Isolation, In Vitro Expansion, And Cryopreservation of Primary Cells Derived from Human Thyroid Carcinoma
DOI:
https://doi.org/10.66266/inajemd.v2i1.35Keywords:
Papillary thyroid carcinoma, primary cell culture, BRAF and RAS mutationsAbstract
Thyroid carcinoma is a malignant neoplasm arising from thyroid parenchymal cells and currently ranks as the fourth most diagnosed cancer in Indonesia. This study aimed to isolate thyroid carcinoma cells for in vitro expansion and long-term preservation as a reliable cell culture stock, including cryopreservation for future research applications. In addition, we sought to identify and characterize cells derived from papillary thyroid carcinoma (PTC) tissue to evaluate the presence of mutations with potential prognostic significance. Primary cell isolation was performed via enzymatic digestion using collagenase, enabling effective separation of tumor cells from adjacent non-malignant thyroid tissue. Cell proliferation was supported using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% fetal bovine serum (FBS), selected for its high concentration of growth-promoting factors that enhance proliferation rates. For biobanking purposes, cryopreservation of the thyroid carcinoma-derived cells was conducted using a standard slow-freezing protocol. Molecular characterization was carried out through PCR amplification, gel electrophoresis, and Sanger sequencing of key oncogenic drivers, specifically the BRAF gene and five RAS gene targets: HRAS exon 2, NRAS exons 2 and 3, and KRAS exons 2 and 3. No pathogenic mutations were identified in the analyzed BRAF or RAS gene regions.
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References
1. Kitahara, C, Schneider, A. 2022. Epidemiology of Thyroid Cancer. Cancer Epidemiology, Biomarkers & Prevention. 2022, 31: 1284-97.
2. Puxeddu, E., Tallini, G, Vanni, R. What is new in thyroid cancer: The special issue of the journal cancers. Cancers (Basel). 2020.
3. Poppe, K, Bisschop, P, Fugazzola, L, Minziori, G., Unuane, D., Weghofer, A. 2021 European Thyroid Association Guideline on Thyroid Disorders prior to and during Assisted Reproduction. Eur Thyroid J. 2021,9: 281-95.
4. Fagin, J.A., Wells, S.A. Biologic and Clinical Perspectives on Thyroid Cancer. New England Journal of Medicine. 2016, 375:1054-67.
5. Ozisik, H., Ozdil, B., Sipahi, M., Erdogan, M., Cetinkalp, S., Ozgen, G., Saygili, F., Oktay, G., Aktug, H. Stemness potency and structural characteristics of thyroid cancer cell lines. Pathol Res Pract. 2023,241:154262.
6. Fallahi, P., Ferrari, S.M., Elia, G., Ragusa, F., Patrizio, A., Paparo, S.R., Marone, G., Galdiero, M.R., Guglielmi, G., Foddis, R., Cristaudo, A., Antonelli, A. Primary cell cultures for the personalized therapy in aggressive thyroid cancer of follicular origin. Semin Cancer Biol. 2022,79:203-16.
7. Wang, Y., Li, W., Phay, J.E., Shen, R., Pellegata, N.S., Saji, M., Ringel, M.D., de la Chapelle, A., He, H. Primary Cell Culture Systems for Human Thyroid Studies. Thyroid. 2016, 26:1131-40.
8. Chew, D., Green, V., Riley, A., England, R.J., Greenman, J. The Changing Face of in vitro Culture Models for Thyroid Cancer Research: A Systematic Literature Review. Front Surg. 2020, 7. Weiskirchen, S., Schröder, S.K., Buhl, E.M., Weiskirchen, R. A Beginner’s Guide to Cell Culture: Practical Advice for Preventing Needless Problems. Cells. 2023.
9. Whaley, D., Damyar, K., Witek, R.P., Mendoza, A., Alexander, M., Lakey, J.R. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplant. 2021, 30, 096368972199961.
10. Xu, X., Quiros, R.M., Gattuso, P., Ain, K.B., Prinz, R.A. High prevalence of BRAF gene mutation in papillary thyroid carcinomas and thyroid tumor cell lines. Cancer Res. 2003, 63: 4561-7
11. Harahap, A.S., Subekti, I., Panigoro, S.S., Asmarinah, Lisnawati, Werdhani, R.A., et al. Profile of BRAFV600E, BRAFK601E, NRAS, HRAS, and KRAS Mutational Status, and Clinicopathological Characteristics of Papillary Thyroid Carcinoma in Indonesian National Referral Hospital, The Application of Clinical Genetics. 2023, 16: 99-110
12. Ferrari, S.M., Fallahi, P., Ruffilli, I., Elia, G., Ragusa, F., Paparo, S.R., Ulisse, S., Baldini, E., Giannini, R., Miccoli, P., Antonelli, A., Basolo, F. Molecular testing in the diagnosis of differentiated thyroid carcinomas.Gland Surg. 2018.
13. Fox, J.M., Wiggins, R.C., Moore, J.W.J., Brewer, C., Andrew, A.C., Martin, F. Methodology for reliable and reproducible cryopreservation of human cervical tissue. Cryobiology. 2017, 77, 14-8.
14. Yang, J., Gao, L., Liu, M., Sui, X., Zhu, Y., Wen, C., Zhang, L. Advanced Biotechnology for Cell Cryopreservation. Transactions of Tianjin University. 2020.
15. Crnčić, T.B., Tomaš, M.I., Girotto, N., Ivanković, S.G. Risk factors for thyroid cancer: What do we know so far? Acta Clin Croat. 2020, 59:66–72.
16. Gavryutina, I., Fordjour, L., Chin, V.L. Genetics of Thyroid Disorders. Endocrines. 2022, 3:198–213.
17. Fagin, J.A., Wells, S.A. Biologic and Clinical Perspectives on Thyroid Cancer. New England Journal of Medicine. 2016, 375, 1054-67.
18. Motoi N, Sakamoto A, Yamochi T, Horiuchi H, Motoi T, Machinami R. Role of ras mutation in the progression of thyroid carcinoma of follicular epithelial origin. Pathol Res Pract. 2000;196 (1):1-7.doi: 10.1016/S0344-0338(00)80015-1.
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