Journal: Biocybernetics and Biomedical Engineering

Article Title: Hollow fiber bioreactor with genetically modified hepatic cells as a model of biologically active function block of the bioartificial liver

doi: 10.1016/j.bbe.2023.11.003

Figure Lengend Snippet: Fig. 2. Comparison of genetically modified cells – C3A_AO and C3A_AO_P2A with their parental C3A cell line – analysis of the expression of selected genes at the mRNA level. Results of RT-qPCR experiments con ducted for C3A, C3A_AO_P2A, and C3A_AO cells cultured with or without 40 mM NH4Cl. To determine the changes in mRNA levels, REST 2009 software and the Pfaffl model were used. (a) Analysis of the expression of ARG1 and OTC transgenes in genetically modified hepatic cells. Data normalization was per formed using the RPL13A and GAPDH genes. Number of biological repeats: 4. (b) Statistical significance (p) for Fig. 2a. (c) Analysis of the expression of ALB, HNF1A, HSP1A1, HNF4A, and NF-κB genes in human HCC cells. Data normal ization was performed using the GAPDH gene. Number of biological repeats: 3. (d) Statistical significance (p) for Fig. 2c. Abbreviations: AO - C3A_AO; P2A - C3A_AO_P2A. All experiments were performed according to the MIQE guide lines. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

Article Snippet: Human hepatocellular carcinoma - C3A (CRL-10741), human osteosarcoma - HOS (CRL-1543), and human embryonic kidney 293 T - HEK293T (CRL-11268) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison, Genetically Modified, Expressing, Quantitative RT-PCR, Cell Culture, Software

Journal: Biocybernetics and Biomedical Engineering

Article Title: Hollow fiber bioreactor with genetically modified hepatic cells as a model of biologically active function block of the bioartificial liver

doi: 10.1016/j.bbe.2023.11.003

Figure Lengend Snippet: Fig. 3. Comparison of genetically modified cells – C3A_AO and C3A_AO_P2A – with their parental C3A cell line at the functional level. (a) Albumin secretion by human HCC cells was measured in media samples using ELISA. The obtained results were expressed as the ratio of albumin mass per number of living cells. Data represent the mean value ± SD (n = 3). Statistical analysis was conducted using multivariate ANOVA with Duncan’s post hoc test. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). (b) Urea production by human HCC cells (after induction with 40 mM NH4Cl) was evaluated both in cell lysates and in conditioned medium using a QuantiChrom Urea Assay Kit. The results were expressed as the ratio of urea mass per number of living cells. Data represent the mean value ± SD (n = 3). Statistical analysis was conducted using multivariate ANOVA with Duncan’s post hoc test. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). (c) Albumin secretion by human HCC cells after 24 h incubation with 20 mM and 40 mM NH4Cl was measured using ELISA. The results were expressed as the ratio of albumin mass per number of living cells. Data represent the mean value ± SD (n = 3). Statistical analysis was conducted using multivariate ANOVA with Duncan’s post hoc test. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). (d) Viability of human HCC cells after treatment with NH4Cl was determined using propidium iodide (PI) staining and flow cytometry analysis (FACS). Cells were cultured with 20 and 40 mM NH4Cl for 24 h. The results are expressed as the per centage of dead cells. Data represent the mean value ± SD (n = 4). Statistical analysis was conducted using multivariate ANOVA with Duncan’s post hoc test. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). (e) Mitochondrial activity in human HCC cells after treatment with different concentrations of NH4Cl was determined using the MTT test. C3A cells and their genetically modified counterparts were treated with 20 mM, 40 mM, and 60 mM NH4Cl for 24 h. Data represent the mean value ± SD (n = 4). Statistical analysis was conducted using multivariate ANOVA with Duncan’s post hoc test. Levels of significance: p < 0.05 (*); p < 0.01 (**); p < 0.001 (***).

Article Snippet: Human hepatocellular carcinoma - C3A (CRL-10741), human osteosarcoma - HOS (CRL-1543), and human embryonic kidney 293 T - HEK293T (CRL-11268) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison, Genetically Modified, Functional Assay, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Flow Cytometry, Cell Culture, Activity Assay

Journal: Biocybernetics and Biomedical Engineering

Article Title: Hollow fiber bioreactor with genetically modified hepatic cells as a model of biologically active function block of the bioartificial liver

doi: 10.1016/j.bbe.2023.11.003

Figure Lengend Snippet: Fig. 6. Comparison of functionality of genetically modified cell lines - C3A_AO and C3A_AO_P2A with their parental C3A cells in dynamic conditions. The final dynamic cultures were conducted using 10 mL/min as the flow rate and 5⋅106/bioreactor as the initial number of seeded cells. Static cultures were set as a control with the same cell density per T25 culture flask. Both types of cultures were conducted simultaneously and lasted for 12 days. (a) Comparison of glucose consumption (left panel) and albumin secretion (right panel) by human HCC cell lines in static and dynamic conditions – first biological repetition. (b) Comparison of glucose consumption (left panel) and albumin secretion (right panel) by human HCC cells in static and dynamic conditions – second biological repetition. Abbreviations: d – dynamic culture; s – static culture.

Article Snippet: Human hepatocellular carcinoma - C3A (CRL-10741), human osteosarcoma - HOS (CRL-1543), and human embryonic kidney 293 T - HEK293T (CRL-11268) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison, Genetically Modified, Control

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 2. In vitro function of islets pre-cultured with exogenous complement component C3a. Insulin release at 2 mmol/L and 20 mmol/L glucose of 1020 replicates of three mouse islets per Eppendorf tube: (A) pre-cultured alone, with 10 nmol/L C3a alone or with 100 nmol/L C3a alone, for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a; (B) pre-cultured alone, with 5 nmol/L ANXA1 alone or with 10 nmol/L C3a alone for 48 h followed by subsequent GSIS assays in the absence of exogenous C3a or ANXA1; (C) pre-cul- tured alone, with 5 nmol/L ANXA1 alone or with a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a for 48 h followed by subse- quent GSIS assays in the absence of exogenous C3a and/or ANXA1, P < 0.05 versus islets pre-cultured alone at the same glucose concentration. (D) Protection from cytokine-induced apoptosis following a 48 h pre-culture with 5 nmol/L ANXA1-alone, 10 nmol/L C3a alone or a dual combination of 5 nmol/L ANXA1 and 10 nmol/L C3a, and the subsequent presence of specified MSC biotherapeutics during the final 20 h cytokine incubation. Eight to 12 replicates of five islets per well in each culture group assayed, *P < 0.05 versus islets pre-cul- tured alone in the presence of cytokines for the final 20 h of the 3-day culture period. The P values (AD) were calculated using two-way ANOVA Bonferroni’s post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vitro, Cell Culture, Concentration Assay, Incubation

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 3. Pre-culturing islets with a cocktail of MSC secretory factors ensures sustained improvements to islet insulin secretory function and protection from cytokine-induced apoptosis. (A, B) Insulin release at 2 and 20 mmol/L glucose of 30 replicates of three mouse islets per Eppendorf tube, pre-cultured alone, with 5 nmol/L ANXA1 alone, with 5 nmol/L ANXA1 and 10 nmol/L SDF-1, with 5 nmol/L ANXA1 and 10 nmol/L C3a, or with a cocktail of 5 nmol/L ANXA1, 10 nmol/L SDF-1 and 10 nmol/L C3a, for 48 h, before removal of the MSC- derived biotherapeutics for 1 day (A) or 3 days (B), *P < 0.05 and **P < 0.01 versus islets cultured alone at the same glucose concentration. (C, D) Protection of islets from cytokine-induced apoptosis after pre-culture with MSC-derived biotherapeutics alone, in dual combination or a cocktail of all three factors (as of legend) for 48 h, before removal of the MSC-derived biotherapeutics for 1 day (C) or 3 days (D), 8 to 12 replicates of five islets per well were assayed, *P < 0.05 and **P < 0.01 versus islets cultured alone with cytokines, +P < 0.05 vs. islets cul- tured alone without cytokines. The P values (AD) were calculated using two-way ANOVA with Bonferroni post hoc test.

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: Cell Culture, Derivative Assay, Concentration Assay

Journal: Cytotherapy

Article Title: Mesenchymal stromal cell secretory factors induce sustained improvements in islet function pre- and post-transplantation.

doi: 10.1016/j.jcyt.2018.07.007

Figure Lengend Snippet: Figure 4. In vivo function of islets pre-cultured alone, with ANXA1 alone or with a cocktail of MSC secretory factors. (A) Average blood glucose concentrations of STZ diabetic mice trans- planted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (repeated-meas- urements ANOVA with Bonferroni post hoc test, n = 79). (B) Area under the curve (AUC) of STZ diabetic mice transplanted with 150 islets pre-cultured for 48 h alone, with ANXA1 alone or with a cocktail of ANXA1/SDF-1/C3a, *P < 0.05 versus mice transplanted with islets pre-cultured alone (one-way ANOVA with Dunn’s post hoc test, n = 79).

Article Snippet: Islets were handpicked into groups of 100 for pre-culture in RPMI supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin plus 0.1 mg/mL streptomycin alone, with recombinant human ANXA1 alone, recombinant mouse SDF-1-alone, recombinant mouse C3a alone or with combinations of these factors (R&D Systems, Abingdon, United Kingdom).

Techniques: In Vivo, Cell Culture