Comparative Study On 2D Or 3D Cultured Rat Primary Hepatocytes And BRL-3A Cells In The Evaluation Of Drug Induced Hepatotoxicity

OBJECTIVE To establish a rat primary hepatocytes isolation and identification system, and carry out studies on comparison in 2D or 3D cultured rat primary hepatocytes and BRL-3A cells on liver toxicity characteristic;METHODS Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and then were identified by PAS staining and its dual-nuclei structure. Using inverted phase contrast microscope and scanning electron microscope to observe the growth of 3D cultured rat primary hepatocytes on cellusponge sponge scaffold. CCK8 method was applied to investigate the 2D cultured rat primary hepatocytes and BRL-3A growth cell growth; IC50 was tested by CCK8 method, at the mean time, hepatocelluar toxicity of acetaminophen to them was studied. Damage to the two kinds of cells from the drug was observed using inverted phase contrast microscope、HE(hematoxylin eosin)stain and transmission electron microscopy(TEM). Automatic biochemical analyzer was used to detected the contents of ALT, AST, ALP, LDH, TP, ALB, and BUN changes in cell supernatants after drug effected on the three cells models.RESULTS Around(100–300)×106 cells were obtained from one rat liver. Cell viability was ranged between 80% and 95%. PAS staining positive and its presence of two nuclei in some cells identified the cells as rat primary hepatocytes. Rat primary hepatocytes grows best with a cell density of 60000/cm2 and a medium depth of 1.5mm.The hepatocytes began to adherent after 4 h, and some hepatocytes attached the wall after 24 h while all the hepatocytes attached in 48 h. Island structure formed by hepatocytes can be seen everywhere, and the number of cells came to a slight decline in the first day. The hepatocytes began to grow fast after 3 days of inoculating, and the logarithmic growth phase is 3-5 days. By the seventh day the cells began to enter a recession. When cultured in cellusponge stent in three-dimensional culture system, rat primary hepatocytes self-aggregated to form cell clusters after 24 hours of culture. Cell aggregation was formed in most cells three days after the cells were cultured. The spheroids growed biger five days after inoculation, up to70mm diameter. The diameter of the spheroids was constant at about 100-150mm after seven days of culture. The incubation period and the number of continuous length of BRL-3A cells viars depending on the density of inoculation. The best cell density we thought was(1-2)×104, and the logarithmic growth phase began at 48 h after inoculation, which last for 3 days. IC50 value of APAP effect on primary rat hepatocytes for 24 h was 18.03 m M, 95%CI=(17.28,18.81)mmol/L; acting on the BRL-3A was 20.05mmol/L,95%CI=(18.99,21.17)mmol/L. The toxicity of APAP on rat primary hepatocytes and BRL-3A cells increased with prolonged action, within the same period, showing a dose-dependent toxicity effects. BRL-3A HE staining showed that low-dose group cells nucleus was round, ovate-orbicular or uniform, the cytoplasma and nucleo-cytoplasmic ratio were uniformly distributed, and nuclear fission; high-dose group cells were uniformly distributed, dye shallow, structure was unelear and the nucleus almost invisible. What’s more, in the high-dose groups, transmission electron microscopy reveal that cells were organelles swelling, nuclear membrane rupture and the nucleus almost invisible.Compared with the control group, with the increase of the drug dose, all concentration groups of AST and Glu of rat primary hepatocytes was significantly increased(P < 0.01), but BRL-3A cells in each dose group of Glu, AST and ALT did not changed significantly. Exposure to low(5mm), medium(20mm) and high concentrations(30mm) of APAP, the ALB, BUN content decreased significantly, AST, Glu and AST value increased significantly. LDH value of both 5m M and 20 m M concentration group increased significantly, low concentration(5m M) of ALP increased significantly, and TP value increased significantly. CONCLUSION Compared with immortalized BRL-3A cells, 2D cultured primary hepatocytes can be more reflective model with the liver toxicity. However, primary hepatocytes cannot live for a long time and BRL-3A cells are lack of extensive metabolic enzymes. Thus, increasing the kinds of liver enzymes of immortalized cells is a better means to boost BRL-3A cells as liver toxicity screening model. The three-dimensional cultured cell model is richer in enzymes value changing in the culture supernatant than 2D cultured cells, which is a relatively good hepatotoxicity evaluation model.