Spatial Proteomics of Single Hepatocytes with Multiplexed Data-Independent Acquisition (mDIA).

Spatially resolved mass spectrometry-based proteomics at single-cell resolution promises to provide insights into biological heterogeneity. We describe a protocol based on multiplexed data-independent acquisition (mDIA) with dimethyl labeling to enhance proteome depth, accuracy, and throughput while minimizing costs. It enables high-quality proteome analysis of single isolated hepatocytes and utilizes liver zonation for single-cell proteomics benchmarking. This adaptable, modular protocol will promote the use of single-cell proteomics in spatial biology.

Microscopic Analysis of Nuclear Speckles in a Viviparous Reptile.

Nuclear speckles are compartments enriched in splicing factors present in the nucleoplasm of eucaryote cells. Speckles have been studied in mammalian culture and tissue cells, as well as in some non-mammalian vertebrate cells and invertebrate oocytes. In mammals, their morphology is linked to the transcriptional and splicing activities of the cell through a recruitment mechanism. In rats, speckle morphology depends on the hormonal cycle. In the present work, we explore whether a similar situation is also present in non-mammalian cells during the reproductive cycle. We studied the speckled pattern in several tissues of a viviparous reptile, the lizard Sceloporus torquatus, during two different stages of reproduction. We used immunofluorescence staining against splicing factors in hepatocytes and oviduct epithelium cells and fluorescence and confocal microscopy, as well as ultrastructural immunolocalization and EDTA contrast in Transmission Electron Microscopy. The distribution of splicing factors in the nucleoplasm of oviductal cells and hepatocytes coincides with the nuclear-speckled pattern described in mammals. Ultrastructurally, those cell types display Interchromatin Granule Clusters and Perichromatin Fibers. In addition, the morphology of speckles varies in oviduct cells at the two stages of the reproductive cycle analyzed, paralleling the phenomenon observed in the rat. The results show that the morphology of speckles in reptile cells depends upon the reproductive stage as it occurs in mammals.

Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system.

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Efficient hepatocyte differentiation of primary human hepatocyte-derived organoids using three dimensional nanofibers (HYDROX) and their possible application in hepatotoxicity research.

Human liver organoids are in vitro three dimensionally (3D) cultured cells that have a bipotent stem cell phenotype. Translational research of human liver organoids for drug discovery has been limited by the challenge of their low hepatic function compared to primary human hepatocytes (PHHs). Various attempts have been made to develop functional hepatocyte-like cells from human liver organoids. However, none have achieved the same level of hepatic functions as PHHs. We here attempted to culture human liver organoids established from cryopreserved PHHs (PHH-derived organoids), using HYDROX, a chemically defined 3D nanofiber. While the proliferative capacity of PHH-derived organoids was lost by HYDROX-culture, the gene expression levels of drug-metabolizing enzymes were significantly improved. Enzymatic activities of cytochrome P450 3A4 (CYP3A4), CYP2C19, and CYP1A2 in HYDROX-cultured PHH-derived organoids (Org-HYDROX) were comparable to those in PHHs. When treated with hepatotoxic drugs such as troglitazone, amiodarone and acetaminophen, Org-HYDROX showed similar cell viability to PHHs, suggesting that Org-HYDROX could be applied to drug-induced hepatotoxicity tests. Furthermore, Org-HYDROX maintained its functions for up to 35 days and could be applied to chronic drug-induced hepatotoxicity tests using fialuridine. Our findings demonstrated that HYDROX could possibly be a novel biomaterial for differentiating human liver organoids towards hepatocytes applicable to pharmaceutical research.

Hepatocytes differentiate into intestinal epithelial cells through a hybrid epithelial/mesenchymal cell state in culture.

Hepatocytes play important roles in the liver, but in culture, they immediately lose function and dedifferentiate into progenitor-like cells. Although this unique feature is well-known, the dynamics and mechanisms of hepatocyte dedifferentiation and the differentiation potential of dedifferentiated hepatocytes (dediHeps) require further investigation. Here, we employ a culture system specifically established for hepatic progenitor cells to study hepatocyte dedifferentiation. We found that hepatocytes dedifferentiate with a hybrid epithelial/mesenchymal phenotype, which is required for the induction and maintenance of dediHeps, and exhibit Vimentin-dependent propagation, upon inhibition of the Hippo signaling pathway. The dediHeps re-differentiate into mature hepatocytes by forming aggregates, enabling reconstitution of hepatic tissues in vivo. Moreover, dediHeps have an unexpected differentiation potential into intestinal epithelial cells that can form organoids in three-dimensional culture and reconstitute colonic epithelia after transplantation. This remarkable plasticity will be useful in the study and treatment of intestinal metaplasia and related diseases in the liver.

A comprehensive review of advances in hepatocyte microencapsulation: selecting materials and preserving cell viability.

Liver failure represents a critical medical condition with a traditionally grim prognosis, where treatment options have been notably limited. Historically, liver transplantation has stood as the sole definitive cure, yet the stark disparity between the limited availability of liver donations and the high demand for such organs has significantly hampered its feasibility. This discrepancy has necessitated the exploration of hepatocyte transplantation as a temporary, supportive intervention. In light of this, our review delves into the burgeoning field of hepatocyte transplantation, with a focus on the latest advancements in maintaining hepatocyte function, co-microencapsulation techniques, xenogeneic hepatocyte transplantation, and the selection of materials for microencapsulation. Our examination of hepatocyte microencapsulation research highlights that, to date, most studies have been conducted in vitro or using liver failure mouse models, with a notable paucity of experiments on larger mammals. The functionality of microencapsulated hepatocytes is primarily inferred through indirect measures such as urea and albumin production and the rate of ammonia clearance. Furthermore, research on the mechanisms underlying hepatocyte co-microencapsulation remains limited, and the practicality of xenogeneic hepatocyte transplantation requires further validation. The potential of hepatocyte microencapsulation extends beyond the current scope of application, suggesting a promising horizon for liver failure treatment modalities. Innovations in encapsulation materials and techniques aim to enhance cell viability and function, indicating a need for comprehensive studies that bridge the gap between small-scale laboratory success and clinical applicability. Moreover, the integration of bioengineering and regenerative medicine offers novel pathways to refine hepatocyte transplantation, potentially overcoming the challenges of immune rejection and ensuring the long-term functionality of transplanted cells. In conclusion, while hepatocyte microencapsulation and transplantation herald a new era in liver failure therapy, significant strides must be made to translate these experimental approaches into viable clinical solutions. Future research should aim to expand the experimental models to include larger mammals, thereby providing a clearer understanding of the clinical potential of these therapies. Additionally, a deeper exploration into the mechanisms of cell survival and function within microcapsules, alongside the development of innovative encapsulation materials, will be critical in advancing the field and offering new hope to patients with liver failure.

Mapping of mitogen and metabolic sensitivity in organoids defines requirements for human hepatocyte growth.

Mechanisms underlying human hepatocyte growth in development and regeneration are incompletely understood. In vitro, human fetal hepatocytes (FH) can be robustly grown as organoids, while adult primary human hepatocyte (PHH) organoids remain difficult to expand, suggesting different growth requirements between fetal and adult hepatocytes. Here, we characterize hepatocyte organoid outgrowth using temporal transcriptomic and phenotypic approaches. FHs initiate reciprocal transcriptional programs involving increased proliferation and repressed lipid metabolism upon initiation of organoid growth. We exploit these insights to design maturation conditions for FH organoids, resulting in acquisition of mature hepatocyte morphological traits and increased expression of functional markers. During PHH organoid outgrowth in the same culture condition as for FHs, the adult transcriptomes initially mimic the fetal transcriptomic signatures, but PHHs rapidly acquire disbalanced proliferation-lipid metabolism dynamics, resulting in steatosis and halted organoid growth. IL6 supplementation, as emerged from the fetal dataset, and simultaneous activation of the metabolic regulator FXR, prevents steatosis and promotes PHH proliferation, resulting in improved expansion of the derived organoids. Single-cell RNA sequencing analyses reveal preservation of their fetal and adult hepatocyte identities in the respective organoid cultures. Our findings uncover mitogen requirements and metabolic differences determining proliferation of hepatocytes changing from development to adulthood.

Establishment of human induced pluripotent stem cell-derived hepatobiliary organoid with bile duct for pharmaceutical research use.

In vitro models of the human liver are promising alternatives to animal tests for drug development. Currently, primary human hepatocytes (PHHs) are preferred for pharmacokinetic and cytotoxicity tests. However, they are unable to recapitulate the flow of bile in hepatobiliary clearance owing to the lack of bile ducts, leading to the limitation of bile analysis. To address the issue, a liver organoid culture system that has a functional bile duct network is desired. In this study, we aimed to generate human iPSC-derived hepatobiliary organoids (hHBOs) consisting of hepatocytes and bile ducts. The two-step differentiation process under 2D and semi-3D culture conditions promoted the maturation of hHBOs on culture plates, in which hepatocyte clusters were covered with monolayered biliary tubes. We demonstrated that the hHBOs reproduced the flow of bile containing a fluorescent bile acid analog or medicinal drugs from hepatocytes into bile ducts via bile canaliculi. Furthermore, the hHBOs exhibited pathophysiological responses to troglitazone, such as cholestasis and cytotoxicity. Because the hHBOs can recapitulate the function of bile ducts in hepatobiliary clearance, they are suitable as a liver disease model and would be a novel in vitro platform system for pharmaceutical research use.

Acquisition of epithelial plasticity in human chronic liver disease.

For many adult human organs, tissue regeneration during chronic disease remains a controversial subject. Regenerative processes are easily observed in animal models, and their underlying mechanisms are becoming well characterized1-4, but technical challenges and ethical aspects are limiting the validation of these results in humans. We decided to address this difficulty with respect to the liver. This organ displays the remarkable ability to regenerate after acute injury, although liver regeneration in the context of recurring injury remains to be fully demonstrated. Here we performed single-nucleus RNA sequencing (snRNA-seq) on 47 liver biopsies from patients with different stages of metabolic dysfunction-associated steatotic liver disease to establish a cellular map of the liver during disease progression. We then combined these single-cell-level data with advanced 3D imaging to reveal profound changes in the liver architecture. Hepatocytes lose their zonation and considerable reorganization of the biliary tree takes place. More importantly, our study uncovers transdifferentiation events that occur between hepatocytes and cholangiocytes without the presence of adult stem cells or developmental progenitor activation. Detailed analyses and functional validations using cholangiocyte organoids confirm the importance of the PI3K-AKT-mTOR pathway in this process, thereby connecting this acquisition of plasticity to insulin signalling. Together, our data indicate that chronic injury creates an environment that induces cellular plasticity in human organs, and understanding the underlying mechanisms of this process could open new therapeutic avenues in the management of chronic diseases.

Generation and characterization of mature hepatocyte organoids for liver metabolic studies.

Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.

Functional mouse hepatocytes derived from interspecies chimeric livers effectively mitigate chronic liver fibrosis.

Liver disease is a major global health challenge. There is a shortage of liver donors worldwide, and hepatocyte transplantation (HT) may be an effective treatment to overcome this problem. However, the present approaches for generation of hepatocytes are associated with challenges, and interspecies chimera-derived hepatocytes produced by interspecies blastocyst complementation (IBC) may be promising donor hepatocytes because of their more comprehensive hepatic functions. In this study, we isolated mouse hepatocytes from mouse-rat chimeric livers using IBC and found that interspecies chimera-derived hepatocytes exhibited mature hepatic functions in terms of lipid accumulation, glycogen storage, and urea synthesis. Meanwhile, they were more similar to endogenous hepatocytes than hepatocytes derived in vitro. Interspecies chimera-derived hepatocytes could relieve chronic liver fibrosis and reside in the injured liver after transplantation. Our results suggest that interspecies chimera-derived hepatocytes are a potentially reliable source of hepatocytes and can be applied as a therapeutic approach for HT.

Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

A Novel Approach to Orthotopic Hepatocyte Transplantation Engineered With Liver Hydrogel for Fibrotic Livers, Enhancing Cell-Cell Interaction and Angiogenesis.

Hepatocyte transplantation (HCT) is a potential bridging therapy or an alternative to liver transplantation. Conventionally, single-cell hepatocytes are injected via the portal vein. This strategy, however, has yet to overcome poor cell engraftment and function. Therefore, we developed an orthotopic HCT method using a liver-derived extracellular matrix (L-ECM) gel. PXB cells (flesh mature human hepatocytes) were dispersed into the hydrogel solution in vitro, and the gel solution was immediately gelated in 37°C incubators to investigate the affinity between mature human hepatocyte and the L-ECM gel. During the 3-day cultivation in hepatocyte medium, PXB cells formed cell aggregates via cell-cell interactions. Quantitative analysis revealed human albumin production in culture supernatants. For the in vivo assay, PXB cells were encapsulated in the L-ECM gel and transplanted between the liver lobes of normal rats. Pathologically, the L-ECM gel was localized at the transplant site and retained PXB cells. Cell survival and hepatic function marker expression were verified in another rat model wherein thioacetamide was administered to induce liver fibrosis. Moreover, cell-cell interactions and angiogenesis were enhanced in the L-ECM gel compared with that in the collagen gel. Our results indicate that L-ECM gels can help engraft transplanted hepatocytes and express hepatic function as a scaffold for cell transplantation.

Delineating the heterogeneity of senescence-induced-functional alterations in hepatocytes.

BACKGROUND AND AIM: Cellular senescence of hepatocytes involves permanent cell cycle arrest, disrupted cellular bioenergetics, resistance to cell death, and the release of pro-inflammatory cytokines. This 'zombie-like' state perpetuates harmful effects on tissues and holds potential implications for liver disease progression. Remarkably, senescence exhibits heterogeneity, stemming from two crucial factors: the inducing stressor and the cell type. As such, our present study endeavors to characterize stressor-specific changes in senescence phenotype, its related molecular patterns, and cellular bioenergetics in primary mouse hepatocytes (PMH) and hepatocyte-derived liver organoids (HepOrgs). METHODS: PMH, isolated by collagenase-perfused mouse liver (C57B6/J; 18-23 weeks), were cultured overnight in William's E-medium supplemented with 2% FBS, L-glutamine, and hepatocyte growth supplements. HepOrgs were developed by culturing cells in a 3D matrix for two weeks. The senescence was induced by DNA damage (doxorubicin, cisplatin, and etoposide), oxidative stress (H2O2, and ethanol), and telomere inhibition (BIBR-1532), p53 activation (nutlin-3a), DNA methyl transferase inhibition (5-azacitidine), and metabolism inhibitors (galactosamine and hydroxyurea). SA-ß galactosidase activity, immunofluorescence, immunoblotting, and senescence-associated secretory phenotype (SASP), and cellular bioenergetics were used to assess the senescence phenotype. RESULTS: Each senescence inducer triggers a unique combination of senescence markers in hepatocytes. All senescence inducers, except hydroxyurea and ethanol, increased SA-ß galactosidase activity, the most commonly used marker for cellular senescence. Among the SASP factors, CCL2 and IL-10 were consistently upregulated, while Plasminogen activator inhibitor-1 exhibited global downregulation across all modes of senescence. Notably, DNA damage response was activated by DNA damage inducers. Cell cycle markers were most significantly reduced by doxorubicin, cisplatin, and galactosamine. Additionally, DNA damage-induced senescence shifted cellular bioenergetics capacity from glycolysis to oxidative phosphorylation. In HepOrgs exposed to senescence inducers, there was a notable increase in γH2A.X, p53, and p21 levels. Interestingly, while showing a similar trend, SASP gene expression in HepOrgs was significantly higher compared to PMH, demonstrating a several-fold increase. CONCLUSION: In our study, we demonstrated that each senescence inducer activates a unique combination of senescence markers in PMH. Doxorubicin demonstrated the highest efficacy in inducing senescence, followed by cisplatin and H2O2, with no impact on apoptosis. Each inducer prompted DNA damage response and mitochondrial dysfunction, independent of MAPK/AKT.

A spatiotemporal atlas of mouse liver homeostasis and regeneration.

The mechanism by which mammalian liver cell responses are coordinated during tissue homeostasis and perturbation is poorly understood, representing a major obstacle in our understanding of many diseases. This knowledge gap is caused by the difficulty involved with studying multiple cell types in different states and locations, particularly when these are transient. We have combined Stereo-seq (spatiotemporal enhanced resolution omics-sequencing) with single-cell transcriptomic profiling of 473,290 cells to generate a high-definition spatiotemporal atlas of mouse liver homeostasis and regeneration at the whole-lobe scale. Our integrative study dissects in detail the molecular gradients controlling liver cell function, systematically defining how gene networks are dynamically modulated through intercellular communication to promote regeneration. Among other important regulators, we identified the transcriptional cofactor TBL1XR1 as a rheostat linking inflammation to Wnt/ß-catenin signaling for facilitating hepatocyte proliferation. Our data and analytical pipelines lay the foundation for future high-definition tissue-scale atlases of organ physiology and malfunction.

Intravital observation of high-scattering and dense-labeling hepatic tissues using multi-photon fluorescence microscopy.

Achieving high-resolution and large-depth microscopic imaging in vivo under conditions characterized by high-scattering and dense-labeling, as commonly encountered in the liver, poses a formidable challenge. Here, through the optimization of multi-photon fluorescence excitation window, tailored to the unique optical properties of the liver, intravital microscopic imaging of hepatocytes and hepatic blood vessels with high spatial resolution was attained. It's worth noting that resolution degradation caused by tissue scattering of excitation light was mitigated by accounting for moderate tissue self-absorption. Leveraging high-quality multi-photon fluorescence microscopy, we discerned structural and functional alterations in hepatocytes during drug-induced acute liver failure. Furthermore, a reduction in indocyanine green metabolism rates associated with acute liver failure was observed using NIR-II fluorescence macroscopic imaging.

Biomimetic hepatic lobules from three-dimensional imprinted cell sheets.

Liver-tissue engineering has proven valuable in treating liver diseases, but the construction of liver tissues with high fidelity remains challenging. Here, we present a novel three-dimensional (3D)-imprinted cell-sheet strategy for the synchronous construction of biomimetic hepatic microtissues with high accuracy in terms of cell type, density, and distribution. To achieve this, the specific composition of hepatic cells in a normal human liver was determined using a spatial proteogenomics dataset. The data and biomimetic hepatic micro-tissues with hexagonal hollow cross-sections indicate that cell information was successfully generated using a homemade 3D-imprinted device for layer-by-layer imprinting and assembling the hepatic cell sheets. By infiltrating vascular endothelial cells into the hollow section of the assembly, biomimetic hepatic microtissues with vascularized channels for nutrient diffusion and drug perfusion can be obtained. We demonstrate that the resultant vascularized biomimetic hepatic micro-tissues can not only be integrated into a microfluidic drug-screening liver-on-a-chip but also assembled into an enlarged physiological structure to promote liver regeneration. We believe that our 3D-imprinted cell sheets strategy will open new avenues for biomimetic microtissue construction.

3D Printing of a Vascularized Mini-Liver Based on the Size-Dependent Functional Enhancements of Cell Spheroids for Rescue of Liver Failure.

The emerging stem cell-derived hepatocyte-like cells (HLCs) are the alternative cell sources of hepatocytes for treatment of highly lethal acute liver failure (ALF). However, the hostile local environment and the immature cell differentiation may compromise their therapeutic efficacy. To this end, human adipose-derived mesenchymal stromal/stem cells (hASCs) are engineered into different-sized multicellular spheroids and co-cultured with 3D coaxially and hexagonally patterned human umbilical vein endothelial cells (HUVECs) in a liver lobule-like manner to enhance their hepatic differentiation efficiency. It is found that small-sized hASC spheroids, with a diameter of ≈50 µm, show superior pro-angiogenic effects and hepatic differentiation compared to the other counterparts. The size-dependent functional enhancements are mediated by the Wnt signaling pathway. Meanwhile, co-culture of hASCs with HUVECs, at a HUVECs/hASCs seeding density ratio of 2:1, distinctly promotes hepatic differentiation and vascularization both in vitro and in vivo, especially when endothelial cells are patterned into hollow hexagons. After subcutaneous implantation, the mini-liver, consisting of HLC spheroids and 3D-printed interconnected vasculatures, can effectively improve liver regeneration in two ALF animal models through amelioration of local oxidative stress and inflammation, reduction of liver necrosis, as well as increase of cell proliferation, thereby showing great promise for clinical translation.

Self-Healing Hydrogel Containing Decellularized Liver Matrix and Endothelial Cell-Covered Hepatocyte Spheroids for Rescue of Injured Hepatocytes.

Liver fibrosis occurs in many chronic liver diseases, while severe fibrosis can lead to liver failure. A chitosan-phenol based self-healing hydrogel (CP) integrated with decellularized liver matrix (DLM) is proposed in this study as a 3D gel matrix to carry hepatocytes for possible therapy of liver fibrosis. To mimic the physiological liver microenvironment, DLM is extracted from pigs and mixed with CP hydrogel to generate DLM-CP self-healing hydrogel. Hepatocyte spheroids coated with endothelial cells (ECs) are fabricated using a customized method and embedded in the hydrogel. Hepatocytes injured by exposure to CCl4-containing medium are used as the in vitro toxin-mediated liver fibrosis model, where the EC-covered hepatocyte spheroids embedded in the hydrogel are co-cultured with the injured hepatocytes. The urea synthesis of the injured hepatocytes reaches 91% of the normal level after 7 days of co-culture, indicating that the hepatic function of injured hepatocytes is rescued by the hybrid spheroid-laden DLM-CP hydrogel. Moreover, the relative lactate dehydrogenase activity of the injured hepatocytes is decreased 49% by the hybrid spheroid-laden DLM-CP hydrogel after 7 days of co-culture, suggesting reduced damage in the injured hepatocytes. The combination of hepatocyte/EC hybrid spheroids and DLM-CP hydrogel presents a promising therapeutic strategy for hepatic fibrosis.