Fig 2: Expression of liver-specific functions in (A) co-culture of HepG2/NIH 3T3 cells compared to only HepG2 cells with Albumin secretion in μg/mL/105 cells (n = 3, * p < 0.05 and ** p < 0.01) and gene expression analysis of (B) HepG2/NIH 3T3 cells (n = 3, * p < 0.05 and ** p < 0.01) compared to (C) HepG2 cells (n = 3, ** p < 0.01, *** p < 0.001 and **** p < 0.0001) at day 1, 3, and 7 for liver-specific genes AFP, ALB, KRT19, and MKI67. Error bars represent the standard error of the mean.

Fig 3: Hepatic function evaluations of 2D and 3D models. (A) Immunostaining of ALB (green) and nuclei (bule, DAPI) of cells on Day 7 during culture period. Determination of the total (B) ALB and (C) urea secretions in the supernatant at days 1, 4 and 7 during culture. The asterisks in figure indicate the level of significant difference (*p < 0.05, **p < 0.01).

Fig 4: Generation and characterization of COs and LOs derived from ASCs of liver tissue(A) Schematic diagram of the workflow of the COs derivation, COs expansion, and LOs differentiation. (BME, basement membrane extract; OIM, organoid initiating medium; OGM, organoid growth medium; ODM, organoid differentiation medium; CO, cholangiocyte organoid; LO, liver organoid).(B) Representative images of generation of COs and LOs derived from ASCs. Scale bar, 200 μm.(C) Morphological structure of the COs and LOs. Scale bar, 50 μm.(D) Representative immunofluorescence images of EpCAM, HNF4A in COs and E-cadherin, ALB in LOs. Scale bar, 100 μm.(E) mRNA expression levels of each specific markers in COs and LOs (3 cases of organoids derived from different patients, and n = 3/each case).(F and G) (F) ALB and (G) AAT production in the COs, LOs, and PHHs (n = 3).(H) Representative images of PAS staining of COs and LOs. Scale bar, 100 μm.(I) Representative images of LOs for the uptake and release of ICG. (ICG, indocyanine green) Scale bar, 100 μm. Results represent mean ± SD, ns, no significant difference, ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. One-way ANOVA was used to compare between different groups.

Fig 5: NACA improves cell survival in NPCs and ameliorates pathophysiological changes related to RILD. Radiation induced cell death and functional markers were measured at 3 days or 7 days post radiation injury. (A) Phase contrast image with DAPI staining to determine cell death at 3 days post 8 Gy radiation, hepatocytes (upper) and NPCs (lower). Briefly, nuclei were counted 3 days after radiation in 4 areas across 4 chips, one-way ANOVA with Tukey’s test, significance at p < 0.05, F(HEP) = 1.647, F(NPCs) = 102.1, DF = 2. (B) LDH effluent measured by ELISA for hepatocytes (left) and NPCs (right). One-way ANOVA with Tukey’s test was performed, significance at p < 0.05, F(HEP) = 2.58, F(NPCs) = 35.52, DF = 2. (C) CD31 is decreased in irradiated samples compared to 8 Gy + NACA, 3 days after radiation. CD31 fluorescence was measured across 3 areas in 3 chips, one-way ANOVA with Tukey’s test, significance at p < 0.05, F = 15.56, DF = 2. (D) α-SMA is increased in irradiated samples compared to 8 Gy + NACA + radiation. 3 days after radiation α-SMA was measured in 5 areas across 3 chips and normalized to cell number (DAPI), one-way ANOVA with Tukey’s test, significance at p < 0.05, F = 8.867, DF = 2. (E) depicts ELISA assays that were performed using NPCs lysates 3 days post radiation injury. These included VCAM-1, ICAM-1, SAA and CRP which were measured in 3 chips, as well as IL-6, Thrombomodulin, PDGFB, level were measured in 4 chips. One-way ANOVA with Tukey’s test was performed for all ELISAs, significance at p < 0.05, F(VCAM-1) = 6.128, F(ICAM-1) = 5.132, F(SAA) = 9.053, F(CRP) = 10.22, F(IL-6) = 8.9, F(Thrombomodulin) = 9.339, F(PDGFB) = 10.84, DF = 2. (F) show RT-PCR validation of TGFB1, TIMP1, TLR4 and CCN2 mRNA levels in NPCs 3 days after radiation, n = 3, one-way ANOVA with Tukey’s test, significance at p < 0.05, F(TGFB1) = 10.61, F(TIMP1) = 24.16, F(TLR4) = 19.19, F(CCN2) = 15.33, DF = 2. (G) depicts ELISA results from hepatocytes. Briefly TG, Urea and albumin were measured 3 days after radiation injury. TG utilized cell lysate while the Urea and Albumin assays relied on effluent. Results are from 4 chips, one-way ANOVA with Tukey’s test, significance at p < 0.05, F (TG) = 8.625, F(Urea) = 6.692, F(Alb) = 0.45, DF = 2. (G) miRNA RT-PCR for hepatocytes relevant to radiation damage. RT-PCR was performed 7 days post radiation injury, n = 3, ANOVA with Tukey’s test, F(miR-34a-5p) = 19.83, F(miR-122-5p) = 6.859, F(miR-21-5p) = 4.634, DF = 2.