Morphological changes in 201B7 cells in hepatocyte selection medium (HSM).

<p>The medium was changed to HSM with or without knockout serum replacement dialysis and insulin, dexamethasone, and aprotinin (IDA) solution (A). Three days after changing the media, the cell numbers decreased to 0 (B). Open circle with solid line: dialysis (−) IDA (+); cross with solid line: dialysis (−) IDA (−); open circle with dashed line: dialysis (+) IDA (+); and cross with dashed line: dialysis (+) IDA (−). Original magnification: ×400; scale bar: 25 µm, n = 3.</p

Culture of 201B7 cells in media without glucose and arginine, but supplemented with galactose and ornithine, followed by that in conventional media.

<p><b>(A)</b> 201B7 cells were cultured in media without glucose and arginine, but supplemented with galactose and ornithine HDI, mWE, or mDF12), for 2 days, followed by 7 days in L15, WE, or DF12. Cells cultured for 9 days in L15, WE, or DF12 only were used to compare with those in HDI, mWE, and mDF12. <b>(B and C)</b> RNA was isolated and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (<i>AFP</i>) <b>(B)</b> and albumin (<i>ALB</i>) <b>(C)</b>. HDI, hepatocyte differentiation inducer; WE, William’s E; DF12, Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; mWE, modified William’s E; mDF12, modified Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; FF, ReproFF; HDI-L15, HDI followed by L15; mWE-L15, mWE followed by L15; mDF12-L15, mDF12 followed by L15; HDI-WE, HDI followed by WE; mWE-WE, mWE followed by WE; mDF12-WE, mDF12 followed by WE; HDI-DF12, HDI followed by DF12; mWE-DF12, mWE followed by DF12; mDF12-DF12, mDF12 followed by DF12. Data are presented as the mean ± standard deviation (error bars). *, P < 0.05 compared with FF (one-way analysis of variance); n = 3.</p

Real-time quantitative polymerase chain reaction (PCR).

<p>Expression levels of GALK1 (A), GALK2 (B), OTC (C), and PAH (D) were analyzed using real-time quantitativePCR. The expression levels were normalized against ribosomal protein L19. The relative expression levels of the 201B7 cells cultured with ReproFF were assigned the value 1. RPL19: ribosomal protein L19; GALK1: galactokinase 1; GALK2: galactokinase 2; OTC: ornithine transcarbamylase; PAH: phenylalanine hydroxylase; FF: 201B7 cells cultured with ReproFF, fetal: fetal liver, adult: adult liver; error bar: standard deviation, n = 3.</p

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining.

<p>201B7 cells were subjected to TUNEL at 1 day after the medium was changed to hepatocyte selection medium (HSM) with or without dialysis of knockout serum replacement or use of insulin, dexamethasone, and aprotinin (IDA) solution. Positive cells were seen in the different HSM conditions (A, B, C, D), whereas no cells were positive in ReproFF (E). The nuclei of the positive cells were condensed (F). A: dialysis (−) IDA (+); B: dialysis (−) IDA (−); C: dialysis (+) IDA (+); D: dialysis (+) IDA (−); E: ReproFF; and F: magnified view of D. Original magnification: ×200, scale bar: 25 µm (A, B, C, D, E) and 2.5 µm (F).</p

Morphology of 201B7 cells cultured in repeated combinations of hepatocyte differentiation inducer (HDI) and William’s E (WE) medium.

<p><b>(A)</b> 201B7 cells were cultured in ReproFF, HDI followed by WE, WE only, or WE followed by HDI. The cells were cultured in HDI for either 12 h or 48 h and then cultured for another 5 days in combination with HID. After three cycles of the culture combinations, the cells were imaged <b>(B)</b>. Original magnification, ×400; scale bar, 100 μm.</p

Hematoxylin and eosin staining.

<p>201B7 cells were stained with hematoxylin and eosin at 1 day after the media change. The cells appeared damaged in the hepatocyte selection medium with or without dialysis of knockout serum replacement or use of insulin, dexamethasone, and aprotinin (IDA) solution (A, B, C, D). The cells appeared to be in comparatively good condition in ReproFF (E). The damaged cells showed condensation (F, arrow) or fragmentation of nuclei (F, arrowhead). A: dialysis (−) IDA (+); B: dialysis (−) IDA (−); C: dialysis (+) IDA (+); D: dialysis (+) IDA (−); E: ReproFF; and F: magnified view of D. Original magnification: ×200; scale bar: 25 µm (A, B, C, D, E) and 2.5 µm (F).</p

Co-culture of human primary hepatocytes and 201B7 in hepatocyte selection medium (HSM).

<p>After establishment of a co-culture of 201B7 and human primary hepatocytes (A), the medium was changed to HSM with dialyzed knockout serum replacement dialysis and without insulin, dexamethasone, or aprotinin. The 201B7 cells (arroheads) disappeared while the primary hepatocyes (arrows) were still alive at day 1 (B), day 2 (C) and day 3 (D). Original magnification: ×400, scale bar: 25 µm, arrow: 201B7 cells, arrowhead: hepatocytes.</p

Repeated cycles of 2d culture of 201B7 cells in hepatocyte differentiation inducer (HDI) either before or after culture in William’s E (WE) medium.

<p><b>(A)</b> 201B7 cells were cultured in three conditions; HDI for 2 days followed by WE for 5 days, WE only for 7 days, and WE for 5 days followed by HDI for 2 days. <b>(B and C)</b> After one, two, or three cycles of these culture conditions, RNA was isolated, and subjected to quantitative polymerase chain reaction to analyze the expression of α-feto protein (<i>AFP</i>) <b>(B)</b> and albumin (<i>ALB</i>) <b>(C)</b>. The thick solid line indicates HDI followed by WE, thick broken line indicates WE only, and thin solid line indicates WE followed by HDI. Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with 0 time of repeat; n = 3.</p

Time-course culture of 201B7 cells in hepatocyte differentiation inducer (HDI).

<p><b>(A)</b> 201B7 cells were cultured in HDI (black bar) for 0, 3, 6, 12, 24, and 48 h, followed by another 7 days of culture in William’s E (WE) medium (gray bar). <b>(B and C)</b> RNA was isolated, and subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (<i>AFP</i>) <b>(B)</b> and albumin (<i>ALB</i>) <b>(C)</b>. Data are presented as the mean ± standard deviation (error bars). *, P <0.05 compared with ReproFF (FF); n = 3.</p

Culture of 201B7 cells in conventional media.

<p>201B7 cells were cultured in different conventional media for 7 days, and then subjected to real-time quantitative polymerase chain reaction to analyze the expression of α-feto protein (AFP). The expression levels of AFP were significantly higher in the cells in WE, DF12 and fetal liver than those in ReproFF (P < 0.05, one-way analysis of variance, n = 3). FF, ReproFF; L15, Leibovitz's-15; DMEM, Dulbecco's Modified Eagle's Medium; RPMI, Roswell Park Memorial Institute 1640; WE, William’s E; DF12, Dulbecco's Modified Eagle's Medium/Nutrient F-12 Ham; MEM, Minimum Essential Medium; GMEM, Glasgow’s Minimum Essential Medium; IMDM, Iscove’s Modified Dulbecco’s Medium; CMRL, Connaught Medical Research Laboratories 1066; BME, Basal Medium Eagle; McCoy, McCoy’s 5A; MCDB, MCDB 131; fetal, fetal liver. Data are presented as the mean ± standard deviation (error bars). *, The expression levels of AFP were higher in the cells in WE and DF12 and fetal liver than those in ReproFF with P <0.05 (one-way analysis of variance).</p