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. 2024 Mar 7;14(1):5606.
doi: 10.1038/s41598-024-56169-0.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture

Melinda Grace Rossan Mathews #  1 , Ravichandran Subramaniam #  1 , Saravanakumar Venkatachalam #  1 , Johnson Retnaraj Samuel Selvan Christyraj  2 , Beryl Vedha Yesudhason  1 , Kalishwaralal Kalimuthu  3 , Manikandan Mohan  4 , Jackson Durairaj Selvan Christyraj  5
Affiliations

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture

Melinda Grace Rossan Mathews et al. Sci Rep. .

Abstract

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Keywords: Perionyx excavatus; Coelomic fluid; Earthworm; Fetal bovine serum; Serum free media.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Vitamins, fatty acids, and trace elements present in heat inactivated coelomic fluid analysed using gas chromatography and mass spectrometry. (A) Vitamin chromatogram; (B) Fatty acids and trace elements chromatogram.
Figure 2
Figure 2
Stability of the elemental quantity of fresh and 6-month-old HI-CF stored in − 20 °C. Data indicate the average value of triplicates (mean ± SD). ***Significant difference p < 0.001.
Figure 3
Figure 3
Mycoplasma detection assay using DAPI staining method for FBS (A) and HI-CF (B) supplemented cells.
Figure 4
Figure 4
Cell lines cultured in 10% DMEM media and f-HICF serum free media.
Figure 5
Figure 5
Cell lines cultured in 10% DMEM media, commercially available serum free media (SFM) and f-HICF serum free media.
Figure 6
Figure 6
Plating efficiency of cell lines (Vero, A549, C2C12 and HeLa) cultured in 10% fetal bovine serum (FBS) containing DMEM media, commercially available serum free media and formulated heat inactivated coelomic fluid (f-HICF) serum free media. Data indicate the average value of triplicates (mean ± SD). *Significant difference from control (p < 0.05), **Significant difference from control (p < 0.01), ***Significant difference from control (p < 0.001) and ****Significant difference from control (p < 0.0001).
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References

    1. Pilbauerova N, Schmidt J, SuchankovaKleplova T, Soukup T, Suchanek J. Effect of human platelet lysate as cultivation nutrient supplement on human natal dental pulp stem cell in vitro expansion. Biomolecules. 2022;12(8):1091. doi: 10.3390/biom12081091. - DOI - PMC - PubMed
    1. Yao T, Asayama Y. Animal-cell culture media: History, characteristics, and current issues. Reprod. Med. Biol. 2017;16(2):99–117. doi: 10.1002/rmb2.12024. - DOI - PMC - PubMed
    1. Van Der Valk J, Bieback K, Buta C, Cochrane B, Dirks W, Fu J, Hickman J, Hohensee C, Kolar R, Liebsch M, Pistollato F. Fetal bovine serum (FBS): Past–present–future. Altex. 2010;35(1):1–20. - PubMed
    1. Pilgrim CR, McCahill KA, Rops JG, Dufour JM, Russell KA, Koch TG. A review of fetal bovine serum in the culture of mesenchymal stromal cells and potential alternatives for veterinary medicine. Front. Vet. Sci. 2022;9:859025. doi: 10.3389/fvets.2022.859025. - DOI - PMC - PubMed
    1. Gstraunthaler G. Alternatives to the use of fetal bovine serum: Serum-free cell culture. ALTEX-Altern. Anim. Exp. 2003;20(4):275–281. - PubMed