Figure 3 - available via license: CC BY-NC-ND
Content may be subject to copyright.
Incision and Preparing for Cannulation The mouse fur and skin are cut in a ''U'' shape, the mouse skin is placed and secured near the head with a needle.
Source publication
Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is...
Context in source publication
Citations
... Following harvest, livers were further processed into single cell suspensions as described above. Hepatocytes were isolated from bulk liver cells by adapting a previously described protocol 28 . Briefly, a sample of liver cells from each animal was centrifuged at 50 × g for 5 min at 4°C. ...
The in vivo efficacy of polymeric nanoparticles (NPs) is dependent on their pharmacokinetics, including time in circulation and tissue tropism. Here we explore the structure-function relationships guiding physiological fate of a library of poly(amine-co-ester) (PACE) NPs with different compositions and surface properties. We find that circulation half-life as well as tissue and cell-type tropism is dependent on polymer chemistry, vehicle characteristics, dosing, and strategic co-administration of distribution modifiers, suggesting that physiological fate can be optimized by adjusting these parameters. Our high-throughput quantitative microscopy-based platform to measure the concentration of nanomedicines in the blood combined with detailed biodistribution assessments and pharmacokinetic modeling provides valuable insight into the dynamic in vivo behavior of these polymer NPs. Our results suggest that PACE NPs—and perhaps other NPs—can be designed with tunable properties to achieve desired tissue tropism for the in vivo delivery of nucleic acid therapeutics. These findings can guide the rational design of more effective nucleic acid delivery vehicles for in vivo applications.
... To identify the effect of MDSCs on the proliferation of hepatocytes. The primary hepatocytes were isolated from BALB/c mice, according to the previous protocol [25]. In briefly, the mouse was anaesthetized and the fur were wiped with 75% ethanol before incising the abdomen. ...
Background
The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury.
Methods
We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b⁺Gr-1⁺) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-β were measured by ELISA assay and followed by being blocked with specific antibodies.
Results
The intrahepatic infiltrations of MDSCs with surface marker of CD11b⁺Gr-1⁺ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b⁺Ly6G⁻Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-β. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs).
Conclusions
The dramatically and quickly infiltrated CD11b⁺Gr-1⁺ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.
... Single cells were isolated and digested using a modified liver collagenase perfusion method from the livers of the control and PH mice [24]. We used a 70-mm filter (BD Biosciences, Franklin Lakes, NJ, USA) to perfuse livers. ...
The regenerative capability of the liver is remarkable, but further research is required to understand the role that neutrophils play in this process. In the present study, we reanalyzed single‐cell RNA sequencing data from a mouse partial hepatectomy (PH) model to track the transcriptional changes in hepatocytes and non‐parenchymal cells. Notably, we unraveled the regenerative capacity of hepatocytes at diverse temporal points after PH, unveiling the contributions of three distinct zones in the liver regeneration process. In addition, we observed that the depletion of neutrophils reduced the survival and liver volume after PH, confirming the important role of neutrophils in liver regeneration. CellChat analysis revealed an intricate crosstalk between neutrophils and macrophages promoting liver regeneration and, using weighted gene correlation network analysis, we identified the most significant genetic module associated with liver regeneration. Our study found that hepatocytes in the periportal zone of the liver are more active than in other zones, suggesting that the crosstalk between neutrophils and macrophages might be a potential target for liver regeneration treatment.
... Hepatocytes were further purified (obtained > 90% viability) by percoll gradient as described [45]. Subsequently, the isolated and purified hepatocytes were seeded onto cell culture plates or glass dishes coated with collagen type 1 (5 µg/ cm 2 ) at a density appropriate for the experimental requirements. ...
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-β 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-β 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.
Graphical Abstract
... Primary hepatocytes and NPCs were isolated according to the protocol [33]. Briefly, the mouse liver was perfused in the inferior vena cava above the kidney with 25 ml perfusion buffer and then with 10 ml digestion buffer. ...
Background
Liver ischemia/reperfusion (I/R) injury is usually caused by hepatic inflow occlusion during liver surgery, and is frequently observed during war wounds and trauma. Hepatocyte ferroptosis plays a critical role in liver I/R injury, however, it remains unclear whether this process is controlled or regulated by members of the DEAD/DExH-box helicase (DDX/DHX) family.
Methods
The expression of DDX/DHX family members during liver I/R injury was screened using transcriptome analysis. Hepatocyte-specific Dhx58 knockout mice were constructed, and a partial liver I/R operation was performed. Single-cell RNA sequencing (scRNA-seq) in the liver post I/R suggested enhanced ferroptosis by Dhx58 hep−/− . The mRNAs and proteins associated with DExH-box helicase 58 (DHX58) were screened using RNA immunoprecipitation-sequencing (RIP-seq) and IP-mass spectrometry (IP-MS).
Results
Excessive production of reactive oxygen species (ROS) decreased the expression of the IFN-stimulated gene Dhx58 in hepatocytes and promoted hepatic ferroptosis, while treatment using IFN-α increased DHX58 expression and prevented ferroptosis during liver I/R injury. Mechanistically, DHX58 with RNA-binding activity constitutively associates with the mRNA of glutathione peroxidase 4 (GPX4), a central ferroptosis suppressor, and recruits the m ⁶ A reader YT521-B homology domain containing 2 (YTHDC2) to promote the translation of Gpx4 mRNA in an m ⁶ A-dependent manner, thus enhancing GPX4 protein levels and preventing hepatic ferroptosis.
Conclusions
This study provides mechanistic evidence that IFN-α stimulates DHX58 to promote the translation of m ⁶ A-modified Gpx4 mRNA, suggesting the potential clinical application of IFN-α in the prevention of hepatic ferroptosis during liver I/R injury.
... Primary mouse hepatocytes: primary hepatocytes were isolated from male, 8-10 weeks-old mice (strain C57BL/6JOlaHsd). Isolation and plating of primary mouse hepatocytes was performed as detailed in our published protocol with no modifications [75]. Three hours after plating, media was changed to Williams E media (ThermoFisher Scientific, cat# 12551032). ...
Background & Aims
During fasting, bodily homeostasis is maintained due to hepatic production of glucose (gluconeogenesis) and ketone bodies (ketogenesis). The main hormones governing hepatic fuel production are glucagon and glucocorticoids that initiate transcriptional programs aimed at supporting gluconeogenesis and ketogenesis.
Methods
Using primary mouse hepatocytes as an ex vivo model, we employed transcriptomic analysis (RNA-seq), genome-wide profiling of enhancer dynamics (ChIP-seq), perturbation experiments (inhibitors, shRNA), hepatic glucose production measurements and computational analyses.
Results
We found that in addition to the known metabolic genes transcriptionally induced by glucagon and glucocorticoids, these hormones induce a set of genes encoding transcription factors (TFs) thereby initiating transcriptional cascades. Upon activation by glucocorticoids, the glucocorticoid receptor (GR) induced the genes encoding two TFs: CCAAT/enhancer-binding protein beta (C/EBPβ) and peroxisome proliferator-activated receptor alpha (PPARα). We found that C/EBPβ mainly serves as an amplifier of hormone-induced gene programs in hepatocytes. C/EBPβ augmented gluconeogenic gene expression and hepatic glucose production. Conversely, the GR-PPARα cascade initiated a secondary transcriptional wave of genes supporting ketogenesis. The cascade led to synergistic induction of ketogenic genes which is dependent on protein synthesis. Genome-wide analysis of enhancer dynamics revealed numerous enhancers activated by the GR-PPARα cascade. These enhancers were proximal to ketogenic genes, enriched for the PPARα response element and showed increased PPARα binding.
Conclusion
This study reveals abundant transcriptional cascades occurring during fasting. These cascades serve two separated purposes: the amplification of the primary gluconeogenic transcriptional program and the induction of a secondary gene program aimed at enhancing ketogenesis.
... Primary hepatocytes were isolated from normal diet-fed Flox mice and Nmnat1 KO mice according to a 2-step perfusion method. 26 After isolation, the primary hepatocytes were cultured overnight in a 6-well cell culture plate (4 Â 10 5 /well) and then treated with 100 nM insulin (Sigma, I2767-100 mg) for 30 minutes. For Nmnat1 knockdown, the isolated hepatocytes were seeded at a density of 1 Â 10 5 in 1 mL standard growth medium per well in a 12-well tissue culture plate. ...
... The hepatocyte sediment was gently resuspended in 15 ml DMEM at room temperature. Then cells were then seeded with DMEM plating media containing 22.5 mM glucose, 10% fetal calf serum, 1% penicillin/streptomycin [30]. The extracted primary hepatocytes were captured by microscope under 40 times as shown in the Supplemental Figure 1A, and we used trypan blue dye to detect the cell viability. ...
Metabolic-associated fatty liver disease (MAFLD) is a globally prevalent chronic hepatic disease. Previous studies have indicated that the activation of the signal transducer and activator of transcription3 (STAT3) plays a vital role in MAFLD progression at the very beginning. However, the specific association between STAT3 and abnormal hepatic metabolism remains unclear. In this study, activated inflammation was observed to induce abnormal glucolipid metabolic disorders in the hepatic tissues of high-fat diet (HFD)-fed ApoE-/- mice. Furthermore, we found that the activation of STAT3 induced by HFD might function as a transcriptional factor to suppress the expression of VAV3, which might participate in intracellular glucolipid metabolism and the regulation of glucose transporter 4 (GLUT4) storage vesicle traffic in the development of MAFLD both in vitro and in vivo. We verified that VAV3 deficiency could retard the GLUT4 membrane translocation and impair the glucose homeostasis. Additionally, VAV3 participates in cholesterol metabolism in hepatocytes, eventually resulting in the accumulation of intracellular cholesterol. Moreover, rAAV8-TBG-VAV3 was conducted to restore the expression of VAV3 in HFD-fed ApoE-/- mice. VAV3 overexpression was observed to improve glucose homeostasis as well as attenuate hepatic cholesterol accumulation in vivo. In conclusion, the STAT3/VAV3 signaling pathway might play a significant role in MAFLD by regulating glucose and cholesterol metabolism, and VAV3 might be a potential therapeutic strategy which could consequently ameliorate MAFLD.
... [10][11][12] week old male Cas9 transgenic mice (Cas9-Ready V2.5) were injected in tail vein with 3x10 10 genome copies (GC) of AAV8 containing Cas9 gRNA against Marc1, Marc2, Marc1/2 (one gRNA targeting both genes) or control gRNA (anti red fluorescent protein (RGF)). 2-4 weeks after virus injection, mice were sacrificed by lethal isoflurane inhalation and cervical dislocation, and primary hepatocytes isolated, counted and plated on poly-D-lysine pre-coated plates [49]. Cells were kept in incubator (37˚C, 5% CO 2 ) in full media (Williams E media with 10% full calf serum (FCS) plus 100 IU/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine) to attach to the plate (4h). ...
Recent human genome-wide association studies have identified common missense variants in MARC1 , p.Ala165Thr and p.Met187Lys, associated with lower hepatic fat, reduction in liver enzymes and protection from most causes of cirrhosis. Using an exome-wide association study we recapitulated earlier MARC1 p.Ala165Thr and p.Met187Lys findings in 540,000 individuals from five ancestry groups. We also discovered novel rare putative loss of function variants in MARC1 with a phenotype similar to MARC1 p.Ala165Thr/p.Met187Lys variants. In vitro studies of recombinant human MARC1 protein revealed Ala165Thr substitution causes protein instability and aberrant localization in hepatic cells, suggesting MARC1 inhibition or deletion may lead to hepatoprotection. Following this hypothesis, we generated Marc1 knockout mice and evaluated the effect of Marc1 deletion on liver phenotype. Unexpectedly, our study found that whole-body Marc1 deficiency in mouse is not protective against hepatic triglyceride accumulation, liver inflammation or fibrosis. In attempts to explain the lack of the observed phenotype, we discovered that Marc1 plays only a minor role in mouse liver while its paralogue Marc2 is the main Marc family enzyme in mice. Our findings highlight the major difference in MARC1 physiological function between human and mouse.
... Mechanistically, it is in part dependent on the regu-of MHC-I in small intestine ( Figure 5, A and B). To confirm that these data were consistent in other GVHD target tissues, we isolated primary hepatocytes from naive mice via collagenase perfusion and Percoll gradient separation as previously described, with minor modifications (33). The B6 Albumin-KO hepatocytes showed significantly increased expression of MHC-I by flow cytometry ( Figure 5C) and by Western blot (Supplemental Figure 5B), demonstrating similar effects in both IECs and hepatocytes. ...
Tissue-intrinsic mechanisms that regulate severity of systemic pathogenic immune-mediated diseases, such as acute graft-versus-host disease (GVHD), remain poorly understood. Following allogeneic hematopoietic stem cell transplantation, autophagy, a cellular stress protective response, is induced in host nonhematopoietic cells. To systematically address the role of autophagy in various host nonhematopoietic tissues, both specific classical target organs of acute GVHD (intestines, liver, and skin) and organs conventionally not known to be targets of GVHD (kidneys and heart), we generated mice with organ-specific knockout of autophagy related 5 (ATG5) to specifically and exclusively inhibit autophagy in the specific organs. When compared with wild-type recipients, animals that lacked ATG5 in the gastrointestinal tract or liver showed significantly greater tissue injury and mortality, while autophagy deficiency in the skin, kidneys, or heart did not affect mortality. Treatment with the systemic autophagy inducer sirolimus only partially mitigated GVHD mortality in intestine-specific autophagy-deficient hosts. Deficiency of autophagy increased MHC class I on the target intestinal epithelial cells, resulting in greater susceptibility to damage by alloreactive T cells. Thus, autophagy is a critical cell-intrinsic protective response that promotes tissue tolerance and regulates GVHD severity.
