Dokument: Etablierung von dreidimensionalen Zellkulturmodellen
| Titel: | Etablierung von dreidimensionalen Zellkulturmodellen | |||||||
| Weiterer Titel: | Establishment of three-dimensional cell culture models | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=72461 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20260318-081407-3 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Deutsch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Görtz, Lotte Anna [Autor] | |||||||
| Dateien: |
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| Beitragende: | Univ.-Prof. Dr. med. Aubin, Hug [Gutachter] Prof. Distler, Jörg [Gutachter] | |||||||
| Stichwörter: | Organoide | |||||||
| Dewey Dezimal-Klassifikation: | 600 Technik, Medizin, angewandte Wissenschaften » 610 Medizin und Gesundheit | |||||||
| Beschreibungen: | Herz-Kreislauf-Erkrankungen sind weltweit die häufigste Todesursache und fordern demnach die Entwicklung neuer Forschungsmodelle zur Untersuchung krankheitsrelevanter Prozesse. Da Krankheitsprozesse in vivo im drei-dimensionalen Raum ablaufen, gewinnen dreidimensionalen Gewebe- und Organstrukturen (Sphäroide/Organoide) als realitätsnahe Modelle für die Forschung zunehmend an Bedeutung. Das Herz konnte bereits als Kardioid aus humanen pluripotenten Stammzellen generiert werden. Ziel dieser Arbeit ist zunächst eine Literaturrecherche bezüglich bereits etablierter Protokolle zur Kardioid-Generierung, ein tabellarischer Vergleich der dazu genutzten Methoden und Materialien und anschließend die Generierung von Kardioiden aus mesenchymalen Stammzellen aus viszeralem Rattenfettgewebe. Eine Gene-rierung dieser Kardioide, basierend auf dem Protokoll von Hofbauer et al. [1], war erfolglos, sodass Anpassungen des Protokolls vorgesehen sind, um es in Zukunft auch auf humane mesenchymale Stammzellen übertragen zu können. Dies könnte personalisierte Medizin ermöglichen, etwa Kardioide aus Zellen herzkranker Patienten zu gewinnen und diese auf Entstehung sowie Therapien von Herz-Kreislauf-Erkrankungen zu untersuchen. Ein weiteres Ziel dieser Arbeit ist die Generierung von Sphäroiden aus Glattmuskelzellen, da Glattmuskelzellen bei Entzündungsprozessen ihren Phänotypen verändern und so die Entstehung von Herz-Kreislauf-Erkrankungen beeinflussen können. Da die entsprechenden Pathomechanismen noch nicht vollständig aufgedeckt sind, sollen diese Glattmuskelzell-Sphäroide als zukünftiges Forschungsmodell dienen. Es ist gelungen, Glattmuskelzell-Sphäroide zu generieren und diese mittels Mikroskopie-Analysen und einer Proteomics-Analyse mit der 2D-Zellkultur zu vergleichen. Es zeigte sich eine unterschiedliche Proteinzusammensetzung je nach Zellkulturmodell. Proteine der extrazellulären Matrix wiesen im 3D-Modell beispielsweise eine signifikant höhere Proteinabundanz auf, was der in vivo-Situation ähnelt. Zusätzlich erfolgte ein Vergleich der Zellkulturmodelle im Hinblick auf deren Proteinzusammensetzung nach Stimulation mit einem inflammatorischen Reiz. In Zukunft müssen weiterführende Experimente durchgeführt werden, um zu analysieren, ob das 3D-Modell die in vivo-Situation adäquater widerspiegelt als das 2D-Modell.Cardiovascular diseases are the leading cause of death worldwide and therefore require the development of new research models for investigating disease-related processes. Since disease processes occur in vivo in three-dimensional space, three-dimensional tissue and organ structures (spheroids/organoids) are becoming increasingly important as realistic models for research. The heart has already been generated as a cardioid from human pluripotent stem cells. The aim of this work is first to conduct a literature search on established protocols for cardioid generation, to compare the methods and materials used in a table, and then to generate cardioids from mesenchymal stem cells from visceral rat adipose tissue. Generation of these cardioids based on the protocol of Hofbauer et al. [1] was unsuccessful, so adjustments to the protocol are planned in order to be able to transfer it to human mesenchymal stem cells in the future. This could enable personalised medicine, for example, obtaining cardioids from cells of patients with heart disease and examining them for the development and treatment of cardiovascular diseases.
Another goal of this work is to generate spheroids from smooth muscle cells, as smooth muscle cells change their phenotype during inflammatory processes and can thus influence the development of cardiovascular diseases. Since the corresponding pathomechanisms have not yet been fully elucidated, these smooth muscle cell spheroids are to serve as a future research model. We succeeded in generating smooth muscle cell spheroids and comparing them with 2D cell culture using microscopy analyses and proteomics analysis. The results showed a different protein composition depending on the cell culture model. For example, extracellular matrix proteins showed significantly higher protein abundance in the 3D model, which is similar to the in vivo situation. In addition, the cell culture models were compared in terms of their protein composition after stimulation with an inflammatory stimulus. Further experiments will need to be conducted in the future to analyse whether the 3D model reflects the in vivo situation more adequately than the 2D model. | |||||||
| Quelle: | 1. Hofbauer, P., et al., Cardioids reveal self-organizing principles of human cardiogenesis. Cell, 2021. 184(12): p. 3299-3317 e22.
2. Jensen, C. and Y. Teng, Is It Time to Start Transitioning From 2D to 3D Cell Culture? Front Mol Biosci, 2020. 7: p. 33. 3. Habanjar, O., et al., 3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages. Int J Mol Sci, 2021. 22(22). 4. Lancaster, M.A. and J.A. Knoblich, Organogenesis in a dish: modeling development and disease using organoid technologies. Science, 2014. 345(6194): p. 1247125. 5. Taylor, J., et al., Generation of immune cell containing adipose organoids for in vitro analysis of immune metabolism. Sci Rep, 2020. 10(1): p. 21104. 6. Kapalczynska, M., et al., 2D and 3D cell cultures - a comparison of different types of cancer cell cultures. Arch Med Sci, 2018. 14(4): p. 910-919. 7. Ryu, N.E., S.H. Lee, and H. Park, Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells. Cells, 2019. 8(12). 8. Ho, B.X., N.M.Q. Pek, and B.S. Soh, Disease Modeling Using 3D Organoids Derived from Human Induced Pluripotent Stem Cells. Int J Mol Sci, 2018. 19(4). 9. Yin, X., et al., Engineering Stem Cell Organoids. Cell Stem Cell, 2016. 18(1): p. 25-38. 10. Valdoz, J.C., et al., The ECM: To Scaffold, or Not to Scaffold, That Is the Question. Int J Mol Sci, 2021. 22(23). 11. Frantz, C., K.M. Stewart, and V.M. Weaver, The extracellular matrix at a glance. J Cell Sci, 2010. 123(Pt 24): p. 4195-200. 12. Nii, T. and Y. Katayama, Biomaterial-Assisted Regenerative Medicine. Int J Mol Sci, 2021. 22(16). 13. Hughes, C.S., L.M. Postovit, and G.A. Lajoie, Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics, 2010. 10(9): p. 1886-90. 14. Caliari, S.R. and J.A. Burdick, A practical guide to hydrogels for cell culture. Nat Methods, 2016. 13(5): p. 405-14. 15. Rajan, S., D.S. Kudryashov, and E. Reisler, Actin Bundles Dynamics and Architecture. Biomolecules, 2023. 13(3). 16. Foth, B.J., M.C. Goedecke, and D. Soldati, New insights into myosin evolution and classification. Proc Natl Acad Sci U S A, 2006. 103(10): p. 3681-6. 17. Odronitz, F. and M. Kollmar, Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species. Genome Biol, 2007. 8(9): p. R196. 18. Pecci, A., et al., MYH9: Structure, functions and role of non-muscle myosin IIA in human disease. Gene, 2018. 664: p. 152-167. 19. Takada, Y., X. Ye, and S. Simon, The integrins. Genome Biol, 2007. 8(5): p. 215. 20. Gordon, M.K. and R.A. Hahn, Collagens. Cell Tissue Res, 2010. 339(1): p. 247-57. 21. Dalton, C.J. and C.A. Lemmon, Fibronectin: Molecular Structure, Fibrillar Structure and Mechanochemical Signaling. Cells, 2021. 10(9). 22. Stenina-Adognravi, O. and E.F. Plow, Thrombospondin-4 in tissue remodeling. Matrix Biol, 2019. 75-76: p. 300-313. 23. Ruzha, Y., et al., Role of Vitronectin and Its Receptors in Neuronal Function and Neurodegenerative Diseases. Int J Mol Sci, 2022. 23(20). 24. Dzobo, K. and C. Dandara, The Extracellular Matrix: Its Composition, Function, Remodeling, and Role in Tumorigenesis. Biomimetics (Basel), 2023. 8(2). 25. Murphy, K.N. and A.J. Brinkworth, Manipulation of Focal Adhesion Signaling by Pathogenic Microbes. Int J Mol Sci, 2021. 22(3). 26. Revach, O.Y., I. Grosheva, and B. Geiger, Biomechanical regulation of focal adhesion and invadopodia formation. J Cell Sci, 2020. 133(20). 27. Nersisyan, S., et al., ECM-Receptor Regulatory Network and Its Prognostic Role in Colorectal Cancer. Front Genet, 2021. 12: p. 782699. 28. Angulo-Urarte, A., T. van der Wal, and S. Huveneers, Cell-cell junctions as sensors and transducers of mechanical forces. Biochim Biophys Acta Biomembr, 2020. 1862(9): p. 183316. 29. Hartsock, A. and W.J. Nelson, Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta, 2008. 1778(3): p. 660-9. 30. Shankaran, A., et al., Advances in development and application of human organoids. 3 Biotech, 2021. 11(6): p. 257. 31. Bora, P. and A.S. Majumdar, Adipose tissue-derived stromal vascular fraction in regenerative medicine: a brief review on biology and translation. Stem Cell Res Ther, 2017. 8(1): p. 145. 32. Minteer, D., K.G. Marra, and J.P. Rubin, Adipose-derived mesenchymal stem cells: biology and potential applications. Adv Biochem Eng Biotechnol, 2013. 129: p. 59-71. 33. Lewis-Israeli, Y.R., et al., Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease. Nat Commun, 2021. 12(1): p. 5142. 34. Miyamoto, M., et al., Heart organoids and tissue models for modeling development and disease. Semin Cell Dev Biol, 2021. 118: p. 119-128. 35. Lewis-Israeli, Y.R., A.H. Wasserman, and A. Aguirre, Heart Organoids and Engineered Heart Tissues: Novel Tools for Modeling Human Cardiac Biology and Disease. Biomolecules, 2021. 11(9). 36. Ozhan, G. and G. Weidinger, Wnt/beta-catenin signaling in heart regeneration. Cell Regen, 2015. 4(1): p. 3. 37. Kruger, M., et al., Insulin signaling regulates cardiac titin properties in heart development and diabetic cardiomyopathy. J Mol Cell Cardiol, 2010. 48(5): p. 910-6. 38. Nakajima, Y., et al., Heart development before beating. Anat Sci Int, 2009. 84(3): p. 67-76. 39. Teo, A.K., et al., Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells, 2012. 30(4): p. 631-42. 40. Asson-Batres, M.A., C. Rochette-Egly, and SpringerLink, The Biochemistry of Retinoid Signaling III : Vitamin A and Retinoic Acid in Embryonic Development. 1st 2020. ed. Subcellular Biochemistry 95. 2020, Cham: Springer International Publishing : Imprint: Springer. 41. Yamazaki, Y. and T. Morita, Molecular and functional diversity of vascular endothelial growth factors. Mol Divers, 2006. 10(4): p. 515-27. 42. Zhao, M., et al., Y-27632 preconditioning enhances transplantation of human-induced pluripotent stem cell-derived cardiomyocytes in myocardial infarction mice. Cardiovasc Res, 2019. 115(2): p. 343-356. 43. Matsumoto, T., M.H. Kim, and M. Kino-Oka, Effect of Rho-Associated Kinase Inhibitor on Growth Behaviors of Human Induced Pluripotent Stem Cells in Suspension Culture. Bioengineering (Basel), 2022. 9(11). 44. Wang, G., et al., Origin and differentiation of vascular smooth muscle cells. J Physiol, 2015. 593(14): p. 3013-30. 45. Shi, J., et al., Metabolism of vascular smooth muscle cells in vascular diseases. Am J Physiol Heart Circ Physiol, 2020. 319(3): p. H613-H631. 46. Sorokin, V., et al., Role of Vascular Smooth Muscle Cell Plasticity and Interactions in Vessel Wall Inflammation. Front Immunol, 2020. 11: p. 599415. 47. Gomez, D. and G.K. Owens, Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res, 2012. 95(2): p. 156-64. 48. Chakraborty, R., et al., Promoters to Study Vascular Smooth Muscle. Arterioscler Thromb Vasc Biol, 2019. 39(4): p. 603-612. 49. Rensen, S.S., P.A. Doevendans, and G.J. van Eys, Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J, 2007. 15(3): p. 100-8. 50. Liu, M. and D. Gomez, Smooth Muscle Cell Phenotypic Diversity. Arterioscler Thromb Vasc Biol, 2019. 39(9): p. 1715-1723. 51. Bennett, M.R., S. Sinha, and G.K. Owens, Vascular Smooth Muscle Cells in Atherosclerosis. Circ Res, 2016. 118(4): p. 692-702. 52. Feil, S., et al., Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis. Circ Res, 2014. 115(7): p. 662-7. 53. Orr, A.W., et al., Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis. J Vasc Res, 2010. 47(2): p. 168-80. 54. Pan, H., et al., Single-Cell Genomics Reveals a Novel Cell State During Smooth Muscle Cell Phenotypic Switching and Potential Therapeutic Targets for Atherosclerosis in Mouse and Human. Circulation, 2020. 142(21): p. 2060-2075. 55. Wong, D., A.W. Turner, and C.L. Miller, Genetic Insights Into Smooth Muscle Cell Contributions to Coronary Artery Disease. Arterioscler Thromb Vasc Biol, 2019. 39(6): p. 1006-1017. 56. Hughes, C.S., et al., Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc, 2019. 14(1): p. 68-85. 57. Aravamudhan, S., et al., Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein. J Mol Cell Cardiol, 2021. 154: p. 41-59. 58. Andersen, P., et al., Precardiac organoids form two heart fields via Bmp/Wnt signaling. Nat Commun, 2018. 9(1): p. 3140. 59. Branco, M.A., et al., Human multilineage pro-epicardium/foregut organoids support the development of an epicardium/myocardium organoid. Nat Commun, 2022. 13(1): p. 6981. 60. Filippo Buono, M., et al., Human Cardiac Organoids for Modeling Genetic Cardiomyopathy. Cells, 2020. 9(7). 61. Feng, W., et al., Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with NKX2-5 deficiency. Commun Biol, 2022. 5(1): p. 399. 62. Drakhlis, L., et al., Human heart-forming organoids recapitulate early heart and foregut development. Nat Biotechnol, 2021. 39(6): p. 737-746. 63. Forsythe, S.D., et al., Environmental Toxin Screening Using Human-Derived 3D Bioengineered Liver and Cardiac Organoids. Front Public Health, 2018. 6: p. 103. 64. Hoang, P., et al., Generation of spatial-patterned early-developing cardiac organoids using human pluripotent stem cells. Nat Protoc, 2018. 13(4): p. 723-737. 65. Hoang, P., et al., Engineering spatial-organized cardiac organoids for developmental toxicity testing. Stem Cell Reports, 2021. 16(5): p. 1228-1244. 66. Kitsuka, T., et al., 2-Cl-C.OXT-A stimulates contraction through the suppression of phosphodiesterase activity in human induced pluripotent stem cell-derived cardiac organoids. PLoS One, 2019. 14(7): p. e0213114. 67. LaBarge, W., et al., Maturation of three-dimensional, hiPSC-derived cardiomyocyte spheroids utilizing cyclic, uniaxial stretch and electrical stimulation. PLoS One, 2019. 14(7): p. e0219442. 68. Lee, J., et al., In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix. Nat Commun, 2020. 11(1): p. 4283. 69. Lee, S.G., et al., Generation of human iPSCs derived heart organoids structurally and functionally similar to heart. Biomaterials, 2022. 290: p. 121860. 70. Lewis-Israeli, Y.R., et al., Generating Self-Assembling Human Heart Organoids Derived from Pluripotent Stem Cells. J Vis Exp, 2021(175). 71. Meier, A.B., et al., Epicardioid single-cell genomics uncovers principles of human epicardium biology in heart development and disease. Nat Biotechnol, 2023. 72. Mills, R.J., et al., Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proc Natl Acad Sci U S A, 2017. 114(40): p. E8372-E8381. 73. Mills, R.J., et al., Drug Screening in Human PSC-Cardiac Organoids Identifies Pro-proliferative Compounds Acting via the Mevalonate Pathway. Cell Stem Cell, 2019. 24(6): p. 895-907 e6. 74. Min, S., et al., Biphasic Electrical Pulse by a Micropillar Electrode Array Enhances Maturation and Drug Response of Reprogrammed Cardiac Spheroids. Nano Lett, 2020. 20(10): p. 6947-6956. 75. Olmsted, Z.T. and J.L. Paluh, A combined human gastruloid model of cardiogenesis and neurogenesis. iScience, 2022. 25(6): p. 104486. 76. Polonchuk, L., et al., Cardiac spheroids as promising in vitro models to study the human heart microenvironment. Sci Rep, 2017. 7(1): p. 7005. 77. Richards, D.J., et al., Inspiration from heart development: Biomimetic development of functional human cardiac organoids. Biomaterials, 2017. 142: p. 112-123. 78. Richards, D.J., et al., Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity. Nat Biomed Eng, 2020. 4(4): p. 446-462. 79. Rossi, G., et al., Capturing Cardiogenesis in Gastruloids. Cell Stem Cell, 2021. 28(2): p. 230-240 e6. 80. Silva, A.C., et al., Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids. Cell Stem Cell, 2021. 28(12): p. 2137-2152 e6. 81. Varzideh, F., et al., Human cardiomyocytes undergo enhanced maturation in embryonic stem cell-derived organoid transplants. Biomaterials, 2019. 192: p. 537-550. 82. Voges, H.K., et al., Development of a human cardiac organoid injury model reveals innate regenerative potential. Development, 2017. 144(6): p. 1118-1127. 83. Yadav, S., S.J. Shire, and D.S. Kalonia, Viscosity analysis of high concentration bovine serum albumin aqueous solutions. Pharm Res, 2011. 28(8): p. 1973-83. 84. Kikuchi, T., K. Matsuura, and T. Shimizu, Non-coating method for non-adherent cell culture using high molecular weight dextran sulfate and bovine serum albumin. J Biosci Bioeng, 2021. 132(5): p. 537-542. 85. Zhang, L., et al., Stem cell therapy in liver regeneration: Focus on mesenchymal stem cells and induced pluripotent stem cells. Pharmacol Ther, 2022. 232: p. 108004. 86. Liu, G.B., et al., Ghrelin promotes cardiomyocyte differentiation of adipose tissue‑derived mesenchymal stem cells by DDX17‑mediated regulation of the SFRP4/Wnt/beta‑catenin axis. Mol Med Rep, 2023. 28(3). 87. Reed, E., et al., Extracellular Matrix Profiling and Disease Modelling in Engineered Vascular Smooth Muscle Cell Tissues. Matrix Biol Plus, 2022. 16: p. 100122. 88. Jiang, D., Y. Yang, and D. Li, Lipopolysaccharide induced vascular smooth muscle cells proliferation: A new potential therapeutic target for proliferative vascular diseases. Cell Prolif, 2017. 50(2). 89. Yang, X., et al., Proinflammatory phenotype of vascular smooth muscle cells: role of efficient Toll-like receptor 4 signaling. Am J Physiol Heart Circ Physiol, 2005. 289(3): p. H1069-76. | |||||||
| Lizenz: |  Dieses Werk ist lizenziert unter einer Creative Commons Namensnennung 4.0 International Lizenz | |||||||
| Bezug: | 2022-2026 | |||||||
| Fachbereich / Einrichtung: | Medizinische Fakultät | |||||||
| Dokument erstellt am: | 18.03.2026 | |||||||
| Dateien geändert am: | 18.03.2026 | |||||||
| Promotionsantrag am: | 01.09.2025 | |||||||
| Datum der Promotion: | 03.03.2026 |
