Volume 25, Issue 5 (9-2021)                   IBJ 2021, 25(5): 349-358 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Habibzadeh S, Doroud D, Taheri T, Seyed N, Rafati S. Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum. IBJ. 2021; 25 (5) :349-358
URL: http://ibj.pasteur.ac.ir/article-1-3410-en.html
Background: Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods: Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (complete serum-free medium [CSFM]) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results: Diff-Quick staining and epi-fluores­cence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion: This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.
Type of Study: Full Length | Subject: Molecular Immunology & Vaccines

1. Steverding D. The history of leishmaniasis. Parasites and vectors 2017; 10(1): 82. [DOI:10.1186/s13071-017-2028-5]
2. WHO. Leishmaniasis. 2020; https://www.who.int/news-room/fact-sheets/detail/leishmaniasis
3. Kevric I, Cappel MA, Keeling JH. New World and Old World Leishmania Infections. Dermatologic clinics 2015; 33(3): 579-593. [DOI:10.1016/j.det.2015.03.018]
4. Ghorbani M, Farhoudi R. Leishmaniasis in humans: drug or vaccine therapy? Drug design, development and therapy 2018; 12: 25-40. [DOI:10.2147/DDDT.S146521]
5. Saunders EC, Naderer T, Chambers J, Landfear SM, McConville MJ. Leishmania mexicana can utilize amino acids as major carbon sources in macrophages but not in animal models. Molecular microbiology 2018; 108(2): 143-158. [DOI:10.1111/mmi.13923]
6. Khademvatan S, Salmanzadeh S, Foroutan-Rad M, Bigdeli S, Hedayati-Rad F, Saki J, Heydari-Gorji E. Spatial distribution and epidemiological features of cutaneous Leishmaniasis in southwest of Iran. Alexandria journal of medicine 2017; 53(1): 93-98. [DOI:10.1016/j.ajme.2016.03.001]
7. Desjeux P. Leishmaniasis: current situation and new perspectives. Comparative immunology, microbiology and infectious diseases 2004; 27(5): 305-318. [DOI:10.1016/j.cimid.2004.03.004]
8. Kohl K, Zangger H, Rossi M, Isorce N, Lye LF, Owens KL, Beverley SM, Mayer A, Fasel N. Importance of polyphosphate in the Leishmania life cycle. Microbial cell 2018; 5(8): 371-384. [DOI:10.15698/mic2018.08.642]
9. Bates PA. Revising Leishmania's life cycle. Nature microbiology 2018; 3(5): 529-530. [DOI:10.1038/s41564-018-0154-2]
10. Lai JY, Klatt S, Lim TS. Potential application of leishmania tarentolae as an alternative platform for antibody expression. Critical reviews in biotechnology 2019; 39(3): 380-394. [DOI:10.1080/07388551.2019.1566206]
11. Doukas A, Karena E, Botou M, Papakostas K, Papadaki A, Tziouvara O, Xingi E, Frillingos S, Boleti H. Heterologous expression of the mammalian sodium-nucleobase transporter rSNBT1 in Leishmania tarentolae. Biochimica et biophysica acta biomembranes 2019; 1861(9): 1546-1557. [DOI:10.1016/j.bbamem.2019.07.001]
12. Klatt S, Simpson L, Maslov DA, Konthur Z. Leishmania tarentolae: Taxonomic classification and its application as a promising biotechnological expression host. PLoS neglected tropical diseases 2019; 13(7): e0007424. [DOI:10.1371/journal.pntd.0007424]
13. Abdossamadi Z, Taheri T, Seyed N, Montakhab Yeganeh H, Zahedifard F, Taslimi Y, Habibzadeh S, Gholami E, Gharibzadeh S, Rafati S. Live Leishmania tarentolae secreting HNP1 as an immunotherapeutic tool against Leishmania infection in BALB/c mice. Journal of immunotherapy 2017; 9(13): 1089-1102. [DOI:10.2217/imt-2017-0076]
14. Pirdel L, Farajnia S. A non-pathogenic recombinant Leishmania expressing Lipophosphoglycan 3 against experimental infection with Leishmania infantum. Scandinavian journal of immunology 2017; 86(1): 15-22. [DOI:10.1111/sji.12557]
15. Katebi A, Gholami E, Taheri T, Zahedifard F, Habibzadeh S, Taslimi Y, Shokri F, Papadopoulou B, Kamhawi S, Valenzuela J, Rafati S. Leishmania tarentolae secreting the sand fly salivary antigen PpSP15 confers protection against Leishmania major infection in a susceptible BALB/c mice model. Molecular immunology 2015; 67(2): 501-511. [DOI:10.1016/j.molimm.2015.08.001]
16. Gholami E, Oliveira F, Taheri T, Seyed N, Gharibzadeh S, Gholami N, Mizbani A, Zali F, Habibzadeh S, Bakhadj DO. DNA plasmid coding for Phlebotomus sergenti salivary protein PsSP9, a member of the SP15 family of proteins, protects against Leishmania tropica. PLoS neglected tropical diseases 2019; 13(1): e0007067. [DOI:10.1371/journal.pntd.0007067]
17. Merlen T, Sereno D, Brajon N, Rostand F, Lemesre JL. Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms. The American journal of tropical medicine and hygiene 1999; 60(1): 41-50. [DOI:10.4269/ajtmh.1999.60.41]
18. de Almeida Rodrigues I, da Silva BA, dos Santos ALS, Vermelho AB, Alviano CS, Rosa MdSS. A new experimental culture medium for cultivation of Leishmania amazonensis: its efficacy for the continuous in vitro growth and differentiation of infective promastigote forms. Parasitology research 2010; 106(5): 1249-1252. [DOI:10.1007/s00436-010-1775-4]
19. Van der Valk J, Mellor D, Brands R, Fischer R, Gruber F, Gstraunthaler G, Hellebrekers L, Hyllner J, Jonker F, Prieto P, Baumans V. The humane collection of fetal bovine serum and possibilities for serum-free cell and tissue culture. Toxicology in vitro 2004; 18(1): 1-12. [DOI:10.1016/j.tiv.2003.08.009]
20. Mesalam A, Lee KL, Khan I, Chowdhury M, Zhang S, Song SH, Joo MD, Lee JH, Jin JI, Kong IK. A combination of bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metalloproteinases. Reproduction, fertility and development 2019; 31(2): 333-346. [DOI:10.1071/RD18162]
21. Barnes D, Sato G. Methods for growth of cultured cells in serum-free medium. Analitical biochemistry metods in the biological sciences 1980; 102(2): 255-270 [DOI:10.1016/0003-2697(80)90151-7]
22. Rauch C, Feifel E, Amann E-M, Spötl HP, Schennach H, Pfaller W, Gstraunthaler G. Alternatives to the use of fetal bovine serum: human platelet lysates as a serum substitute in cell culture media. Alternatives to animal experimentation 2011; 28(4): 305-316. [DOI:10.14573/altex.2011.4.305]
23. Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G. The serum-free media interactive online database. Alternatives to animal experimentation 2010; 27(1): 53-62. [DOI:10.14573/altex.2010.1.53]
24. Gstraunthaler G. Alternatives to the use of fetal bovine serum: serum-free cell culture. Alternatives to animal experimentation 2003; 20(4): 275-281.
25. Barnes D, Sato G. Serum-free cell culture: a unifying approach. Cell 1980; 22(3): 649-655. [DOI:10.1016/0092-8674(80)90540-1]
26. Muniaraj M, Lal C, Kumar S, Sinha P, Das P. Milk of cow (Bos taurus), buffalo (Bubalus bubalis), and goat (Capra hircus): a better alternative than fetal bovine serum in media for primary isolation, in vitro cultivation, and maintenance of Leishmania donovani promastigotes. Journal of clinical microbiology 2007; 45(4): 1353-1356. [DOI:10.1128/JCM.01761-06]
27. Belford DA, Rogers ML, Regester GO, Francis GL, Smithers GW, Liepe IJ, Priebe IK, Ballard FJ. Milk-derived growth factors as serum supplements for the growth of fibroblast and epithelial cells. In vitro cellular and developmental biology-animal 1995; 31(10): 752-760. [DOI:10.1007/BF02634116]
28. Sidana A, Alam A, Farooq U. Soy protein isolate: A substitute of fetal bovine serum for the in vitro cultivation of Leishmania donovani. Legume research-an international journal 2018; 41(2): 218-221. [DOI:10.18805/LR-3730]
29. Fritsche C, Sitz M, Weiland N, Breitling R, Pohl HD. Characterization of the growth behavior of Leishmania tarentolae-a new expression system for recombinant proteins. Journal of basic microbiology 2007; 47(5): 384-393. [DOI:10.1002/jobm.200710111]
30. Sharief AH, Khalil EAG, Omer SA, Abdalla HS. Innovative serum-free medium for in vitro cultivation of promastigote forms of Leishmania species. Parasitology international 2008; 57(2): 138-142. [DOI:10.1016/j.parint.2007.10.003]
31. Ali SA, Iqbal J, Ahmad B, Masoom M. A semisynthetic fetal calf serum-free liquid medium for in vitro cultivation of Leishmania promastigotes. The American journal of tropical medicine and hygiene 1998; 59(1): 163-165. [DOI:10.4269/ajtmh.1998.59.163]
32. Bolhassani A, Taheri T, Taslimi Y, Zamanilui S, Zahedifard F, Seyed N, Torkashvand F, Vaziri B, Rafati S. Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Experimental parasitology 2011; 127(3): 637-645. [DOI:10.1016/j.exppara.2010.12.006]
33. Nasiri V. An overview of the recent findings in the cultivation of Leishmania. Reviews in medical microbiology 2017; 28(1): 34-42. [DOI:10.1097/MRM.0000000000000093]
34. Trager W. Nutrition of a hemoflagellate (Leishmania tarentolae) having an interchangeable requirement for choline or pyridoxal. The journal of protozoology 1957; 4(4): 269-276 [DOI:10.1111/j.1550-7408.1957.tb02519.x]
35. Kar K, Mukerji K, Naskar K, Bhattacharya A, Ghosh DK. Leishmania donovani: a chemically defined medium suitable for cultivation and cloning of promastigotes and transformation of amastigotes to promastigotes. The journal of protozoology 1990; 37(4): 277-279. [DOI:10.1111/j.1550-7408.1990.tb01147.x]
36. Van der Valk J, Brunner D, De Smet K, Svenningsen AF, Honegger P, Knudsen LE, Lindl T, Noraberg J, Price A, Scarino M. Optimization of chemically defined cell culture media-replacing fetal bovine serum in mammalian in vitro methods. Toxicology in vitro 2010; 24(4): 1053-1063. [DOI:10.1016/j.tiv.2010.03.016]
37. Nayak A, Akpunarlieva S, Barrett M, Burchmore R. A defined medium for Leishmania culture allows definition of essential amino acids. Experimental parasitology 2018; 185: 39-52. [DOI:10.1016/j.exppara.2018.01.009]
38. Laranjeira-Silva MF, Hamza I, Pérez-Victoria JM. Iron and Heme Metabolism at the Leishmania-Host Interface. Trends in Parasitology 2020; 36(3): 279-289. [DOI:10.1016/j.pt.2019.12.010]
39. Toh SQ, Glanfield A, Gobert GN, Jones MK. Heme and blood-feeding parasites: friends or foes? Parasites and vectors 2010; 3(1): 108. [DOI:10.1186/1756-3305-3-108]
40. Schuster FL, Sullivan JJ. Cultivation of clinically significant hemoflagellates. Clinical microbiology reviews 2002; 15(3): 374-389. [DOI:10.1128/CMR.15.3.374-389.2002]
41. Santarem N, Cunha J, Silvestre R, Silva C, Moreira D, Ouellette M, Cordeiro-Da-Silva A. The impact of distinct culture media in Leishmania infantum biology and infectivity. Parasitology 2014; 141(2): 192-205. [DOI:10.1017/S0031182013001388]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2022 CC BY-NC 4.0 | Iranian Biomedical Journal

Designed & Developed by : Yektaweb