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Abstract
Cardiovascular disease become one of the leading factors of death in the world. Thus, research is urgently needed to discover newer drugs or therapeutical agents and biological plausibility. The H9C2 cardiomyoblast originated from embryonic BDIX rat ventricular cells and was previously used in numerous in vitro studies because of its similar nature to cardiomyocytes. However, to our knowledge, there are still limited studies on the basic procedure for culturing H9C2 cardiomyoblast and arranging the best strategy to perform a suitable timeline. Here, we shared our experience in culturing the H9C2 cardiomyoblast, including harvesting and subculturing the cells. We also demonstrated the change of cell confluency, depending on the seeding number, serum concentration, and culture flask through days 1, 3, and 6, to determine their doubling-time population. H9C2 cardiomyoblasts’ doubling time is around 48-54 days with Mean±SD 2.38±0.41. However, seeding density, different culture flasks, and serum concentration have become independent factors in determining specific measures to harvest the cells for further experiments.
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References
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References
Yun JS, Ko SH. Current trends in epidemiology of cardiovascular disease and cardiovascular risk management in type 2 diabetes. Metabolism 2021;123. https://doi.org/10.1016/j.metabol.2021.154838.
Majnaric LT, Bosnic Z, Kurevija T, Wittlinger T. Cardiovascular risk and aging: The need for a more comprehensive understanding. Journal of Geriatric Cardiology 2021;18:462–78. https://doi.org/10.11909/j.issn.1671-5411.2021.06.004.
Fernández-Rojas B, Vázquez-Cervantes GI, Pedraza-Chaverri J, Gutiérrez-Venegas G. Lipoteichoic acid reduces antioxidant enzymes in H9c2 cells. Toxicol Rep 2020;7:101–8. https://doi.org/10.1016/j.toxrep.2019.12.007.
Xu X, Ruan L, Tian X, pan F, Yang C, Liu G. Calcium inhibitor inhibits high glucose-induced hypertrophy of H9C2 cells. Mol Med Rep 2020;22:1783–92. https://doi.org/10.3892/mmr.2020.11275.
Boccellino M, Galasso G, Ambrosio P, Stiuso P, Lama S, Di Zazzo E, et al. H9c2 Cardiomyocytes under Hypoxic Stress: Biological Effects Mediated by Sentinel Downstream Targets. Oxid Med Cell Longev 2021;2021. https://doi.org/10.1155/2021/6874146.
Feng D, Guo L, Liu J, Song Y, Ma X, Hu H, et al. DDX3X deficiency alleviates LPS induced H9c2 cardiomyocytes pyroptosis by suppressing activation of NLRP3 inflammasome. Exp Ther Med 2021;22. https://doi.org/10.3892/etm.2021.10825.
Chang C-C, Cheng H-C, Chou W-C, Huang Y-T, Hsieh P-L, Chu P-M, et al. Sesamin suppresses angiotensin-II-enhanced oxidative stress and hypertrophic markers in H9c2 cells. Environ Toxicol 2023;38:2165–72. https://doi.org/10.1002/tox.23853.
Yang Y, Xie Y, Zhao X, Qi M, Liu X. Pogostone alleviates angiotensin II-induced cardiomyocyte hypertrophy in H9c2 cells through MAPK and Nrf2 pathway. Tropical Journal of Pharmaceutical Research 2023;22:1373–8. https://doi.org/10.4314/tjpr.v22i7.3.
Sulistiyorini I, Safitri R, Lesmana R, Syamsunarno MRAA. The viability test of sappan wood (Caesalpinia sappan L.) ethanol extract in the H9C2 cell line. International Journal of Applied Pharmaceutics 2020;12:76–8. https://doi.org/10.22159/ijap.2020.v12s3.39479.
Green MR, Sambrook J. Estimation of cell number by hemocytometry counting. Cold Spring Harb Protoc 2019;2019:732–4. https://doi.org/10.1101/pdb.prot097980.
Data sheet of H9C2 (2-1). European Collection of Autheticated Cell Culture n.d. https://www.culturecollections.org.uk/nop/product/h9c2-2-1 (accessed January 3, 2024).
Mehrabani D, Mahdiyar P, Torabi K, Robati R, Zare S, Dianatpour M, et al. Growth kinetics and characterization of human dental pulp stem cells: Comparison between third molar and first premolar teeth. J Clin Exp Dent 2017;9:e172–7. https://doi.org/10.4317/jced.52824.
Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G. Morphological, Biochemical, and Electrophysiological Characterization of a Clonal Cell (H9c2) Line From Rat Heart. 1991.
Dott W, Mistry P, Wright J, Cain K, Herbert KE. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity. Redox Biol 2014;2:224–33. https://doi.org/10.1016/j.redox.2013.12.028.
Kankeu C, Clarke K, Van Haver D, Gevaert K, Impens F, Dittrich A, et al. Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a “function follows form” model of differentiation. Mol Omics 2018;14:181–96. https://doi.org/10.1039/c8mo00036k.
Kaynak Bayrak G, Gümüşderelioğlu M. Construction of cardiomyoblast sheets for cardiac tissue repair: comparison of three different approaches. Cytotechnology 2019;71:819–33. https://doi.org/10.1007/s10616-019-00325-2.
Branco AF, Pereira SP, Gonzalez S, Gusev O, Rizvanov AA, Oliveira PJ. Gene expression profiling of H9c2 myoblast differentiation towards a cardiac-like phenotype. PLoS One 2015;10. https://doi.org/10.1371/journal.pone.0129303.
Salih ARC, Farooqi HMU, Kim YS, Lee SH, Choi KH. Impact of serum concentration in cell culture media on tight junction proteins within a multiorgan microphysiological system. Microelectron Eng 2020;232. https://doi.org/10.1016/j.mee.2020.111405.
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. https://doi.org/10.3389/fvets.2022.859025.
Lee DY, Lee SY, Yun SH, Jeong JW, Kim JH, Kim HW, et al. Review of the Current Research on Fetal Bovine Serum and the Development of Cultured Meat. Food Sci Anim Resour 2022;42:775–99. https://doi.org/10.5851/kosfa.2022.e46.
Liu S, Yang W, Li Y, Sun C. Fetal bovine serum, an important factor affecting the reproducibility of cell experiments. Sci Rep 2023;13. https://doi.org/10.1038/s41598-023-29060-7.
Lehrich BM, Liang Y, Fiandaca MS. Foetal bovine serum influence on in vitro extracellular vesicle analyses. J Extracell Vesicles 2021;10. https://doi.org/10.1002/jev2.12061.