Elimination of Mycoplasma Contamination from Infected Human Hepatocyte C3A Cells by Intraperitoneal Injection in BALB/c Mice

Abstract

Background/Aims: The use of antibiotics to eliminate Mycoplasma contamination has some serious limitations. Mycoplasma contamination can be eliminated by intraperitoneal injection of BALB/c mice with contaminated cells combined with screening monoclonal cells. However, in vivo passage in mice after injection with contaminated cells requires a long duration (20-54 days). Furthermore, it is important to monitor for cross-contamination of mouse and human cells, xenotropic murine leukemia virus-related virus (XMRV) infection, and altered cell function after the in vivo treatment. The present study aimed to validate a reliable and simplified method to eliminate mycoplasma contamination from human hepatocytes. BALB/c mice were injected with paraffin oil prior to injection with cells, in order to shorten duration of intraperitoneal passage. Cross-contamination of mouse and human cells, XMRV infection and cell function-related genes and proteins were also evaluated. Methods: PCR and DNA sequencing were used to confirm Mycoplasma hyorhinis (M. hyorhinis) contamination in human hepatocyte C3A cells. Five BALB/c mice were intraperitoneally injected with 0.5 ml paraffin oil 1 week before injection of the cells. The mice were then intraperitoneally injected with C3A hepatocytes (5.0 × 10⁶/ml) contaminated with M. hyorhinis (6.2 ± 2.2 × 10⁸ CFU/ml). Ascites were collected for monoclonal cell screening on the 14th day after injection of contaminated cells. Elimination of mycoplasma from cells was determined by PCR and Transmission Electron Microscopy (TEM). Human-mouse cell and XMRV contamination were also detected by PCR. Quantitative reverse transcription PCR and western blotting were used to compare the expression of genes and proteins among treated cells, non-treated infected cells, and uninfected cells. Results: Fourteen days after injection with cells, 4 of the 5 mice had ascites. Hepatocyte colonies extracted from the ascites of four mice were all mycoplasma-free. There was no cell cross-contamination or XMRV infection in treated cell cultures. Elimination of Mycoplasma resulted in partial or complete recovery in the expression of ALB, TF, and CYP3A4 genes as well as proteins. Proliferation of the treated cells was not significantly affected by this management. Conclusion: The method of elimination of Mycoplasma contamination in this study was validated and reproducible. Success was achieved in four of five cases examined. Compared to the previous studies, the duration of intraperitoneal passage in this study was significantly shorter.

Keywords: Mycoplasma; cell cross-contamination; elimination; intraperitoneal inoculation; monoclonal cells.

Figure 1

Flowchart of Mycoplasma elimination. Aseptic inflammation was induced in BALB/c mice by intraperitoneal injection (i.p) with 0.5 ml sterile paraffin oil 7 days before cells injection. 1d M(+) cell were intraperitoneal injected with a 0.5 ml of cell suspension (4 × 10⁶/ml) in mice in day 7. Cells from the ascites were collected and inoculated in 96-well plate in day 21. After 12 h, the single attached cells were screened by microscopy and cultured for 27 days. Cells were harvested in 25-cm² culture flasks in day 49 for further experiments.

Figure 2

Detection of Mycoplasma contamination in infected C3A cells. Lane 1: day 2 culture; lane 2: day 4 culture; lane 3: day 6 culture; lane 4: positive control; lane 5, negative control. (B) Mycoplasma detection in Tc and M(+) cell groups. Lane 1: 1d M(+) cell; lane 2: 8d M(+) cell; lane 3: 15d M(+) cell; lane 4: 22d M(+) cell; lane 5: 27d M(+) cell; lane 6: 36d M(+) cell; lane 7: 43d M(+) cell; lane 8: 1d Tc; lane 9: 8d Tc; lane 10: 15d Tc; lane 11: 22d Tc; lane 12: 29d Tc; lane 13: positive control; lane 14: negative control. (C) Monoclonal human–mice cell contamination. Lane 1: Tc+ human COX-I primers; lane 2: M(−) cell+ human COX-I primers; lane 3: ML12+human COX-I primers; lane 4: ddH₂0+human COX-I primers; lane 5: Tc+ mice COX-I primers; lane 6: M(−) cell+mice COX-I primers; lane 7: AML12+mice COX-I primers; lane 8: ddH₂0+mice COX-I primers; lane M, 1-2000-bp DNA marker (Takara).

Figure 3

Selection and identification of monoclonal cells (Tc). Data are time after inoculation: (A) 12 h, (B) 2 d, (C) 5 d, (D) 8 d, (E) 27 d (25-cm² tissue flask).

Figure 4

Transmission electron microscopy (TEM) of (A1–A4), 1d M(+) cell; (B1–B4), 43d M(+) cell; and (C1–C4), Tc. M, Mycoplasma; AB, autophagy body; APB, apoptotic body; Mit, mitochondria; ER, endoplasmic reticulum; V, vacuoles.

Figure 5

Capillary electrophoresis of Tc (D5S818, TH01, D13S317, D7S820, D16S539, CSF1PO, vWA, TPOX, Amelogenin, D3S1358, D21S11, D18S51, Penta E, Penta D, D8S1179, FGA).

Figure 6

Hoechst 33258 staining (A) Tc, (B,C) 1d M(+) cell, (D) 43d M(+) cell. Mycoplasma (arrow). Tc exhibited distinct nuclei with no evidence of Mycoplasma; 1d M(+) cell exhibited filamentous or small granular blue fluorescence around the nuclei which were evidence of Mycoplasma (White arrow). 43d M(+) cell exhibited broken and fragmented nuclei with almost no intact nuclei observed. Nuclear fragmentation and intense staining was due to DNA condensation during apoptosis1d M(+) cell and 43d M(+) cell cells.

Figure 7

XMRV PCR detection. Tc cells were negative for XMRV expression. The positive control was 22RV1. The negative controls were AML12 and HepG2 cells. Human and Murine GAPDH gene primers served as the loading controls.

Figure 8

qRT-PCR detection of (A) ALB, (B) CPS1, (C) TF, (D) CYP3A4, (E) CYP2D6. ΔP < 0.05, ΔΔP < 0.01 vs. 1d M(+) cell; #P < 0.05, ##P < 0.01 vs. 43d M(+) cell; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. M(−) cell.

Figure 9

Western-Blot detection of (A) protein bands (B) ALB, (C) CPS1, (D) TF, (E) CYP3A4, (F) CYP2D6. ΔP < 0.05, ΔΔP < 0.01, ΔΔΔP < 0.001 vs. 1d M(+) cell; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. 43d M(+) cell; ▲P < 0.05, ▲▲P < 0.01, ▲▲▲P < 0.001 vs. M(−) cell.

Figure 10

Cell proliferation evaluated by MTT assay. ^*P < 0.001 compared with 1dM(+) cell.