Journal: Biomaterials Research

Article Title: Genetically Modified Hepatocytes Targeting Bilirubin and Ammonia Metabolism for the Construction of Bioartificial Liver System

doi: 10.34133/bmr.0043

Figure Lengend Snippet: Generation of SynHeps cells. (A) Schematic illustration of bilirubin and ammonia detoxification pathway. SynHeps-I cell line was generated by overexpression of UGT1A1 and OATP1B1 in HepG2 cell line, aiming to detoxify bilirubin. SynHeps-II cell line was generated by overexpression of CPS1, OTC, and ARG1 in SynHeps-I cell, aiming to detoxify bilirubin and ammonia. (B) Western blot analysis on the expression of UGT1A1, OATP1B1, OTC, ARG1, and CPS1 in HepG2, SynHeps-I, and SynHeps-II cells. Cell lysates were separated by 10% SDS-PAGE under reducing conditions, with 50 μg of total protein per lane. (C) Quantification of band intensity from (B). n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant; statistical significance was analyzed using unpaired Student’s t test. β-Actin was used for normalization. (D) Representative pathways by KEGG analysis that were enriched in SynHeps-I and SynHeps-II cells. HepG2 cells were used as comparison cell. (E) Venn diagram showing the number of differentially expressed genes in HepG2, SynHeps-I, and SynHeps-II. (F) Removal of unconjugated bilirubin from 80% patient plasma (PP) by different amount of HepG2 and SynHeps-I cells. Different numbers of HepG2 and SynHeps-I cells were attached to the surface of T-25 cell culture flask for 12 h, and the medium was replaced with plasma [DMEM supplemented with 80% patient plasma (PP)] from ALF patients. After 6 and 12 h of incubation, the concentration of unconjugated bilirubin (IBIL) in the plasma was measured. n = 3; * P < 0.05, **** P < 0.0001; statistical significance was analyzed using one-way ANOVA. (G) Comparison of different cell lines for their ability to remove bilirubin from patient plasma. The ratio of cells (in millions) to plasma (in milliliters) was fixed at 2:1. Plasma from 10 ALF patients was processed in the same way as in (F). * P < 0.05, **** P < 0.0001; statistical significance was analyzed using one-way ANOVA. (H) Comparison of different cell lines for their ability to remove ammonia from patient plasma. Experimental settings were the same as in (F). (I) Oxygen and GLC consumption data for different cell lines. n = 3; * P < 0.05, ** P < 0.01; statistical significance was analyzed using unpaired Student’s t test.

Article Snippet: The membranes were subsequently probed with rabbit monoclonal antibodies against β-actin, UGT1A1, OATP1B1, OTC, ARG1, and CPS1 from Proteintech (Wuhan, China) and P21, Cyclin D1, HGF, NF-κB p65, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), phospho-NF-κB p65, phospho-ERK1/2, and phospho-STAT3 from ABclonal (Wuhan, China) at 4 °C overnight.

Techniques: Generated, Over Expression, Western Blot, Expressing, SDS Page, Comparison, Clinical Proteomics, Cell Culture, Incubation, Concentration Assay

Journal: Biomaterials Research

Article Title: Genetically Modified Hepatocytes Targeting Bilirubin and Ammonia Metabolism for the Construction of Bioartificial Liver System

doi: 10.34133/bmr.0043

Figure Lengend Snippet: Proliferation properties of SynHeps cells. (A) Doubling time of HepG2, SynHeps-I, and SynHeps-II cells in DMEM growth medium or DMEM supplemented with 80% patient plasma (PP). The cell doubling time (DT) was calculated as DT = Δ t *[lg2/(lgNt − lgNo)], where Δ t is the incubation time in hours, No is the number of cells counted for the first time after cell inoculation, and Nt is the cell number after Δ t . Cell number was counted with automated cell counter Countess 3 (Thermo Fisher, Carlsbad, CA). Each time point was counted 5 times and then averaged, and the experiment was repeated 3 times. * P < 0.05; statistical significance was analyzed using unpaired Student’s t test. (B) Proliferation rates of HepG2, SynHeps-I, and SynHeps-II cells in DMEM growth medium and DMEM supplemented with 80% patient plasma. Cell proliferation was measured using the CCK-8 kit following the manufacture’s protocol, and the experiment was repeated 3 times. * P < 0.05, ** P < 0.01, *** P < 0.001; statistical significance was analyzed using unpaired Student’s t test. (C) Proliferation of HepG2, SynHeps-I, and SynHeps-II cells in DMEM supplemented with increasing concentrations of bilirubin (left) or NH 4 Cl (middle) or in a mixture of 80% patient plasma (PP) supplemented with 100 μM bilirubin and increasing concentrations of NH 4 Cl (right). Cell proliferation was measured with a CCK-8 kit and expressed as A450 values determined by multimode microplate reader. Statistical analyses were based on comparisons between SynHeps-II and HepG2 at the same time period and processing conditions. n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001; statistical significance was analyzed using unpaired Student’s t test. (D) Western blot analysis on the expression of UGT1A1, OATP1B1, OTC, ARG1, and CPS1 in HepG2, SynHeps-I, and SynHeps-II cells. Cells were grown in DMEM or DMEM supplemented with 80% PP for 24 h prior to the assay ( n = 3). (E) Quantification of band intensity in (D). β-Actin was used for normalization. * P < 0.05, ** P < 0.01, *** P < 0.001; statistical significance was analyzed using unpaired Student’s t test. (F) Expression of Bax and Bcl-2 genes in HepG2, SynHeps-I, and SynHeps-II cells cultured in DMEM or DMEM supplemented with 80% patient plasma (PP) for 6 and 12 h ( n = 3). * P < 0.05, ** P < 0.01; statistical significance was analyzed using unpaired Student’s t test. The assay was performed by qPCR. Bcl-2/Bax was the ratio of Bcl-2 to Bax.

Article Snippet: The membranes were subsequently probed with rabbit monoclonal antibodies against β-actin, UGT1A1, OATP1B1, OTC, ARG1, and CPS1 from Proteintech (Wuhan, China) and P21, Cyclin D1, HGF, NF-κB p65, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), phospho-NF-κB p65, phospho-ERK1/2, and phospho-STAT3 from ABclonal (Wuhan, China) at 4 °C overnight.

Techniques: Clinical Proteomics, Incubation, CCK-8 Assay, Western Blot, Expressing, Cell Culture

Journal: Biomaterials Research

Article Title: Genetically Modified Hepatocytes Targeting Bilirubin and Ammonia Metabolism for the Construction of Bioartificial Liver System

doi: 10.34133/bmr.0043

Figure Lengend Snippet: 3D expansion of hepatocytes in Cytopore. (A) Schematic diagram of large-scale cell expansion strategy. Cells were initially grown in cell culture flasks (T-175) and then expanded in a multilayer cell culture system to approximately 2 × 10 9 . Thereafter, cells were transferred to shake flasks or fermenter for further expansion. (B) Expansion curves of HepG2, SynHeps-I, and SynHeps-II cells from approximately 2.5 × 10 7 to approximately 1.5 × 10 10 cells within 17-day period ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; statistical significance was analyzed using unpaired Student’s t test. (C) Microscope images of SynHeps-II cells cultured on Cytopore microcarrier (left) and stained with 0.1% crystal violet (middle) or DAPI (right). Scale bar, 25 μm. (D) Fluorescence imaging of SynHeps-II cells transfected with GFP coding gene, grown in Cytopore, and stained with DAPI (blue, left) or imaged by endogenous fluorescence of GFP (green, middle). Scale bar, 100 μm. (E) Protein expression levels of UGT1A1, OATP1B1, OTC, ARG1, and CPS1 determined by Western blot after 3 days of plate culturing (2D) and microcarrier culturing (3D). (F) Synthesis of ALB and urea in HepG2, SynHeps-I, and SynHeps-II cells after 3 days of plate culturing (2D) and microcarrier culturing (3D). n = 3; * P < 0.05, ** P < 0.01, *** P < 0.001; statistical significance was analyzed by two-way ANOVA. (G) Schematic illustration of extracorporeal blood purification method using ALF patient plasma. The diagram showed the plasma separator (OP-08), membrane oxygenator, micro-BAL (loaded hepatocytes), and blood pump. (H) Continuous measurement of unconjugated bilirubin [indirect bilirubin (IBIL)] and NH3 during the extracorporeal blood purification process ( n = 3 per group). **** P < 0.0001; statistical significance was analyzed by one-way ANOVA.

Article Snippet: The membranes were subsequently probed with rabbit monoclonal antibodies against β-actin, UGT1A1, OATP1B1, OTC, ARG1, and CPS1 from Proteintech (Wuhan, China) and P21, Cyclin D1, HGF, NF-κB p65, extracellular signal-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (STAT3), phospho-NF-κB p65, phospho-ERK1/2, and phospho-STAT3 from ABclonal (Wuhan, China) at 4 °C overnight.

Techniques: Cell Culture, Microscopy, Staining, Fluorescence, Imaging, Transfection, Expressing, Western Blot, Purification, Clinical Proteomics, Membrane