Sağlam Metiner Pelin, Can Hüseyin, Ayyıldız Tamiş Duygu, Karakavuk Muhammet, Kımız Geboloğlu Ilgın, Gülçe İz Sultan, Atalay Şahar Esra, Değirmenci Döşkaya Aysu, Gürüz Yüksel, Deliloğlu Gürhan Saime İsmet, Döşkaya Mert
Department of Bioengineering, Faculty of Engineering, Ege University, Bornova, İzmir, Turkey.
Department of Biology, Molecular Biology Section, Faculty of Science, Ege University, Bornova, İzmir, Turkey.
Cytotechnology. 2019 Feb;71(1):91-105. doi: 10.1007/s10616-018-0269-6. Epub 2019 Jan 3.
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 × 10 cells/ml and 2 × 10cells/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 × 10cells/ml using RPMI 1640, and the cell:tachyzoite ratio to obtain the highest tachyzoite yield (17.1 × 10) 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.
刚地弓形虫可感染几乎所有温血动物，包括人类。在弓形虫病的实验室诊断中，血清学检测对于检测抗体反应具有重要意义。传统上，体内培养的刚地弓形虫速殖子被用作血清学检测的抗原来源。目前，为了减少动物使用，正在测试体外产生的速殖子作为抗原来源。微载体技术使我们能够在短时间内在微载体悬浮液中培养依赖贴壁的宿主细胞，且产量比传统培养瓶技术高约10倍。刚地弓形虫速殖子在附着于微载体的宿主细胞中生长的能力尚未得到分析。在本研究中，我们旨在开发一种新的体外培养方法，使用附着于Cytodex 1微载体的HeLa细胞大量生产刚地弓形虫速殖子。最初，使用RPMI 1640、DMEM和EMEM分析附着于Cytodex 1的HeLa细胞的生长情况。接下来，将浓度为1×10⁶个细胞/ml和2×10⁶个细胞/ml的HeLa细胞附着于Cytodex 1，并在转瓶中培养。然后，以1:1和2:1的细胞:速殖子比例将刚地弓形虫速殖子接种到附着于转瓶中微载体的HeLa细胞上。在转瓶中连续生产过程中，在培养的第2天、第4天和第7天收获速殖子，并使用弓形虫病患者的血清通过ELISA和Western Blotting检测这些速殖子产生的抗原质量。优化研究表明，使用RPMI 1640时，最佳的HeLa接种值为2×10⁶个细胞/ml，接种后第4天获得最高速殖子产量（17.1×10⁶）的细胞:速殖子比例为1:1。根据比较HeLa细胞和小鼠来源抗原的ELISA结果，在HeLa细胞培养第4天，HeLa:速殖子比例为1:1时，与小鼠抗原的相关性最高（P < 0.0001）。ELISA的敏感性和特异性比率均为100%。此外，HeLa细胞培养第4天，HeLa:速殖子比例为1:1时产生的抗原的Western印迹条带模式与小鼠来源抗原相当。总体而言，这种新方法可以成为诊断检测中抗原的替代来源，减少抗原生产中的动物使用，并有助于解决伦理和经济问题。
