Development of Low-Serum Culture Media for the in Vitro Cultivation of Theileria annulata S15 Cell Line
-
Gholamreza Habibi
,
Asghar Afshari
,
Amin Shahedi
,
Mousa Hashemlo
,
Ali Es-Haghi
,
Mohamad Hosein Fallah Mehrabadi
,
Saeid Fathi
Abstract
Background: The production of bovine theileriosis vaccine involves in vitro cultivation of Theileria annulata schizont-infected cell lines. Fetal bovine serum (FBS) is commonly used in animal cell culture, including the Theileria cell line. However, we aimed to reduce the amount of serum needed for cell culture by modifying the Stoker culture medium with supplements such as excretion factor and serum substitutes.
Methods: To evaluate the effectiveness of these modifications, techniques such as cell counting, cell viability assays, and genomic analysis were employed in the Parasitic Vaccines Production Department of Razi institute of Iran, from 2020 to 2022. Statistical analysis was used to compare the results of different experimental conditions.
Results: The three experimental media were as effective as the commonly used 10% Stoker medium in supporting the growth and viability of cells.
Conclusion: The significant reduction in the required amount of serum and the remarkable cell growth achieved by using defined serum replacements for the production of cell culture media is a significant step towards the preparation of a proper cell culture medium for the production of bovine Theileriosis vaccine.
1. Subbiahanadar Chelladurai K, Selvan Christyraj JD, et al. Alternative to FBS in animal cell culture - An overview and future perspective. Heliyon. 2021;7(8):e07686.
2. Gstraunthaler G, Lindl T, van der Valk J. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65(5):791-3.
3. Ota M, Takagaki K, Takaoka S, Tanemura H, Urushihata N. A new method to confirm the absence of human and animal serum in mesenchymal stem cell culture media. Int J Med Sci. 2019;16(8):1102-1106.
4. Stout AJ, Mirliani AB, Rittenberg ML, et al. Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Commun Biol. 2022;(5):466.
5. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture ALTEX. 2003;20(4): 275-81.
6. Brown DJ, Campbell JD, Russell GC, Hopkins J, Glass EJ. T cell activation by Theileria annulata-infected macrophages correlates with cytokine production. Clin Exp Immunol. 1995; 102(3):507-14.
7. Marsolier J, Perichon M, DeBarry J, et al. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation. Nature. 2015;520:378–382.
8. Eagle H. Nutrition Needs of Mammalian Cells in Tissue Culture. Science. 1955;122:501-514.
9. Hashemi-Fesharki R. Control of Theileria annulata in Iran. Parasitol Today. 1988;4(2):36-40.
10. Davami F, Baldi L, Rajendra Y, M Wurm F. Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells. Int J Mol Cell Med. 2014;3(3):146-56.
11. Dessels C, Potgieter M, Pepper MS. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion. Front Cell Dev Biol. 2016;4:115.
12. Freshney RI. Culture of animal cells: A manual of basic technique and specialized applications. John Wiley & Sons. 2010.
13. Strober W. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 2015; 111:A3.B.1-A3.B.3.
14. Żuławińska, J. "Cell Doubling Time Calculator". Available at: https://www.omnicalculator.com/biology/cell-doubling-time. Accessed: 15 April 2023.
15. Wittekind DH. On the nature of Romanowsky--Giemsa staining and its significance for cytochemistry and histochemistry: an overall view. Histochem J. 1983;15(10):1029-47.
16. Waymouth C. Osmolality of Mammalian Blood and of Media for Culture of Mammalian Cells. In Vitro, 1970;6(2):109–27.
17. Habibi GR, Esmaeil Nia K, Bozorgi S, Hashemi-Fesharki R, Bordbar N. Semi-quantitative Analysis of Expression of Various Genes in relation to Possible Markers for Theileria annulata Attenuation. Arch Razi Inst. 2009;64(1):9-17.
18. Habibi GR, Safavie S, Afshari A, et al. The Identity of mother and working seed of Bovine Theileriosis vaccine S15 strain (2018) Project ID# 2-18-18-073- 950535.
19. Modirrousta H, Habibi G, Shayan P, Afshari A, Mirjalili A, Abdigoudarzi M. Determination of the Infectivity of Cryopreserved Theileria annulata Sporozoites in Tick Derived Stabilates Iran Ak-93 Strain, by in vivo and in vitro Methods. Iran J Parasitol. 2019;14(4):572-583.
20. Afshari A, Tavassoli M, Esmaeilnejad B, Habibi G, Esmaeilnia K. Molecular Characterization and Phylogenetic Analysis of Pathogenic Theileria spp. Isolated from Cattle and Sheep based on Cytochrome b Gene in Iran. Arch Razi Inst. 2021;76(2):243-252.
21. Hosios AM, Hecht VC, Laura V, et al. Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells. Developmental Cell. 2016;36(5):540-9.
22. Limoncu M, Özbilgin A, Balcioḡlu İ, Özbel Y. Evaluation of three new culture media for the cultivation and isolation of Leishmania parasites. J Basic Microbiol. 2004;44(3):197-202.
23. Davami F, Eghbalpour F, Nematollahi L, Barkhordari F, Mahboudi F. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles. Iran Biomed J. 2015;19(4):194-205.
24. Lezin C, Mauduit P, Uzan G, Abdelgawad ME. An Evaluation of Different Types of Peptone as Partial Substitutes for Animal-derived Serum in Vero Cell Culture. Altern Lab Anim. 2022;50(5):339-348.
25. Xu W, Wang J, Li W, et al. Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers. Theriogenology. 2023;196:37-49.
2. Gstraunthaler G, Lindl T, van der Valk J. A plea to reduce or replace fetal bovine serum in cell culture media. Cytotechnology. 2013;65(5):791-3.
3. Ota M, Takagaki K, Takaoka S, Tanemura H, Urushihata N. A new method to confirm the absence of human and animal serum in mesenchymal stem cell culture media. Int J Med Sci. 2019;16(8):1102-1106.
4. Stout AJ, Mirliani AB, Rittenberg ML, et al. Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat. Commun Biol. 2022;(5):466.
5. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture ALTEX. 2003;20(4): 275-81.
6. Brown DJ, Campbell JD, Russell GC, Hopkins J, Glass EJ. T cell activation by Theileria annulata-infected macrophages correlates with cytokine production. Clin Exp Immunol. 1995; 102(3):507-14.
7. Marsolier J, Perichon M, DeBarry J, et al. Theileria parasites secrete a prolyl isomerase to maintain host leukocyte transformation. Nature. 2015;520:378–382.
8. Eagle H. Nutrition Needs of Mammalian Cells in Tissue Culture. Science. 1955;122:501-514.
9. Hashemi-Fesharki R. Control of Theileria annulata in Iran. Parasitol Today. 1988;4(2):36-40.
10. Davami F, Baldi L, Rajendra Y, M Wurm F. Peptone Supplementation of Culture Medium Has Variable Effects on the Productivity of CHO Cells. Int J Mol Cell Med. 2014;3(3):146-56.
11. Dessels C, Potgieter M, Pepper MS. Making the Switch: Alternatives to Fetal Bovine Serum for Adipose-Derived Stromal Cell Expansion. Front Cell Dev Biol. 2016;4:115.
12. Freshney RI. Culture of animal cells: A manual of basic technique and specialized applications. John Wiley & Sons. 2010.
13. Strober W. Trypan Blue Exclusion Test of Cell Viability. Curr Protoc Immunol. 2015; 111:A3.B.1-A3.B.3.
14. Żuławińska, J. "Cell Doubling Time Calculator". Available at: https://www.omnicalculator.com/biology/cell-doubling-time. Accessed: 15 April 2023.
15. Wittekind DH. On the nature of Romanowsky--Giemsa staining and its significance for cytochemistry and histochemistry: an overall view. Histochem J. 1983;15(10):1029-47.
16. Waymouth C. Osmolality of Mammalian Blood and of Media for Culture of Mammalian Cells. In Vitro, 1970;6(2):109–27.
17. Habibi GR, Esmaeil Nia K, Bozorgi S, Hashemi-Fesharki R, Bordbar N. Semi-quantitative Analysis of Expression of Various Genes in relation to Possible Markers for Theileria annulata Attenuation. Arch Razi Inst. 2009;64(1):9-17.
18. Habibi GR, Safavie S, Afshari A, et al. The Identity of mother and working seed of Bovine Theileriosis vaccine S15 strain (2018) Project ID# 2-18-18-073- 950535.
19. Modirrousta H, Habibi G, Shayan P, Afshari A, Mirjalili A, Abdigoudarzi M. Determination of the Infectivity of Cryopreserved Theileria annulata Sporozoites in Tick Derived Stabilates Iran Ak-93 Strain, by in vivo and in vitro Methods. Iran J Parasitol. 2019;14(4):572-583.
20. Afshari A, Tavassoli M, Esmaeilnejad B, Habibi G, Esmaeilnia K. Molecular Characterization and Phylogenetic Analysis of Pathogenic Theileria spp. Isolated from Cattle and Sheep based on Cytochrome b Gene in Iran. Arch Razi Inst. 2021;76(2):243-252.
21. Hosios AM, Hecht VC, Laura V, et al. Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells. Developmental Cell. 2016;36(5):540-9.
22. Limoncu M, Özbilgin A, Balcioḡlu İ, Özbel Y. Evaluation of three new culture media for the cultivation and isolation of Leishmania parasites. J Basic Microbiol. 2004;44(3):197-202.
23. Davami F, Eghbalpour F, Nematollahi L, Barkhordari F, Mahboudi F. Effects of Peptone Supplementation in Different Culture Media on Growth, Metabolic Pathway and Productivity of CHO DG44 Cells; a New Insight into Amino Acid Profiles. Iran Biomed J. 2015;19(4):194-205.
24. Lezin C, Mauduit P, Uzan G, Abdelgawad ME. An Evaluation of Different Types of Peptone as Partial Substitutes for Animal-derived Serum in Vero Cell Culture. Altern Lab Anim. 2022;50(5):339-348.
25. Xu W, Wang J, Li W, et al. Exogenous pyruvate and recombinant human basic fibroblast growth factor maintain pluripotency and enhance global metabolic activity of bovine embryonic stem cells grown on low-density feeder layers. Theriogenology. 2023;196:37-49.
| Files | ||
| Issue | Vol 19 No 1 (2024) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijpa.v19i1.15206 | |
| Keywords | ||
| Stoker Cell culture medium Theileria annulata Serum | ||
| Rights and permissions | |
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Habibi G, Afshari A, Shahedi A, Hashemlo M, Es-Haghi A, Fallah Mehrabadi MH, Fathi S. Development of Low-Serum Culture Media for the in Vitro Cultivation of Theileria annulata S15 Cell Line. Iran J Parasitol. 2024;19(1):75-85.
