Department of Parasitology, Ege University Medical School, Bornova/Izmir, Turkey.
Exp Parasitol. 2011 May;128(1):1-8. doi: 10.1016/j.exppara.2011.01.019. Epub 2011 Feb 4.
Toxoplasma gondii is one of the most researched parasite due to its easy growth both in vitro and in vivo. Tachyzoites, derived from mouse or rat peritoneum encounters ethical and economical problems when used for research or diagnostic purposes. Currently, research has focused on determining the most suitable cell culture environment to reach highest amount of viable tachyzoites with least host cell contamination. However, gene expression changes that take place throughout the adaptation of evolving T. gondii strains to continuous cell cultures appear as a problem. The present study aimed to determine a novel cell culture strategy for T. gondii RH Ankara strain tachyzoites to harvest abundant tachyzoites with least host cell contamination and minimal antigenic variation at predetermined dates to use as an antigen source in serological assays that will facilitate reduction in animal use. To achieve this purpose, T. gondii RH Ankara strain tachyzoites were incubated with HeLa cell at different ratios for two or three days. In all flasks incubated for two days, viability rate reached to 100% and HeLa cell contamination decreased to levels between 0.12-0.5×10(6)/ml. In the flasks with HeLa-tachyzoite ratio 1/8, the tachyzoite yield and viability ratio were 3×10(6)/ml and 100%, respectively, with accompanying 10 fold decrease (0.12×10(6)/ml) in HeLa contamination. During continuous production, highest tachyzoite yield was obtained from the first passage (3.55×10(6)/ml) and until the end of third subculture viability rates and HeLa cell contaminations were between 98.2-99.4% and 0.31-0.37×10(6)/ml, respectively. ELISA, IFA and Western blot analyses showed that the quality, specificity and sensitivity of the antigen harvested from the first passage of cell culture performed at two days intervals were comparable to the antigen harvested from mice and decreased in the following subcultures. Overall, these results demonstrated that T. gondii RH Ankara strain is still evolving to adapt to cell culture environment and therefore such strains continuously produced in cell cultures should be avoided for serological assays. However, the two day short interval cell culture method described herein offers a chance to reduce the animal use intended for the preparation of serological assays' antigen from local evolving strains.
刚地弓形虫是研究最多的寄生虫之一，因为它在体外和体内都很容易生长。速殖子来源于小鼠或大鼠腹膜，用于研究或诊断目的时存在伦理和经济问题。目前，研究的重点是确定最合适的细胞培养环境，以达到最高数量的活速殖子，同时宿主细胞污染最少。然而，随着不断进化的弓形虫株适应连续细胞培养，基因表达的变化似乎成为一个问题。本研究旨在确定一种新的细胞培养策略，用于收获大量 RH 虫株速殖子，同时宿主细胞污染最少，抗原变异最小，以预定日期作为血清学检测的抗原来源，从而减少动物使用。为了达到这个目的，将 RH 虫株速殖子与 HeLa 细胞以不同比例孵育 2 或 3 天。在所有孵育 2 天的培养瓶中，存活率达到 100%，HeLa 细胞污染降低至 0.12-0.5×10(6)/ml。在 HeLa-速殖子比例为 1/8 的培养瓶中，速殖子产量和存活率分别为 3×10(6)/ml 和 100%，同时 HeLa 污染降低 10 倍（0.12×10(6)/ml）。在连续生产过程中，从第一传代（3.55×10(6)/ml）中获得最高的速殖子产量，直到第三亚培养结束时，存活率和 HeLa 细胞污染率分别在 98.2-99.4%和 0.31-0.37×10(6)/ml 之间。ELISA、IFA 和 Western blot 分析表明，从 2 天间隔的细胞培养的第一传代收获的抗原的质量、特异性和敏感性与从小鼠中收获的抗原相当，并且在随后的亚培养中降低。总的来说，这些结果表明，RH 虫株仍在进化以适应细胞培养环境，因此，应避免在血清学检测中使用连续培养的此类虫株。然而，本文所述的两天短间隔细胞培养方法为减少用于制备血清学检测抗原的本地进化株的动物使用提供了机会。
