Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas
Los organoides son cultivos 3D desarrollados a partir de células madre creando un ambiente artificial propicio, en el cual las células pueden crecer e interactuar en un entorno tridimensional similar a las condiciones de un estado in vivo, imitando su funcionamiento molecular y celular lo cual permi...
- Autores:
-
Quesada Quintero, Fabio Andrés
Gutiérrez Burgos, Isabella
- Tipo de recurso:
- Trabajo de grado de pregrado
- Fecha de publicación:
- 2022
- Institución:
- Universidad Autónoma de Occidente
- Repositorio:
- RED: Repositorio Educativo Digital UAO
- Idioma:
- spa
- OAI Identifier:
- oai:red.uao.edu.co:10614/13944
- Acceso en línea:
- https://hdl.handle.net/10614/13944
https://red.uao.edu.co/
- Palabra clave:
- Ingeniería Biomédica
Células madre
Cultivo de células
Stem cells
Cell culture
Matriz de colágeno
Diferenciación celular
Organoides
Viabilidad celular
- Rights
- openAccess
- License
- Derechos reservados - Universidad Autónoma de Occidente, 2022
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dc.title.spa.fl_str_mv |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
title |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
spellingShingle |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas Ingeniería Biomédica Células madre Cultivo de células Stem cells Cell culture Matriz de colágeno Diferenciación celular Organoides Viabilidad celular |
title_short |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
title_full |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
title_fullStr |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
title_full_unstemmed |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
title_sort |
Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas |
dc.creator.fl_str_mv |
Quesada Quintero, Fabio Andrés Gutiérrez Burgos, Isabella |
dc.contributor.advisor.none.fl_str_mv |
Neuta Arciniegas, Paola Andrea |
dc.contributor.author.none.fl_str_mv |
Quesada Quintero, Fabio Andrés Gutiérrez Burgos, Isabella |
dc.subject.spa.fl_str_mv |
Ingeniería Biomédica |
topic |
Ingeniería Biomédica Células madre Cultivo de células Stem cells Cell culture Matriz de colágeno Diferenciación celular Organoides Viabilidad celular |
dc.subject.armarc.spa.fl_str_mv |
Células madre Cultivo de células |
dc.subject.armarc.eng.fl_str_mv |
Stem cells Cell culture |
dc.subject.proposal.spa.fl_str_mv |
Matriz de colágeno Diferenciación celular Organoides Viabilidad celular |
description |
Los organoides son cultivos 3D desarrollados a partir de células madre creando un ambiente artificial propicio, en el cual las células pueden crecer e interactuar en un entorno tridimensional similar a las condiciones de un estado in vivo, imitando su funcionamiento molecular y celular lo cual permite tener un resultado en investigación de enfermedades y fármacos muy preciso. Para obtener un organoidese deben cultivar células madre en un medio favorable que permita su crecimiento y desarrollo. El propósito de esta investigación fue realizar el diseño de un método de cultivo que permita un adecuado entorno de crecimiento para el desarrollo de organoides de miocardio a partir de un cultivo de células madre mesenquimales (MSCs). Las células MSCs primero pasaron por un proceso de diferenciación acélulas endoteliales y cardiomiocitos que tarda entre 15 y 21 días respectivamente y posteriormente fueron utilizadas junto con hidrogel compuesto de colágeno como medio de cultivo para el desarrollo del organoides de miocardio. Los protocolos implementados para la gelificación del hidrogel fueron evaluados mediante la viabilidad celular del organoide al pasar 7 y 15 días de incubación, donde se realizó la observación de componentes de células vivas con pruebas de tinción supravital. |
publishDate |
2022 |
dc.date.accessioned.none.fl_str_mv |
2022-06-02T15:16:52Z |
dc.date.available.none.fl_str_mv |
2022-06-02T15:16:52Z |
dc.date.issued.none.fl_str_mv |
2022-05-18 |
dc.type.spa.fl_str_mv |
Trabajo de grado - Pregrado |
dc.type.coarversion.fl_str_mv |
http://purl.org/coar/version/c_71e4c1898caa6e32 |
dc.type.coar.eng.fl_str_mv |
http://purl.org/coar/resource_type/c_7a1f |
dc.type.content.eng.fl_str_mv |
Text |
dc.type.driver.eng.fl_str_mv |
info:eu-repo/semantics/bachelorThesis |
dc.type.redcol.eng.fl_str_mv |
https://purl.org/redcol/resource_type/TP |
format |
http://purl.org/coar/resource_type/c_7a1f |
dc.identifier.uri.none.fl_str_mv |
https://hdl.handle.net/10614/13944 |
dc.identifier.instname.spa.fl_str_mv |
Universidad Autónoma de Occidente |
dc.identifier.reponame.spa.fl_str_mv |
Repositorio Educativo Digital |
dc.identifier.repourl.spa.fl_str_mv |
https://red.uao.edu.co/ |
url |
https://hdl.handle.net/10614/13944 https://red.uao.edu.co/ |
identifier_str_mv |
Universidad Autónoma de Occidente Repositorio Educativo Digital |
dc.language.iso.spa.fl_str_mv |
spa |
language |
spa |
dc.relation.cites.spa.fl_str_mv |
Quesada Quintero, F.A. y Gutiérrez Burgos, I. (2022). Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas. (Pasantía de investigación). Universidad Autónoma de Occidente. Cali. Colombia |
dc.relation.references.none.fl_str_mv |
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Manzini, “Declaración De Helsinki: Principios Éticos Para La Investigación Médica Sobre Sujetos Humanos,” Acta Bioeth, vol. 6, no. 2, pp. 321–334, 2000, doi: 10.4067/s1726-569x2000000200010. [7] K. E. Sung, X. Su, E. Berthier, C. Pehlke, A. Friedl, and D. J. Beebe, “Understanding the Impact of 2D and 3D Fibroblast Cultures on In Vitro Breast Cancer Models,” PLoS ONE, vol. 8, no. 10, pp. 1–14, 2013, doi: 10.1371/journal.pone.0076373. [8] “Boletin Observatorio Nacional de Salud.” https://www.ins.gov.co/Direcciones/ONS/Boletines/boletin_web_ONS/boletin 1.html (accedido Feb. 28, 2022). [9] R. E. Hynds and A. Giangreco, “Concise review: The relevance of human stem cell-derived organoid models for epithelial translational medicine,” Stem Cells, vol. 31, no. 3, pp. 417–422, 2013, doi: 10.1002/stem.1290. [10] B. Nugraha, M. F. Buono, and M. Y. Emmert, “Modelling human cardiac diseases with 3D organoid,” European Heart Journal, vol. 39, no. 48, pp. 4234–4237, 2018, doi: 10.1093/eurheartj/ehy765. [11] H. K. Voges, R. J. Mills, D. A. Elliott, R. G. Parton, E. R. Porrello, and J. E. Hudson, “Development of a human cardiac organoid injury model reveals innate regenerative potential,” Development (Cambridge), vol. 144, no. 6, pp. 1118–1127, 2017, doi: 10.1242/dev.143966. [12] H. D. Devalla and R. Passier, “Cardiac differentiation of pluripotent stem cells and implications for modeling the heart in health and disease,” Science Translational Medicine, vol. 10, no. 435, pp. 1–14, 2018, doi: 10.1126/scitranslmed.aah5457. [13] M. Zamani, E. Karaca, and N. F. Huang, “Multicellular Interactions in 3D Engineered Myocardial Tissue,” Frontiers in Cardiovascular Medicine, vol. 5, no. October, pp. 1–7, 2018, doi: 10.3389/fcvm.2018.00147. [14] A. I. Hoch and J. K. Leach, “Concise Review: Optimizing Expansion of Bone Marrow Mesenchymal Stem/Stromal Cells for Clinical Applications,” Stemcells Translational Medicine, vol. 3, no. 5, pp. 1–13, 2014, doi: 10.5966/sctm.2013- 0196. [15] M. F. Hoes, N. Bomer, and P. van der Meer, “Concise Review: The Current State of Human In Vitro Cardiac Disease Modeling: A Focus on Gene Editing and Tissue Engineering,” Stem Cells Translational Medicine, vol. 8, no. 1, pp. 66–74, 2019, doi: 10.1002/sctm.18-0052. [16] J. Kim, B. K. Koo, and K. J. Yoon, “Modeling host-virus interactions in viral infectious diseases using stem-cell-derived systems and CRISPR/Cas9 technology,” Viruses, vol. 11, no. 2, 2019, doi: 10.3390/v11020124. [17] S. D. Forsythe et al., “Environmental toxin screening using human-derived 3D bioengineered liver and cardiac organoids,” Frontiers in Public Health, vol. 6, no. April, 2018, doi: 10.3389/fpubh.2018.00103. [18] X. Yin, B. E. Mead, H. Safaee, R. Langer, J. M. Karp, and O. Levy, “Engineering Stem Cell Organoids,” Cell Stem Cell, vol. 18, no. 1, pp. 25–38, 2016, doi: 10.1016/j.stem.2015.12.005. [19] X. Qian, H. N. Nguyen, F. Jacob, H. Song, and G. L. Ming, “Using brain organoids to understand Zika virus-induced microcephaly,” Development (Cambridge), vol. 144, no. 6, pp. 952–957, 2017, doi: 10.1242/dev.140707. [20] K. Takahashi and S. Yamanaka, “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors,” Cell, vol. 126, no. 4, pp. 663–676, 2006, doi: 10.1016/j.cell.2006.07.024. [21] D. E. Rodríguez-Fuentes, L. E. Fernández-Garza, J. A. Samia-Meza, S. A. Barrera-Barrera, A. I. Caplan, and H. A. Barrera-Saldaña, “Mesenchymal Stem Cells Current Clinical Applications: A Systematic Review,” Archives of Medical Research, vol. 52, no. 1, pp. 93–101, 2021, doi: 10.1016/j.arcmed.2020.08.006. [22] C. Bock et al., “The Organoid Cell Atlas,” Nature Biotechnology, vol. 39, no. 1, pp. 13–17, 2021, doi: 10.1038/s41587-020-00762-x. [23] S. Yamanaka, “Induced pluripotent stem cells: Past, present, and future,” Cell Stem Cell, vol. 10, no. 6, pp. 678–684, 2012, doi: 10.1016/j.stem.2012.05.005. [24] B. Nugraha, M. F. Buono, L. von Boehmer, S. P. Hoerstrup, and M. Y. Emmert, “Human Cardiac Organoids for Disease Modeling,” Clinical Pharmacology and Therapeutics, vol. 105, no. 1, pp. 79–85, 2019, doi: 10.1002/cpt.1286. [25] S. Bartfeld and H. Clevers, “Stem cell-derived organoids and their application for medical research and patient treatment,” Journal of Molecular Medicine, vol. 95, no. 7, pp. 729–738, 2017, doi: 10.1007/s00109-017-1531-7. [26] K. Thygesen et al., “Fourth universal definition of myocardial infarction (2018),” European Heart Journal, vol. 40, no. 3, pp. 237–269, 2019, doi: 10.1093/eurheartj/ehy462. [27] C. C. Veerman, G. Kosmidis, C. L. Mummery, S. Casini, A. O. 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Haubner et al., “Functional Recovery of a Human Neonatal Heart after Severe Myocardial Infarction,” Circulation Research, vol. 118, no. 2, pp. 216– 221, 2016, doi: 10.1161/CIRCRESAHA.115.307017. [36] H. K. Voges, R. J. Mills, D. A. Elliott, R. G. Parton, E. R. Porrello, and J. E. Hudson, “Development of a human cardiac organoid injury model reveals innate regenerative potential,” Development (Cambridge), vol. 144, no. 6, pp. 1118–1127, 2017, doi: 10.1242/dev.143966. [37] E. R. Porrello and E. N. Olson, “A neonatal blueprint for cardiac regeneration,” Stem Cell Research, vol. 13, no. 3, pp. 556–570, 2014, doi: 10.1016/j.scr.2014.06.003. [38] M. Telsnig, “3-D Audio für Casinospielgeräte mittels Binauralsynthese und Übersprechkompensation Interner Projektbericht,” Audio, vol. 331, no. September, pp. 1078–1080, 2008, doi: 10.1126/science.1200708.Transient. [39] S. Schaaf et al., “Human engineered heart tissue as a versatile tool in basic research and preclinical toxicology,” PLoS ONE, vol. 6, no. 10, 2011, doi: 10.1371/journal.pone.0026397. [40] E. R. Porrello et al., “Regulation of neonatal and adult mammalian heart regeneration by the miR-15 family,” Proc Natl Acad Sci U S A, vol. 110, no. 1, pp. 187–192, 2013, doi: 10.1073/pnas.1208863110. [41] Y. Petrenko, E. Syková, and Š. Kubinová, “The therapeutic potential of threedimensional multipotent mesenchymal stromal cell spheroids,” Stem Cell Research and Therapy, vol. 8, no. 1, pp. 1–9, 2017, doi: 10.1186/s13287-017- 0558-6. [42] I. A. Potapova, P. R. Brink, I. S. Cohen, and S. v. Doronin, “Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells,” Journal of Biological Chemistry, vol. 283, no. 19, pp. 13100–13107, 2008, doi: 10.1074/jbc.M800184200. [43] I. A. Potapova et al., “Mesenchymal Stem Cells Support Migration, Extracellular Matrix Invasion, Proliferation, and Survival of Endothelial Cells In Vitro,” Stem Cells, vol. 25, no. 7, pp. 1761–1768, 2007, doi: 10.1634/stemcells.2007-0022. [44] S. Acosta and V. Andrade, Manual de Esterilización para Centros de Salud. 2016. [Online]. Available: http://www1.paho.org/PAHOUSAID/ dmdocuments/AMR-Manual_Esterilizacion_Centros_Salud_2008.pdf [45] M. I. Melo, “Extracción, cultivo y caracterización de células mesenquimales de médula ósea en biodispositivos para la regeneración del miocardio infartado,” Trabajo de investigación. Fac. Ingeniería, Dpto de Electrónica y Automática, Prog. Ing Biomédica. Univ. Autonoma de occidente. Santiago de Cali, Valle, 2019 [46] “Collagen Type I, Rat Tail | 3D Cell Culture Gels & Coating | ibidi.” https://ibidi.com/cell-culture-microscopy/107-collagen-type-i-rat-tail.html (accedido Apr. 26, 2022). [47] O. Pagliarosi, V. Picchio, I. Chimenti, E. Messina, and R. Gaetani, “Building an Artificial Cardiac Microenvironment: A Focus on the Extracellular Matrix,” Frontiers in Cell and Developmental Biology, vol. 8, no. September, pp. 1–8, 2020, doi: 10.3389/fcell.2020.559032. [48] C. Gardin, L. Ferroni, C. Latremouille, J. C. Chachques, D. Mitrečić, and B. Zavan, Recent Applications of Three Dimensional Printing in Cardiovascular Medicine, vol. 9, no. 3. 2020. doi: 10.3390/cells9030742. |
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Derechos reservados - Universidad Autónoma de Occidente, 2022 |
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Universidad Autónoma de Occidente |
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Neuta Arciniegas, Paola Andreab23ceaec23223a94087e311e0de3e934Quesada Quintero, Fabio Andrésc84898049d9aaea99d0f74d2cc935ac6Gutiérrez Burgos, Isabella564ad661e4150eba02e6ebc4b91047a4Universidad Autónoma de Occidente, Cll 25 # 115-85 Km 2 Vía Cali - Jamundi2022-06-02T15:16:52Z2022-06-02T15:16:52Z2022-05-18https://hdl.handle.net/10614/13944Universidad Autónoma de OccidenteRepositorio Educativo Digitalhttps://red.uao.edu.co/Los organoides son cultivos 3D desarrollados a partir de células madre creando un ambiente artificial propicio, en el cual las células pueden crecer e interactuar en un entorno tridimensional similar a las condiciones de un estado in vivo, imitando su funcionamiento molecular y celular lo cual permite tener un resultado en investigación de enfermedades y fármacos muy preciso. Para obtener un organoidese deben cultivar células madre en un medio favorable que permita su crecimiento y desarrollo. El propósito de esta investigación fue realizar el diseño de un método de cultivo que permita un adecuado entorno de crecimiento para el desarrollo de organoides de miocardio a partir de un cultivo de células madre mesenquimales (MSCs). Las células MSCs primero pasaron por un proceso de diferenciación acélulas endoteliales y cardiomiocitos que tarda entre 15 y 21 días respectivamente y posteriormente fueron utilizadas junto con hidrogel compuesto de colágeno como medio de cultivo para el desarrollo del organoides de miocardio. Los protocolos implementados para la gelificación del hidrogel fueron evaluados mediante la viabilidad celular del organoide al pasar 7 y 15 días de incubación, donde se realizó la observación de componentes de células vivas con pruebas de tinción supravital.Organoids are 3D cultures developed from stem cells, creating a favorable artificial environment in which cells can grow and interact in a three-dimensional environment similar to the conditions of an in vivo state, mimicking their molecular and cellular functioning, which allows for more accurate results in disease and drug research. To obtain an organoid, stem cells must be cultured in a favorable environment that allows their growth and development. The purpose of this research was to design a culture method that allows a suitable growth environment for the development of myocardial organoids from a culture of mesenchymal stem cells (MSCs). MSCs cells first undergo a differentiation process to endothelial cells and cardiomyocytes that can range from 15 to 21 days respectively, which are subsequently used together with collagen composite hydrogel as the culture medium for myocardial organoid development. The protocols implemented for the gelation of the hydrogel were evaluated by the cell viability of the organoid after 7 and 15 days of incubation, where the observation of live cell components was performed with supravital cell staining.Pasantía de investigación (Ingeniero Biomédico)-- Universidad Autónoma de Occidente, 2022PregradoIngeniero(a) Biomédico(a)61 páginasapplication/pdfspaUniversidad Autónoma de OccidenteIngeniería BiomédicaDepartamento de Automática y ElectrónicaFacultad de IngenieríaCaliDerechos reservados - Universidad Autónoma de Occidente, 2022https://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessAtribución-NoComercial-SinDerivadas 4.0 Internacional (CC BY-NC-ND 4.0)http://purl.org/coar/access_right/c_abf2Ingeniería BiomédicaCélulas madreCultivo de célulasStem cellsCell cultureMatriz de colágenoDiferenciación celularOrganoidesViabilidad celularDiseño de un método de cultivo de organoides de miocardio derivados de células madre adultasTrabajo de grado - Pregradohttp://purl.org/coar/resource_type/c_7a1fTextinfo:eu-repo/semantics/bachelorThesishttps://purl.org/redcol/resource_type/TPhttp://purl.org/coar/version/c_71e4c1898caa6e32Quesada Quintero, F.A. y Gutiérrez Burgos, I. 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Zavan, Recent Applications of Three Dimensional Printing in Cardiovascular Medicine, vol. 9, no. 3. 2020. doi: 10.3390/cells9030742.Comunidad generalPublicationLICENSElicense.txtlicense.txttext/plain; charset=utf-81665https://dspace7-uao.metacatalogo.com/bitstreams/3b28c82d-049e-43f4-9727-6855ae0ec107/download20b5ba22b1117f71589c7318baa2c560MD53ORIGINALT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdfT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdfTexto archivo completo del trabajo de grado, PDFapplication/pdf827333https://dspace7-uao.metacatalogo.com/bitstreams/810ca8a2-e854-407c-ad47-45f39b0fe38e/download8537ec22f1428d021dd17a732d09557dMD54TA10225_Autorización trabajo de grado.pdfTA10225_Autorización trabajo de grado.pdfAutorización publicación del trabajo de gradoapplication/pdf634578https://dspace7-uao.metacatalogo.com/bitstreams/863b7ec1-db00-423a-bd4f-8587631936b7/downloadc22bb4e1136cba041ffa0615c32d0cd8MD55TEXTT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdf.txtT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdf.txtExtracted texttext/plain101531https://dspace7-uao.metacatalogo.com/bitstreams/fa628ad2-cbf9-43b1-9a58-22032316eaca/download02aad38d7649e9454eeb227899cdc5f2MD56TA10225_Autorización trabajo de grado.pdf.txtTA10225_Autorización trabajo de grado.pdf.txtExtracted texttext/plain4080https://dspace7-uao.metacatalogo.com/bitstreams/05317a73-f657-4769-8792-897c88e6a395/download55c7f632528328801392f2ed91ed337bMD58THUMBNAILT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdf.jpgT10225_Diseño de un método de cultivo de organoides de miocardio derivados de células madre adultas.pdf.jpgGenerated Thumbnailimage/jpeg6147https://dspace7-uao.metacatalogo.com/bitstreams/9396fd06-b190-46a4-b717-bc1218df03b5/download45a7f2fe43b8a6e80927dede7fd525d9MD57TA10225_Autorización trabajo de grado.pdf.jpgTA10225_Autorización trabajo de grado.pdf.jpgGenerated Thumbnailimage/jpeg13507https://dspace7-uao.metacatalogo.com/bitstreams/a7975445-c9a6-40a4-ad3b-be0e0cc2045e/download3a5d22aff0174bd7ca22411d550bd547MD5910614/13944oai:dspace7-uao.metacatalogo.com:10614/139442024-01-19 16:29:30.219https://creativecommons.org/licenses/by-nc-nd/4.0/Derechos reservados - Universidad Autónoma de Occidente, 2022open.accesshttps://dspace7-uao.metacatalogo.comRepositorio UAOrepositorio@uao.edu.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 |