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. 2020 May 30;9(6):1357. doi: 10.3390/cells9061357

Figure 3.

Figure 3

Human liver cell culture on a solubilized HL-ECM coating. (A–D) Hepatocytes (HEPs) from 3 donors (32, 155 and 158) were seeded on rat tail collagen I (Collagen I)- and solubilized human liver extracellular matrix (ECM)-coated tissue culture plastic and maintained in culture for up to 5 days. (A) Plating was evaluated after 24 h and 5 days of culture by DNA dosage. (B) Viability was evaluated after 24 h and 5 days of culture by resazurin reduction. (C) Urea was measured in culture supernatant after 5 days of incubation and normalized to viable HEP number. (D) Expression of key markers of hepatocyte functions was assessed by qPCR after 24 h and 5 days of culture (right panel: only statistically significant differences are indicated). (E,F) HSCs from 3 donors (94, 97 and 98) were grown on tissue culture plastic surface (i) uncoated (Ctr) or (ii) coated with solubilized human liver ECM or (iii) with a rat tail collagen I solution. (E) The number of viable HSCs over time was evaluated by resazurin reduction. (F) Gene expression was evaluated after 24 h and 7 days of culture by qPCR for markers of activated HSCs. (G,H) HSCs from 3 donors (94, 97 and 98) were grown on a tissue culture plastic surface in culture medium (i) non-supplemented (Ctr) or (ii) supplemented with 100 μg/mL solubilized human liver ECM. (G) The number of viable HSCs over time was evaluated by resazurin reduction. (H) Gene expression was evaluated after 24 h and 7 days of culture by qPCR for markers of activated HSCs. (I) (LS)ECs from 3 donors (156, 164 and 173) were seeded at the same density on rat tail collagen I- and solubilized human liver ECM-coated tissue culture plastic and maintained in culture for up to 3 days. Gene expression was evaluated after 24 h and 3 days of culture by qPCR for EC and LSEC markers. All: n = 3, results were analyzed per donor, graphs show mean ± SEM, results are expressed as % Collagen I or % Ctr, Student’s t-test (All except E and G) or 2-way ANOVA with Bonferroni’s post-hoc test (E and G) versus Collagen I or Ctr value, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.005. ACTA2: actin α2, ALB: albumin, APOB: apolipoprotein B, ARG1: arginase 1, ASL: argininosuccinate lyase, COL1A1: collagen type 1 α1 chain, CYP: cytochrome P450, FCGR2B: Fcγ receptor 2b, G6PC: glucose-6-phosphatase catalytic subunit, HNF4A: hepatocyte nuclear factor 4α, HNF1A: hepatocyte nuclear factor 1α, LYVE1: lymphatic vessel endothelial hyaluronan receptor 1, NOS3: NO synthase 3, PDGFRB: PDGF receptor β, STAB: stabilin.