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SH-SY5Y Hücrelerinin Kültürlenmesinde Kollajen Kaplama, Fetal Sığır Serum Konsantrasyonu, Diferansiyasyon Ajanları ve Nörotoksin Uygulamasının Parkinson Hastalığının İn vitro Modellemesine Etkileri

Year 2024, Volume: 8 Issue: 2, 105 - 109, 30.08.2024
https://doi.org/10.30565/medalanya.1459470

Abstract

Amaç: Bu çalışma, Parkinson hastalığı (PH) araştırmaları için doğru in vitro hastalık modelleri geliştirmek amacıyla SH-SY5Y kültür koşullarını optimize etmeyi amaçlamaktadır. Kollajen kaplama, fetal sığır serum (FSS) konsantrasyonu, diferansiyasyon ajanları ve nörotoksin tedavileri gibi çeşitli faktörlerin hücresel davranış ve hastalık modellemesi üzerindeki etkilerini araştırmayı hedeflemektedir.

Yöntem: İnsan nöroblastoma SH-SY5Y hücre hattı, ısı ile inaktive edilmiş fetal sığır serumu, penisilin-streptomisin ve L-glutamin ile desteklenmiş DMEM/F12 içinde kültürlenmiştir. Hücre diferansiyasyonu üzerindeki etkisini değerlendirmek için kollajen kaplama uygulanmış, ideal hücre yoğunluğu ve serum oranı ise deneysel olarak belirlenmiştir. Parakuat'ın sitotoksik etkilerini değerlendirmek için MTT testi kullanılmış, dopamin seviyeleri ELISA ile ölçülmüştür. Gen ekspresyonu gerçek zamanlı qPCR ile analiz edilmiştir. Parkinson modelini doğrulamak ve hücresel morfolojiyi değerlendirmek için immünofloresan boyama ve nörit uzunluğu ölçümleri yapılmıştır.

Bulgular: 5x103 hücre/cm2 yoğunluğunda kültürlenmiş hücreler, kollajen ve %2 FSS ile retinoik asit maruziyetinde dopaminerjik nöron özellikleri sergilemiştir. Bununla birlikte, parakuat tedavisi nörotoksisiteye neden olmuş, dopamin seviyelerinde azalma ve nörit büyümesinde bozulma gözlenmiştir.

Sonuç: Bu çalışma, PH modellemesi için SH-SY5Y hücre kültürü koşullarınının optimizasyonu araştırmıştır. Temel bulgular arasında optimal hücre yoğunluğu, FSS konsantrasyonu ve kolajen kaplamanın faydalı etkileri yer almaktadır. Ek olarak, nöronal farklılaşma ve dejenerasyon konusunda gelecekteki araştırmalar için sağlam bir çerçeve sağlayan etkili bir parakuat nörotoksisite protokolü oluşturulmuştur.

Project Number

220S745

References

  • 1. Hoffmann LF, Martins A, Majolo F, Contini V, Laufer S, Goettert MI. Neural regeneration research model to be explored: SH-SY5Y human neuroblastoma cells. Neural Regen Res 2022;18:1265–6. https://doi.org/10.4103/1673-5374.358621.
  • 2. Ross RA, Spengler BA, Biedler JL. Coordinate Morphological and Biochemical Interconversion of Human Neuroblastoma Cells2. JNCI: Journal of the National Cancer Institute 1983;71:741–7. https://doi.org/10.1093/jnci/71.4.741.
  • 3. Påhlman S, Hoehner JC, Nånberg E, Hedborg F, Fagerström S, Gestblom C, et al. Differentiation and survival influences of growth factors in human neuroblastoma. European Journal of Cancer 1995;31:453–8. https://doi.org/10.1016/0959-8049(95)00033-F.
  • 4. Adem A, Mattsson MEK, Nordberg A, Påhlman S. Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation. Developmental Brain Research 1987;33:235–42. https://doi.org/10.1016/0165-3806(87)90156-8.
  • 5. Melino G, Thiele CJ, Knight RA, Piacentini M. Retinoids and the control of growth/death decisions in human neuroblastoma cell lines. J Neurooncol 1997;31:65–83. https://doi.org/10.1023/A:1005733430435.
  • 6. Ivankovic-Dikic I, Grönroos E, Blaukat A, Barth B-U, Dikic I. Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins. Nat Cell Biol 2000;2:574–81. https://doi.org/10.1038/35023515.
  • 7. Hahn M, Glass T, Koke J. Extracellular matrix effects on a neuroblastoma cell line. Cytobios 2000;102:7–19.
  • 8. Kumar S, Weaver VM. Mechanics, malignancy, and metastasis: The force journey of a tumor cell. Cancer Metastasis Rev 2009;28:113–27. https://doi.org/10.1007/s10555-008-9173-4.
  • 9. Discher DE, Janmey P, Wang Y. Tissue Cells Feel and Respond to the Stiffness of Their Substrate. Science 2005;310:1139–43. https://doi.org/10.1126/science.1116995.
  • 10. Ali SA, Pappas IS, Parnavelas JG. Collagen type IV promotes the differentiation of neuronal progenitors and inhibits astroglial differentiation in cortical cell cultures. Developmental Brain Research 1998;110:31–8. https://doi.org/10.1016/S0165-3806(98)00091-1.
  • 11. Pekel NB. Diabetes Mellitusun Parkinson Hastalığında Non-Motor Semptomlar Üzerine Etkisi. Acta Med Alanya 2019;3:293–9. https://doi.org/10.30565/medalanya.569168.
  • 12. Hawkes CH, Del Tredici K, Braak H. A timeline for Parkinson’s disease. Parkinsonism & Related Disorders 2010;16:79–84. https://doi.org/10.1016/j.parkreldis.2009.08.007.
  • 13. Erdem NŞ, Gencer GYG, Özkaynak SS, Uçar T. Effect of Subthalamic Nucleus Deep Brain Stimulation Treatment on Non-motor Symptoms and Sleep Quality in Parkinson’s Disease Patients. Acta Med Alanya 2023;7:59–65. https://doi.org/10.30565/medalanya.1221314.
  • 14. Maria S, Helle B, Tristan L, Gaynor S, Arnar A, Michele M, et al. Improved cell therapy protocol for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC and non-human primate iPSC-derived DA neurons. Stem Cells 2013;31:1548–62. https://doi.org/10.1002/stem.1415.
  • 15. Gantner C, Cota A, Thompson L, Parish C. An Optimized Protocol for the Generation of Midbrain Dopamine Neurons under Defined Conditions. STAR Protocols 2020;1:100065. https://doi.org/10.1016/j.xpro.2020.100065.
  • 16. Ferrari E, Cardinale A, Picconi B, Gardoni F. From cell lines to pluripotent stem cells for modelling Parkinson’s Disease. Journal of Neuroscience Methods 2020;340:108741. https://doi.org/10.1016/j.jneumeth.2020.108741.
  • 17. Dwane S, Durack E, Kiely PA. Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration. BMC Res Notes 2013;6:366. https://doi.org/10.1186/1756-0500-6-366.
  • 18. Moretto E, Stuart S, Surana S, Vargas JNS, Schiavo G. The Role of Extracellular Matrix Components in the Spreading of Pathological Protein Aggregates. Front Cell Neurosci 2022;16. https://doi.org/10.3389/fncel.2022.844211.
  • 19. Xicoy H, Wieringa B, Martens GJM. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegeneration 2017;12:10. https://doi.org/10.1186/s13024-017-0149-0.
  • 20. Zhai Y, Quanwei W, Zhu Z, Zheng W, Ma S, Hao Y, et al. Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis. Biomedical Materials 2022;17. https://doi.org/10.1088/1748-605X/ac4089.
  • 21. Fiore NJ, Tamer-Mahoney JD, Beheshti A, Nieland TJF, Kaplan DL. 3D biocomposite culture enhances differentiation of dopamine-like neurons from SH-SY5Y cells: A model for studying Parkinson’s disease phenotypes. Biomaterials 2022;290:121858. https://doi.org/10.1016/j.biomaterials.2022.121858.
  • 22. Grosman-Dziewiszek P, Wiatrak B, Dziewiszek W, Jawień P, Mydlikowski R, Bolejko R, et al. Influence of 40 Hz and 100 Hz Vibration on SH-SY5Y Cells Growth and Differentiation—A Preliminary Study. Molecules 2022;27:3337. https://doi.org/10.3390/molecules27103337.
  • 23. Simões RF, Ferrão R, Silva MR, Pinho SLC, Ferreira L, Oliveira PJ, et al. Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research. Food and Chemical Toxicology 2021;149:111967. https://doi.org/10.1016/j.fct.2021.111967.
  • 24. Liu Q, Lü L, Sun H, Zhang J, Ma W, Zhang T. [Effect of serum on the differentiation of neural stem cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2018;32:223–7. https://doi.org/10.7507/1002-1892.201710113.
  • 25. Biffi E, Regalia G, Menegon A, Ferrigno G, Pedrocchi A. The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study. PLOS ONE 2013;8:e83899. https://doi.org/10.1371/journal.pone.0083899.
  • 26. Kaya ZB, Santiago-Padilla V, Lim M, Boschen SL, Atilla P, McLean PJ. Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability. Sci Rep 2024;14:4775. https://doi.org/10.1038/s41598-024-55516-5.
  • 27. Singh S, Somvanshi RK, Kumar U. Somatostatin-Mediated Regulation of Retinoic Acid-Induced Differentiation of SH-SY5Y Cells: Neurotransmitters Phenotype Characterization. Biomedicines 2022;10:337. https://doi.org/10.3390/biomedicines10020337.
  • 28. Tanner CM, Kamel F, Ross GW, Hoppin JA, Goldman SM, Korell M, et al. Rotenone, Paraquat, and Parkinson’s Disease. Environmental Health Perspectives 2011;119:866–72. https://doi.org/10.1289/ehp.1002839.
  • 29. Izumi Y, Ezumi M, Takada-Takatori Y, Akaike A, Kume T. Endogenous Dopamine Is Involved in the Herbicide Paraquat-Induced Dopaminergic Cell Death. Toxicological Sciences 2014;139:466–78. https://doi.org/10.1093/toxsci/kfu054.
  • 30. Shi G, Zhang C, Bai X, Sun J, Wang K, Meng Q, et al. A potential mechanism clue to the periodic storm from microglia activation and progressive neuron damage induced by paraquat exposure. Environmental Toxicology 2024;39:1874–88. https://doi.org/10.1002/tox.24053.

Effects of Collagen Coating, Fetal Bovine Serum Concentration, Differentiation Agents, and Neurotoxin Application on In Vitro Modeling of Parkinson's Disease Using SH-SY5Y Cell Culture

Year 2024, Volume: 8 Issue: 2, 105 - 109, 30.08.2024
https://doi.org/10.30565/medalanya.1459470

Abstract

Aim: This study aims to optimize SH-SY5Y culture conditions to develop precise in vitro disease models for Parkinson's disease (PD) research. It seeks to investigate the effects of various factors such as collagen coating, fetal bovine serum concentration, differentiation agents, and neurotoxin treatments on cellular behavior and disease modeling.

Materials and Methods: The human neuroblastoma SH-SY5Y cell line was cultured in DMEM/F12 supplemented with heat-inactivated fetal bovine serum (FBS), penicillin-streptomycin, and L-glutamine. Collagen coating was applied to assess its impact on cell differentiation, while the ideal cell density and serum ratio for generating neurite-like cells were determined through experimentation. The MTT assay was employed to evaluate the cytotoxic effects of paraquat, while dopamine levels were quantified using ELISA. Gene expression was analyzed via real-time qPCR. Immunofluorescence staining and neurite length measurements were conducted to validate the PD model and assess cellular morphology.

Results: Cells cultured at a density of 5x103 cells/cm2 with collagen and 2% FBS exhibited characteristics of dopaminergic neurons upon exposure to retinoic acid. Conversely, paraquat treatment induced neurotoxicity, resulting in decreased dopamine levels and impaired neurite outgrowth.

Conclusion: This study investigated the optimization of SH-SY5Y cell culture conditions for PD modeling. Key findings include optimal cell density, FBS concentration, and beneficial effects of collagen coating. Additionally, an effective paraquat neurotoxicity protocol has been established, providing a solid framework for future research on neuronal differentiation and degeneration.

Ethical Statement

Since our study involves in vitro cell culture techniques, ethics committee permission is not required.

Supporting Institution

TUBİTAK-220S745

Project Number

220S745

References

  • 1. Hoffmann LF, Martins A, Majolo F, Contini V, Laufer S, Goettert MI. Neural regeneration research model to be explored: SH-SY5Y human neuroblastoma cells. Neural Regen Res 2022;18:1265–6. https://doi.org/10.4103/1673-5374.358621.
  • 2. Ross RA, Spengler BA, Biedler JL. Coordinate Morphological and Biochemical Interconversion of Human Neuroblastoma Cells2. JNCI: Journal of the National Cancer Institute 1983;71:741–7. https://doi.org/10.1093/jnci/71.4.741.
  • 3. Påhlman S, Hoehner JC, Nånberg E, Hedborg F, Fagerström S, Gestblom C, et al. Differentiation and survival influences of growth factors in human neuroblastoma. European Journal of Cancer 1995;31:453–8. https://doi.org/10.1016/0959-8049(95)00033-F.
  • 4. Adem A, Mattsson MEK, Nordberg A, Påhlman S. Muscarinic receptors in human SH-SY5Y neuroblastoma cell line: regulation by phorbol ester and retinoic acid-induced differentiation. Developmental Brain Research 1987;33:235–42. https://doi.org/10.1016/0165-3806(87)90156-8.
  • 5. Melino G, Thiele CJ, Knight RA, Piacentini M. Retinoids and the control of growth/death decisions in human neuroblastoma cell lines. J Neurooncol 1997;31:65–83. https://doi.org/10.1023/A:1005733430435.
  • 6. Ivankovic-Dikic I, Grönroos E, Blaukat A, Barth B-U, Dikic I. Pyk2 and FAK regulate neurite outgrowth induced by growth factors and integrins. Nat Cell Biol 2000;2:574–81. https://doi.org/10.1038/35023515.
  • 7. Hahn M, Glass T, Koke J. Extracellular matrix effects on a neuroblastoma cell line. Cytobios 2000;102:7–19.
  • 8. Kumar S, Weaver VM. Mechanics, malignancy, and metastasis: The force journey of a tumor cell. Cancer Metastasis Rev 2009;28:113–27. https://doi.org/10.1007/s10555-008-9173-4.
  • 9. Discher DE, Janmey P, Wang Y. Tissue Cells Feel and Respond to the Stiffness of Their Substrate. Science 2005;310:1139–43. https://doi.org/10.1126/science.1116995.
  • 10. Ali SA, Pappas IS, Parnavelas JG. Collagen type IV promotes the differentiation of neuronal progenitors and inhibits astroglial differentiation in cortical cell cultures. Developmental Brain Research 1998;110:31–8. https://doi.org/10.1016/S0165-3806(98)00091-1.
  • 11. Pekel NB. Diabetes Mellitusun Parkinson Hastalığında Non-Motor Semptomlar Üzerine Etkisi. Acta Med Alanya 2019;3:293–9. https://doi.org/10.30565/medalanya.569168.
  • 12. Hawkes CH, Del Tredici K, Braak H. A timeline for Parkinson’s disease. Parkinsonism & Related Disorders 2010;16:79–84. https://doi.org/10.1016/j.parkreldis.2009.08.007.
  • 13. Erdem NŞ, Gencer GYG, Özkaynak SS, Uçar T. Effect of Subthalamic Nucleus Deep Brain Stimulation Treatment on Non-motor Symptoms and Sleep Quality in Parkinson’s Disease Patients. Acta Med Alanya 2023;7:59–65. https://doi.org/10.30565/medalanya.1221314.
  • 14. Maria S, Helle B, Tristan L, Gaynor S, Arnar A, Michele M, et al. Improved cell therapy protocol for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC and non-human primate iPSC-derived DA neurons. Stem Cells 2013;31:1548–62. https://doi.org/10.1002/stem.1415.
  • 15. Gantner C, Cota A, Thompson L, Parish C. An Optimized Protocol for the Generation of Midbrain Dopamine Neurons under Defined Conditions. STAR Protocols 2020;1:100065. https://doi.org/10.1016/j.xpro.2020.100065.
  • 16. Ferrari E, Cardinale A, Picconi B, Gardoni F. From cell lines to pluripotent stem cells for modelling Parkinson’s Disease. Journal of Neuroscience Methods 2020;340:108741. https://doi.org/10.1016/j.jneumeth.2020.108741.
  • 17. Dwane S, Durack E, Kiely PA. Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration. BMC Res Notes 2013;6:366. https://doi.org/10.1186/1756-0500-6-366.
  • 18. Moretto E, Stuart S, Surana S, Vargas JNS, Schiavo G. The Role of Extracellular Matrix Components in the Spreading of Pathological Protein Aggregates. Front Cell Neurosci 2022;16. https://doi.org/10.3389/fncel.2022.844211.
  • 19. Xicoy H, Wieringa B, Martens GJM. The SH-SY5Y cell line in Parkinson’s disease research: a systematic review. Mol Neurodegeneration 2017;12:10. https://doi.org/10.1186/s13024-017-0149-0.
  • 20. Zhai Y, Quanwei W, Zhu Z, Zheng W, Ma S, Hao Y, et al. Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis. Biomedical Materials 2022;17. https://doi.org/10.1088/1748-605X/ac4089.
  • 21. Fiore NJ, Tamer-Mahoney JD, Beheshti A, Nieland TJF, Kaplan DL. 3D biocomposite culture enhances differentiation of dopamine-like neurons from SH-SY5Y cells: A model for studying Parkinson’s disease phenotypes. Biomaterials 2022;290:121858. https://doi.org/10.1016/j.biomaterials.2022.121858.
  • 22. Grosman-Dziewiszek P, Wiatrak B, Dziewiszek W, Jawień P, Mydlikowski R, Bolejko R, et al. Influence of 40 Hz and 100 Hz Vibration on SH-SY5Y Cells Growth and Differentiation—A Preliminary Study. Molecules 2022;27:3337. https://doi.org/10.3390/molecules27103337.
  • 23. Simões RF, Ferrão R, Silva MR, Pinho SLC, Ferreira L, Oliveira PJ, et al. Refinement of a differentiation protocol using neuroblastoma SH-SY5Y cells for use in neurotoxicology research. Food and Chemical Toxicology 2021;149:111967. https://doi.org/10.1016/j.fct.2021.111967.
  • 24. Liu Q, Lü L, Sun H, Zhang J, Ma W, Zhang T. [Effect of serum on the differentiation of neural stem cells]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi 2018;32:223–7. https://doi.org/10.7507/1002-1892.201710113.
  • 25. Biffi E, Regalia G, Menegon A, Ferrigno G, Pedrocchi A. The Influence of Neuronal Density and Maturation on Network Activity of Hippocampal Cell Cultures: A Methodological Study. PLOS ONE 2013;8:e83899. https://doi.org/10.1371/journal.pone.0083899.
  • 26. Kaya ZB, Santiago-Padilla V, Lim M, Boschen SL, Atilla P, McLean PJ. Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability. Sci Rep 2024;14:4775. https://doi.org/10.1038/s41598-024-55516-5.
  • 27. Singh S, Somvanshi RK, Kumar U. Somatostatin-Mediated Regulation of Retinoic Acid-Induced Differentiation of SH-SY5Y Cells: Neurotransmitters Phenotype Characterization. Biomedicines 2022;10:337. https://doi.org/10.3390/biomedicines10020337.
  • 28. Tanner CM, Kamel F, Ross GW, Hoppin JA, Goldman SM, Korell M, et al. Rotenone, Paraquat, and Parkinson’s Disease. Environmental Health Perspectives 2011;119:866–72. https://doi.org/10.1289/ehp.1002839.
  • 29. Izumi Y, Ezumi M, Takada-Takatori Y, Akaike A, Kume T. Endogenous Dopamine Is Involved in the Herbicide Paraquat-Induced Dopaminergic Cell Death. Toxicological Sciences 2014;139:466–78. https://doi.org/10.1093/toxsci/kfu054.
  • 30. Shi G, Zhang C, Bai X, Sun J, Wang K, Meng Q, et al. A potential mechanism clue to the periodic storm from microglia activation and progressive neuron damage induced by paraquat exposure. Environmental Toxicology 2024;39:1874–88. https://doi.org/10.1002/tox.24053.
There are 30 citations in total.

Details

Primary Language English
Subjects Geriatrics and Gerontology
Journal Section Research Article
Authors

Fatma Gonca Koçancı 0000-0002-7248-7933

Project Number 220S745
Publication Date August 30, 2024
Submission Date March 27, 2024
Acceptance Date August 15, 2024
Published in Issue Year 2024 Volume: 8 Issue: 2

Cite

Vancouver Koçancı FG. Effects of Collagen Coating, Fetal Bovine Serum Concentration, Differentiation Agents, and Neurotoxin Application on In Vitro Modeling of Parkinson’s Disease Using SH-SY5Y Cell Culture. Acta Med. Alanya. 2024;8(2):105-9.

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