The use of Toxoplasma gondii tachyzoites produced in HeLa cells adhered to Cytodex 1 microcarriers as antigen in serological assays: an application of microcarrier technology

Abstract

Toxoplasma gondii can infect nearly all warm-blooded animals, including humans. In the laboratory diagnosis of toxoplasmosis, serological tests have importance in detecting antibody response. Traditionally T. gondii tachyzoites grown in vivo are being used as an antigen source in serological assays. Currently, tachyzoites produced in vitro are being tested as an antigen source in order to decrease animal use. Microcarrier technology allowed us to grow anchorage-dependent host cells on microcarrier suspension in short time and approximately 10 times more than traditional flask technique. The ability of T. gondii tachyzoites to grow in host cells adhered to microcarriers has not been analyzed yet. In this study, we aimed to develop a novel in vitro culture method to produce T. gondii tachyzoites abundantly using HeLa cells adhered to Cytodex 1 microcarriers. Initially, the growth of HeLa cells adhered to Cytodex 1 was analyzed using RPMI 1640, DMEM, and EMEM. Next, HeLa cells with a concentration of 1 × 10⁵ cells/ml and 2 × 10⁵ cells/ml were adhered to Cytodex 1 and grown in spinner flasks. Then, T. gondii tachyzoites were inoculated with 1:1 and 2:1 cell:tachyzoite ratios to HeLa cells adhered to microcarriers in spinner flaks. During continuous production in spinner flasks, tachyzoites were harvested at the 2nd, 4th, and 7th day of culture and the quality of antigens produced from these tachyzoites were tested in ELISA and Western Blotting using sera of patients with toxoplasmosis. The optimization studies showed that finest HeLa inoculation value was 2 × 10⁵ cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 10⁷) was 1:1 at the 4th day of inoculation. According to the results of ELISA comparing HeLa cell and mouse derived antigens, the highest correlation with mouse antigen was achieved at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio (P < 0.0001). The sensitivity and specificity ratios of ELISA were 100%. In addition, Western blotting banding patterns of the antigen derived at the 4th day of HeLa cell culture with 1:1 HeLa:tachyzoite ratio was comparable with mouse derived antigen. Overall, this novel methodology can be an alternative source of antigen in diagnostic assays, decrease animal use for antigen production, and contribute to the solution of ethical and economic problems.

Keywords: Antigen; HeLa cell; Microcarriers; Toxoplasma gondii.

Fig. 1

a Growth kinetics and b cell viability percentage of HeLa cells incubated in RPMI 1640, EMEM and DMEM media

Fig. 2

a Cell adhesion time and b MTT assay results of HeLa cells incubated in RPMI 1640, EMEM and DMEM media

Fig. 3

The adhesion ratio percentage of HeLa cells on Cytodex 1 microcarriers using RPMI 1640, DMEM and EMEM media

Fig. 4

The pH of the HeLa cell cultures in RPMI 1640, DMEM and EMEM media in Erlenmeyer flasks

Fig. 5

SEM images of HeLa cells adhered on Cytodex 1 in a RPMI 1640, b DMEM and c EMEM media (×1000, LEoL JSM-5200, Japan). The observed samples were taken from the 3rd day of culture

Fig. 6

The pH change of HeLa cell cultures in spinner flasks with two different concentrations (1 × 10⁵ cell/ml and 2 × 10⁵ cell/ml)

Fig. 7

SEM images of HeLa cells inoculated to Cytodex 1 at a 1 × 10⁵ cells/ml, b 2 × 10⁵ cells/ml concentrations (× 1000, LEoL JSM-5200, Japan). The observed samples were taken from the 3rd day of culture

Fig. 8

Pearson correlation and linear regression analyses of ELISA absorbance values (AV) of antigens derived from three subsequent HeLa subcultures at 2nd, 4th, and 7th days of culture and Swiss Webster outbred mice. Cut-off value: the mean AV + S.D. of the negative control sera

Fig. 9

The banding patterns observed by Western blotting. a Positive and b Negative serum sample pools. Lanes 1–3–5 HeLa cell culture derived antigens obtained at 2nd, 4th, and 7th days with 2:1 HeLa:tachyzoite ratio. Lanes 2–4–6 HeLa cell culture derived antigens obtained at 2nd, 4th, and 7th days with 1:1 HeLa:tachyzoite ratio. Lane 7 Mouse derived antigen. *Cross reactive bands, red arrows show the low molecular weight bands. (Color figure online)