, Volume 71, Issue 1, pp 91–105 | Cite as

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

  • Pelin Sağlam Metiner
  • Hüseyin CanEmail author
  • Duygu Ayyıldız Tamiş
  • Muhammet Karakavuk
  • Ilgın Kımız Geboloğlu
  • Sultan Gülçe İz
  • Esra Atalay Şahar
  • Aysu Değirmenci Döşkaya
  • Yüksel Gürüz
  • Saime İsmet Deliloğlu Gürhan
  • Mert Döşkaya
Original Article


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.


Toxoplasma gondii HeLa cell Microcarriers Antigen 



This study was supported by the grant given by the Scientific Research Projects Branch Directorate of Ege University, Turkey (Grant No: 2014-TIP-042) to M.D. In addition, access to the Animal Cell & Tissue Engineering laboratories of Ege University, Department of Bioengineering are highly appreciated.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Armstrong D (1966) Serial cultivation of human leukemic cells. Proc Soc Exp Biol Med 122:475–481CrossRefGoogle Scholar
  2. Ashburn D, Evans R, Chatterton JM, Joss AW, Ho-Yen DO (2000) Toxoplasma dye test using cell culture derived tachyzoites. J Clin Pathol 53:630–633CrossRefGoogle Scholar
  3. Ashburn D, Chatterton JM, Evans R, Joss AW, Ho-Yen DO (2001) Success in the toxoplasma dye test. J Infect 42:16–19CrossRefGoogle Scholar
  4. Ayyildiz-Tamis D, Avcı K, Deliloglu-Gurhan SI (2014) Comparative investigation of the use of various commercial microcarriers as a substrate for culturing mammalian cells. In Vitro Cell Dev Biol Anim 50:221–231CrossRefGoogle Scholar
  5. Blaker GJ, Birch JR, Pirt SJ (1971) The glucose, insulin and glutamine requirements of suspension cultures of HeLa cells in a defined culture medium. J Cell Sci 9:529–537Google Scholar
  6. Bleckwenn NA, Bentley WE, Shiloach J (2005) Evaluation of production parameters with the vaccinia virus expression system using microcarrier attached HeLa cells. Biotechnol Prog 21:554–561CrossRefGoogle Scholar
  7. Boudreault P, Tremblay JP, Pépin MF, Garnier A (2001) Scale-up of a myoblast culture process. J Biotechnol 91:63–74CrossRefGoogle Scholar
  8. Buddhirongawatr R, Tungsudjai S, Chaichoune K, Sangloung C, Tantawiwattananon N, Phonaknguen R, Sukthana Y (2006) Detection of Toxolasma gondii in captive wild felids. Southeast Asian J Trop Med Public Health 37:15–17Google Scholar
  9. Butler M (2005) Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 68:283–291CrossRefGoogle Scholar
  10. Chatterton JM, Evans R, Ashburn D, Joss AW, Ho-Yen DO (2002) Toxoplasma gondii in vitro culture for experimentation. J Microbiol Methods 51:331–335CrossRefGoogle Scholar
  11. Chen AK, Reuveny S, Oh SK (2013) Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: achievements and future direction. Biotechnol Adv 31:1032–1046CrossRefGoogle Scholar
  12. Contini C, Fainardi E, Cultrera R, Canipari R, Peyron F, Delia S, Paolino E, Granieri E (1998) Advanced laboratory techniques for diagnosing Toxoplasma gondii encephalitis in AIDS patients: significance of intrathecal production and comparison with PCR and ECL-western blotting. J Neuroimmunol 92:29–37CrossRefGoogle Scholar
  13. Crowell RL, Syverton JT (1960) The mammalian cell-virus relationship VI. sustained ınfectıon of HeLa cells by coxsackie B3 virus and effect on superinfection. J Exp Med 113:419–435CrossRefGoogle Scholar
  14. da Costa-Silva TA, da Silva MC, Frazzatti-Gallina N, Pereira-Chioccola VL (2012) Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium. Exp Parasitol 130:463–469CrossRefGoogle Scholar
  15. Değirmenci A, Döşkaya M, Caner A, Ciçek C, Korkmaz M, Gürüz Y, Uner A (2011) Toxoplasma gondii RH Ankara: production of evolving tachyzoites using a novel cell culture method. Exp Parasitol 128:1–8CrossRefGoogle Scholar
  16. Diab MR, El-Bahy MM (2008) Toxoplasma gondii: virulence of tachyzoites in serum free media at different temperatures. Exp Parasitol 118:75–79CrossRefGoogle Scholar
  17. Döşkaya M, Degirmenci A, Ciçek C, Ak M, Korkmaz M, Gürüz Y, Uner A (2006) Behaviour of Toxoplasma gondii RH Ankara strain tachyzoites during continuous production in various cell lines. Parasitology 132:315–319CrossRefGoogle Scholar
  18. Döşkaya M, Caner A, Can H, Gülce İz S, Değirmenci A, Gürüz Y (2013) Cryopreservation of Toxoplasma gondii tachyzoites and tissue cysts. Turk J Parasitol 37:44–46CrossRefGoogle Scholar
  19. Döşkaya M, Caner A, Can H, Gülçe İz S, Gedik Y, Değirmenci Döşkaya A, Kalantari-Dehaghi M, Gürüz Y (2014) Diagnostic value of a Rec-ELISA using Toxoplasma gondii recombinant SporoSAG, BAG1, and GRA1 proteins in murine models infected orally with tissue cysts and oocysts. PLoS ONE 9:e108329CrossRefGoogle Scholar
  20. Evans R, Chatterton JM, Ashburn D, Joss AW, Ho-Yen DO (1999) Cell culture system for continuous production of Toxoplasma gondii tachyzoites. Eur J Clin Microbiol Infect Dis 18:879–884CrossRefGoogle Scholar
  21. Fliedl L, Kaisermayer C (2011) Transient gene expression in HEK293 and Vero cells immobilised on microcarriers. J Biotechnol 153:15–21CrossRefGoogle Scholar
  22. Francis JM, Payne RA, Joynson DHM (1988) Rapid indirect enzyme-linked immunosorbent assay (ELISA) for detecting antitoxoplasma IgG: comparison with the dye test. J Clin Pathol 41:802–805CrossRefGoogle Scholar
  23. Franck J, Garin YJ, Dumon H (2008) LDBio-Toxo II immunoglobulin G Western blot confirmatory test for anti-toxoplasma antibody detection. J Clin Microbiol 46:2334–2338CrossRefGoogle Scholar
  24. Freshney RI (2010) Culture of animal cells, 6th edn. Wiley, LondonCrossRefGoogle Scholar
  25. Fritz H, Barr B, Packham A, Melli A, Conrad PA (2012) Methods to produce and safely work with large numbers of Toxoplasma gondii oocysts and bradyzoite cysts. J Microbiol Methods 88:47–52CrossRefGoogle Scholar
  26. Gallego-Marín C, Henao AC, Gómez-Marín JE (2006) Clinical validation of a western blot assay for congenital toxoplasmosis and newborn screening in a hospital in Armenia (Quindio) Colombia. J Trop Pediatr 52:107–112CrossRefGoogle Scholar
  27. Gürhan Sİ, Ünal N, Kiremitçi M (1994) Relationship of inactivation techniques of the sera to cellular attachment on PHEMA and polypropylene surfaces. Anim Cell Technol Basic Appl Asp 6:389–394CrossRefGoogle Scholar
  28. Gürüz AY, Ok UZ, Korkmaz M (1996) Assessment of latex indirect agglutination test (Toxolatex Fumouze) for the detection of toxoplasma specific antibodies in human sera in Turkey. J Egypt Soc Parasitol 26:367–374Google Scholar
  29. Hellman A, Regan JD, Martin DH (1967) Large-scale cultivation of mammalian cells in vitro. Appl Microbiol 15:201–202Google Scholar
  30. Ho-Yen DO (2010) Annual reports, 2005–2009, Scottish Toxoplasma Reference Laboratory. Active 2 Aug 2010
  31. Jakob PH, Kehrer J, Flood P, Wiegel C, Haselmann U, Meissner M, Stelzer EH, Reynaud EG (2016) A 3-D cell culture system to study epithelia functions using microcarriers. Cytotechnology 68:1813–1825CrossRefGoogle Scholar
  32. Kahraman E, Deliloğlu Gürhan İ, Korkmaz M (2013) Investigation of the potential cytotoxic and genotoxic effects of different boron compounds on the CCL 62 (HeLa contaminant) human amniotic epithelial cell lines. Med Sci 2:454–468CrossRefGoogle Scholar
  33. Kato D, Takeuchi M, Sakurai T, Furukawa S, Mizokami H, Sakata M, Hirayama C, Kunitake M (2003) The design of polymer microcarrier surfaces for enhanced cell growth. Biomaterials 24:4253–4264CrossRefGoogle Scholar
  34. Koethe M, Straubinger RK, Pott S, Bangoura B, Geuthner AC, Daugschies A, Ludewig M (2015) Quantitative detection of Toxoplasma gondii in tissues of experimentally infected turkeys and in retail turkey products by magnetic-capture PCR. Food Microbiol 52:11–17CrossRefGoogle Scholar
  35. Kurokawa M, Sato S (2011) Growth and poliovirus production of Vero cells on a novel microcarrier with artificial cell adhesive protein under serum-free conditions. J Biosci Bioeng 111:600–604CrossRefGoogle Scholar
  36. Liang L, Döşkaya M, Juarez S, Caner A, Jasinskas A, Tan X, Hajagos BE, Bradley PJ, Korkmaz M, Gürüz Y, Felgner PL, Davies DH (2011) Identification of potential serodiagnostic and subunit vaccine antigens by antibody profiling of toxoplasmosis cases in Turkey. Mol Cell Proteom 10:M110.006916CrossRefGoogle Scholar
  37. Masatani T, Takashima Y, Takasu M, Matsuu A, Amaya T (2016) Prevalence of anti-Toxoplasma gondii antibody in domestic horses in Japan. Parasitol Int 65:146–150CrossRefGoogle Scholar
  38. Merten OW (2015) Advances in cell culture: anchorage dependence. Philos Trans R Soc Lond B Biol Sci 370:20140040CrossRefGoogle Scholar
  39. Microcarrier Cell Culture Prenciples & Methods (2005) Accessed 13 Dec 2016
  40. Oyeleye OO, Ogundeji ST, Ola SI, Omitogun OG (2016) Basics of animal cell culture: foundation for modern science. Biotechnol Mol Biol Rev 11:6–16CrossRefGoogle Scholar
  41. Payne RA, Joynson DH, Balfour AH, Harford JP, Fleck DG, Mythen M, Saunders RJ (1987) Public health laboratory service enzyme linked immunosorbent assay for detecting Toxoplasma specific IgM antibody. J Clin Pathol 40:276–281CrossRefGoogle Scholar
  42. Remington JS, Thulliez P, Montoya JG (2004) Recent developments for diagnosis of toxoplasmosis. J Clin Microbiol 42:941–945CrossRefGoogle Scholar
  43. Robert-Gangneux F, Amrein C, Lavarde V, Botterel F, Dupouy-Camet J (2000) Neosynthesized IgG detected by western blotting in Toxoplasma-seropositive heart or lung transplant recipients. Transpl Int 13:448–452Google Scholar
  44. Rourou S, van der Ark A, van der Velden T, Kallel H (2007) A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine 25:3879–3889CrossRefGoogle Scholar
  45. Souza MC, Freire MS, Schulze EA, Gaspar LP, Castilho LR (2009) Production of yellow fever virus in microcarrier-based Vero cell cultures. Vaccine 27:6420–6423CrossRefGoogle Scholar
  46. Sun LY, Lin SZ, Li YS, Harn HJ, Chiou TW (2011) Functional cells cultured on microcarriers for use in regenerative medicine research. Cell Transpl 20:49–62CrossRefGoogle Scholar
  47. Toulah FH, Sayed Al-Ahl SA, Amin DM, Hamouda MH (2011) Toxoplasma gondii: ultrastructure study of the entry of tachyzoites into mammalian cells. Saudi J Biol Sci 18:151–156CrossRefGoogle Scholar
  48. van Wezel AL (1967) Growth of cell-strains and primary cells on micro-carriers in homogeneous culture. Nature 216:64–65CrossRefGoogle Scholar
  49. Wu SC, Liu CC, Lian WC (2004) Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 22:3858–3864CrossRefGoogle Scholar
  50. Wu L, Chen SX, Jiang XG, Fu XL, Shen YJ, Cao JP (2011) Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems. Exp Parasitol 130:91–94CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Pelin Sağlam Metiner
    • 1
  • Hüseyin Can
    • 2
    Email author
  • Duygu Ayyıldız Tamiş
    • 1
  • Muhammet Karakavuk
    • 3
  • Ilgın Kımız Geboloğlu
    • 1
  • Sultan Gülçe İz
    • 1
  • Esra Atalay Şahar
    • 3
  • Aysu Değirmenci Döşkaya
    • 3
  • Yüksel Gürüz
    • 3
  • Saime İsmet Deliloğlu Gürhan
    • 1
  • Mert Döşkaya
    • 3
  1. 1.Department of Bioengineering, Faculty of EngineeringEge UniversityBornovaTurkey
  2. 2.Department of Biology, Molecular Biology Section, Faculty of ScienceEge UniversityBornovaTurkey
  3. 3.Department of Parasitology, Faculty of MedicineEge UniversityBornovaTurkey

Personalised recommendations