Fig 1.
Procedure for preparation of concentrated F-CM.
(A). Fibroblasts were rinsed with PBS and refreshed with DMEM supplemented with 10% depleted BCS media. The cells were incubated at 37°C for 24 h to condition the media with fibroblasts. The media were then collected and pre-cleaned by serial centrifugation and further concentrated using centrifugal concentrator units (3-kD Centripreps). Isolated hepatocytes were cultured with a medium that consisted of 75% normal hepatocyte culture media and 25% F-CM (final F-CM concentration: 55 mg/ml). (B) Conditioned medium was pre-cleaned by serial centrifugation. First, the medium was centrifuged at 500 x g for 10 min to remove cells. Supernatants were centrifuged again at 3000 x g for 20 min to remove cell debris and then concentrated by a factor of ~80 using 3-kD Centripreps.
Fig 2.
Size exclusion chromatography results of cultured F-CM and non-cultured F-CM.
Qualitative composition of concentrated F-CM (red line) and concentrated medium (unconditioned, blue line) were both analyzed using size exclusion chromatography. Contents > 50 kDa were removed before analysis to prevent clogging of columns. Black arrows: reference sizes.
Fig 3.
Effect of F-CM on maintaining hepatocyte morphology.
Hepatocyte monoculture (left), co-culture with NIH-3T3 J2 (middle) and monoculture with F-CM (55 mg/ml, right) were observed under phase contrast microscope daily for 7 d. Cells with distinct nuclei and bright boundaries are heathy hepatocytes. Scale bars: 200 μm
Fig 4.
Immuno-histochemistry of intracellular albumin on 9th day of hepatocyte cultures.
Hepatocyte monoculture (left), co-culture with NIH-3T3 J2 (middle) and monoculture with F-CM (55 mg/ml, right) on day 9 were fixed and permeabilized using paraformaldehyde and 0.1% Triton X-100, respectively. The fixed samples were then stained with anti-rat albumin primary antibody and Alexa Fluor 488 conjugated secondary antibody (green). Nucleus was counter-stained with DAPI (blue). Scale bars: 200 μm
Fig 5.
Quantitative analysis of hepatocyte functions.
(A) Albumin secretion and (B) urea synthesis of cultured hepatocytes on day 9 were measured using ELISA and colorimetric assay, respectively. “Hepatocyte co-culture” indicates NIH-3T3 J2 co-cultured samples, “(+) F-CM”: hepatocyte monoculture supplemented with F-CM (55 mg/ml); “(+) concentrated media”: hepatocyte monoculture supplemented with concentrated media (unconditioned). Results were analyzed using one-way ANOVA and Tukey’s post hoc test. Bars: ± 1 SD, n = 9. Bars labeled with different letters (a,b,c and d) are significantly different (Tukey’s test, P<0.01)
Fig 6.
Effects of F-CM on the RNA levels of hepatocytes.
(A) PCR for identification of mRNA related to actin, albumin and TAT in hepatocyte; isolated hepatocyte on day 0, J2; NIH-3T3 J2 fibroblast, only-H; hepatocyte monoculture on day 7, Co; Hepatocyte co-culture with NIH-3T3 J2 on day 7, and (+) FCM; hepatocyte monoculture with F-CM (55 mg/ml) on day 7. (B) qPCR for albumin and TAT. the data was analyzed using one-way ANOVA and Tukey’s post hoc test. Bars: ± 1 SD, n = 3. Bars labeled with different letters (a,b, and c) are significantly different (Tukey’s test, P<0.01)
Fig 7.
Concentration dependent effects of F-CM on morphology and functions of hepatocytes.
Hepatocytes monoculture and monocultures with F-CMs (5.5, 27.5 and 55 mg/ml) were culture for 9 d. (A) Morphologies of one monoculture and three different concentrations of F-CM added samples on day 9 of culture. Scale bars: 200 μm. (B) Albumin secretion and (C) urea synthesis on the same day were measured using ELISA and colorimetric assay, respectively. The results were analyzed using one-way ANOVA and Tukey’s post hoc test. Bars: ± 1 SD, n > 4. Bars labeled with different letters (a,b,c and d) are significantly different (Tukey’s test, p<0.01)