemia-reperfusion injury based on sphere differentiation culture from human pluripotent stem cells.

Skeletal muscle ischemia-reperfusion (IR) injury poses significant challenges due to its local and systemic complications. Traditional studies relying on two-dimensional (2D) cell culture or animal models often fall short of faithfully replicating the human in vivo environment, thereby impeding the translational process from animal research to clinical applications. Three-dimensional (3D) constructs, such as skeletal muscle spheroids with enhanced cell-cell interactions from human pluripotent stem cells (hPSCs) offer a promising alternative by partially mimicking human physiological cellular environment in vivo processes. This study aims to establish an innovative in vitro model, human skeletal muscle spheroids based on sphere differentiation from hPSCs, to investigate human skeletal muscle developmental processes and IR mechanisms within a controlled laboratory setting. By eticulously recapitulating embryonic myogenesis through paraxial mesodermal differentiation of neuro-mesodermal progenitors, we successfully established 3D skeletal muscle spheroids that mirror the dynamic colonization observed during human skeletal muscle development. Co-culturing human skeletal muscle spheroids with spinal cord spheroids facilitated the formation of neuromuscular junctions, providing functional relevance to skeletal muscle spheroids. Furthermore, through oxygen-glucose deprivation/re-oxygenation treatment, 3D skeletal muscle spheroids provide insights into the molecular events and pathogenesis of IR injury. The findings presented in this study significantly contribute to our understanding of skeletal muscle development and offer a robust platform for in vitro studies on skeletal muscle IR injury, holding potential applications in drug testing, therapeutic development, and personalized medicine within the realm of skeletal muscle-related pathologies.

Vascular microphysiological systems.

PURPOSE OF REVIEW: This review summarizes innovations in vascular microphysiological systems (MPS) and discusses the themes that have emerged from recent works. RECENT FINDINGS: Vascular MPS are increasing in complexity and ability to replicate tissue. Many labs use vascular MPS to study transport phenomena such as analyzing endothelial barrier function. Beyond vascular permeability, these models are also being used for pharmacological studies, including drug distribution and toxicity modeling. In part, these studies are made possible due to exciting advances in organ-specific models. Inflammatory processes have also been modeled by incorporating immune cells, with the ability to explore both cell migration and function. Finally, as methods for generating vascular MPS flourish, many researchers have turned their attention to incorporating flow to more closely recapitulate in vivo conditions. SUMMARY: These models represent many different types of tissue and disease states. Some devices have relatively simple geometry and few cell types, while others use complex, multicompartmental microfluidics and integrate several cell types and origins. These 3D models enable us to observe model evolution in real time and perform a plethora of functional assays not possible using traditional cell culture methods.

Comparing the BAD Protein Interactomes in 2D and 3D Cell Culture Using Proximity Labeling.

Protein-protein interaction studies using proximity labeling techniques, such as biotin ligase-based BioID, have become integral in understanding cellular processes. Most studies utilize conventional 2D cell culture systems, potentially missing important differences in protein behavior found in 3D tissues. In this study, we investigated the protein-protein interactions of a protein, Bcl-2 Agonist of cell death (BAD), and compared conventional 2D culture conditions to a 3D system, wherein cells were embedded within a 3D extracellular matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase miniTurbo (BirA*), we identified both overlapping and distinct BAD interactomes under 2D and 3D conditions. The known BAD binding proteins 14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D. Of the 131 BAD-interactors identified, 56% were specific to 2D, 14% were specific to 3D, and 30% were common to both conditions. Interaction network analysis demonstrated differential associations between 2D and 3D interactomes, emphasizing the impact of the culture conditions on protein interactions. The 2D-3D overlap interactome encapsulated the apoptotic program, which is a well-known role of BAD. The 3D unique pathways were enriched in ECM signaling, suggestive of hitherto unknown functions for BAD. Thus, exploring protein-protein interactions in 3D provides novel clues into cell behavior. This exciting approach has the potential to bridge the knowledge gap between tractable 2D cell culture and organoid-like 3D systems.

Characterization of ERα Signaling to Cell Proliferation Induced by Chronic and Pulsatile E2 Stimulation in 2D and 3D Cell Cultures.

17ß-estradiol is a hormone that plays a vital role in human physiology. It acts through estrogen receptors, specifically estrogen receptor α and estrogen receptor ß, and its action is determined by the pulsatile secretion in the bloodstream. 17ß-estradiol affects cell proliferation, and dysregulation of 17ß-estradiol:estrogen receptor α signaling contribute to the development of breast cancer. Previous research on 17ß-estradiol:estrogen receptor α signaling has primarily used two-dimensional cell cultures, which do not fully recapitulate the complexity of tumors that exist in a three-dimensional environment and do not consider the pulsatile nature of this hormone. To address these limitations, we studied 17ß-estradiol:estrogen receptor α signaling in cell proliferation using both two-dimensional and three-dimensional breast cancer cell culture models under continuous and pulsatile stimulation conditions. Results revealed that breast cancer cells grown in an alginate-based three-dimensional matrix exhibited similar responsiveness to 17ß-estradiol compared with cells grown in conventional two-dimensional culture plates. 17ß-estradiol induced the expression of proteins containing estrogen response element in the three-dimensional model. The efficacy of the antiestrogen drugs fulvestrant (ICI182,280) and 4OH-tamoxifen was also demonstrated in the three-dimensional model. These results support the use of the three-dimensional culture model for studying tumor response to drugs and provide a more realistic microenvironment for such studies. Furthermore, the study revealed that a brief 5-min exposure to 17ß-estradiol triggered a physiological response comparable with continuous hormone exposure, suggesting that the cellular response to 17ß-estradiol is more important than the continuous presence of the hormone. In conclusion, the study demonstrates that the alginate-based three-dimensional culture model is suitable for studying the effects of 17ß-estradiol and antiestrogen drugs on breast cancer cells, offering a more realistic representation of tumor-microenvironment interactions. The results also highlight the importance of considering the physiological importance of the temporal dynamics in studying 17ß-estradiol signaling and cellular responses.

Simple 3D spheroid cell culture model for studies of prion infection.

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 µm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, ß-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

Full-Combinatorial Concentration Gradient Array with 3D Micro/Nanofluidics for Antibiotic Susceptibility Testing.

Recent advancements in micro/nanofluidics have facilitated on-chip microscopy of cellular responses in a high-throughput and controlled microenvironment with the desired physicochemical properties. Despite its potential benefits to combination drug discovery, generating a complete combinatorial set of concentration gradients for multiple reagents in an array format remains challenging. The main reason is limited layouts of conventional micro/nanofluidic systems based on two-dimensional channel networks. In this paper, we present a device with three-dimensional (3D) interconnection of micro/nanochannels capable of generating a complete combinatorial set of concentration gradients for two reagents. The device was readily fabricated by laminating a pair of multilayered monolithic films containing a Christmas tree-like mixer, a cell culture chamber array, and through-holes, all within each single film. We assessed the reliable generation of a full-combinatorial concentration gradient array and validated it by using numerical analysis. We applied the proposed device to test the antibiotic susceptibility of bacterial cells in a convenient one-step manner. Furthermore, we explored the potential of the device to accommodate the arrayed complete combinatorial set for two or more drugs, while extending the capabilities of our laminated object manufacturing method for realizing 3D micro/nanofluidic systems.

Microarray Platforms Based on 3D Printing.

This paper introduces an innovative method for the fabrication and infusion of microwell arrays based on digital light processing (DLP) 3D printing. A low-cost DLP 3D printer is employed to fabricate microstructures rapidly with a broad dynamic range while maintaining high precision and fidelity. We constructed microwell arrays with varying diameters, from 200 to 2000 µm and multiple aspect ratios, in addition to microchannels with widths ranging from 45 to 1000 µm, proving the potential and flexibility of this fabrication method. The superimposition of parallel microchannels onto the microwell array, facilitated by positive or negative pressure, enabled the transfer of liquid to the microwells. Upon removal of the microchannel chip, a dispensed microdroplet array was obtained. This array can be modulated by adjusting the volume of the microwells and the inflow fluid. The filled microwell array allows chip-to-chip dispensing to the microreactor array through binding and centrifugation, facilitating multistep and multireagent assays. The 3D printing approach also enables the fabrication of intricate cavity designs, such as micropyramid arrays, which can be integrated with parallel microchannels to generate spheroid flowcells. This device demonstrated the ability to generate spheroids and manipulate their environment. We have successfully utilized precise modulation of spheroids size and performed parallel drug dose-response assays to evaluate its effectiveness. Furthermore, we managed to execute dynamic drug combinations based on a compact spheroids array, utilizing two orthogonal parallel microchannels. Our findings suggest that both the combination and temporal sequence of drug administration have a significant impact on therapeutic outcomes.

Toolbox for creating three-dimensional liver models.

Research within the hepato-biliary system and hepatic function is currently experiencing heightened interest, this is due to the high frequency of relapse rates observed in chronic conditions, as well as the imperative for the development of innovative therapeutic strategies to address both inherited and acquired diseases within this domain. The most commonly used sources for studying hepatocytes include primary human hepatocytes, human hepatic cancer cell lines, and hepatic-like cells derived from induced pluripotent stem cells. However, a significant challenge in primary hepatic cell culture is the rapid decline in their phenotypic characteristics, dedifferentiation and short cultivation time. This limitation creates various problems, including the inability to maintain long-term cell cultures, which can lead to failed experiments in drug development and the creation of relevant disease models for researchers' purposes. To address these issues, the creation of a powerful 3D cell model could play a pivotal role as a personalized disease model and help reduce the use of animal models during certain stages of research. Such a cell model could be used for disease modelling, genome editing, and drug discovery purposes. This review provides an overview of the main methods of 3D-culturing liver cells, including a discussion of their characteristics, advantages, and disadvantages.