Bioimpressão 3D de neuroesferas murinas em microambiente contendo oligômero beta amiloide para modelar a Doença de Alzheimer
Data
2024-12-10
Autores
Ferreira, Natalia Dall´Agnol 
Orientadores
Porcionatto, Marimelia Aparecida
Tipo
Trabalho de conclusão de curso
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Globalmente, estima-se que a demência mais comum na sociedade, a Doença de Alzheimer (DA), poderá acometer cerca de 75 milhões de pessoas em 2030 e mais de 130 milhões, em 2050. Os principais mecanismos fisiopatológicos da DA são a formação extracelular de placas amiloides (A ) e de emaranhados neurofibrilares β βintracelulares. Ambas as alterações causam extrema neurotoxicidade, resultando em dano sináptico e aumento do estresse oxidativo, com resposta inflamatória local seguida de morte neuronal. A neuroinflamação é também responsável por inibir a neurogênese, contribuindo com a aceleração da senescência de células-tronco neurais, perda de memória e comprometimento cognitivo. Modelos in vivo e in vitro são fundamentais para o entendimento das patologias neurodegenerativas, incluindo a DA. Recentemente, o desenvolvimento de novos biomateriais e da tecnologia de bioimpressão tem contribuído com a proposição de modelos in vitro mais complexos. A combinação de células e materiais biocompatíveis, além de minimizar o uso de animais de experimentação permite a simulação in vitro dos principais eventos patogênicos de doenças. Deste modo, a bioimpressão e a cultura tridimensional (3D) representam alternativas para mimetizar o microambiente 3D do tecido cerebral e estudar interações célula-célula e célula-microambiente extracelular. O objetivo deste projeto foi produzir um modelo in vitro que mimetizasse o microambiente da DA, por meio da bioimpressão 3D utilizando biotinta contendo oligômeros A (AβOs) e neuroesferas originadas de β células-tronco neurais, extraídas de zonas neurogênicas do cérebro de camundongos wild type (C57bl/6) e de modelos transgênicos da DA, sendo esses o APPswe/PSEN1dE9 (APP/PS1) e o 5xFAD. Os biomateriais foram desenvolvidos, caracterizados e padronizados para serem utilizados na bioimpressão 3D. Os parâmetros biológicos avaliados foram: diferenciação das células nas neuroesferas, estresse oxidativo e morte celular. Os construtos 3D produzidos pelo protocolo desenvolvido durante a execução deste projeto, são modelos in vitro promissores para o estudo da DA. A composição de biomaterial que utilizamos proporcionou condições favoráveis para a bioimpressão, mantendo a organização e integridade dos construtos, evidenciando sua adequação para o cultivo celular. As neuroesferas bioimpressas do grupo controle proliferaram e apresentaram resultados similares aos observados em culturas em suspensão, indicando que o processo de bioimpressão não foi deletério para as células. Além disso, a adição de AβOs à biotinta permitiu reproduzir características-chave da DA, como diminuição da viabilidade celular, indução de estresse oxidativo e diferenciação das células-tronco neurais, reforçando o potencial do modelo para estudos de mecanismos patológicos. Análises comparativas entre culturas 3D contendo neuroesferas de camundongos transgênicos APP/PS1 e 5xFAD, modelos da DA, indicam o potencial do modelo para estudos in vitro. Esses resultados destacam a relevância dos modelos 3D bioimpressos que produzimos como ferramentas para investigar a DA, permitindo avanços em estudos futuros envolvendo mecanismos moleculares da doença, bem como possíveis alvos terapêuticos.
Globally, it is estimated that the most common form of dementia in society, Alzheimer’s disease (AD), will affect around 75 million people by 2030 and over 130 million by 2050. The primary pathophysiological mechanisms of AD include the extracellular formation of amyloid plaques (A) and intracellular neurofibrillary tangles. Both alterations cause extreme neurotoxicity, resulting in synaptic damage and increased oxidative stress, accompanied by a local inflammatory response followed by neuronal death. Neuroinflammation also inhibits neurogenesis, contributing to accelerated neural stem cell senescence, memory loss, and cognitive impairment. In vivo and in vitro models are essential for understanding neurodegenerative pathologies, including AD. The development of new biomaterials and bioprinting technology has recently contributed to the proposal of more complex in vitro models. Combining cells with biocompatible materials minimizes the use of experimental animals and allows for the in vitro simulation of key pathogenic events of diseases. Thus, bioprinting and three-dimensional (3D) culture represent alternative approaches to mimic the 3D microenvironment of brain tissue and study cell-cell and cell-extracellular microenvironment interactions. The objective of this project was to produce an in vitro model mimicking the AD microenvironment using 3D bioprinting with bioink containing A oligomers (AβOs) and neurospheres derived from neural stem cells extracted from neurogenic zones of the brains of wild-type mice (C57bl/6) and transgenic AD models, including APPswe/PSEN1dE9 (APP/PS1) and 5xFAD. Biomaterials were developed, characterized, and standardized for use in 3D bioprinting. The biological parameters evaluated included cell differentiation within neurospheres, oxidative stress, and cell death. The 3D constructs produced using the protocol developed during this project are promising in vitro models for studying AD. The biomaterial composition we used provided favorable conditions for bioprinting, maintaining the constructs' organization and integrity highlighting their suitability for cell culture. Neurospheres bioprinted in the control group increased and showed results comparable to those observed in suspension culture, indicating that the bioprinting process was not detrimental to the cells. Moreover, adding AβOs to the bioink allowed us to reproduce key AD characteristics, such as reduced cell viability, induction of oxidative stress, and differentiation of neural stem cells (NSCs), reinforcing the model's potential for studying pathological mechanisms. Comparative analyses of 3D cultures containing neurospheres from APP/PS1 and 5xFAD transgenic AD mouse models demonstrate the model’s potential for in vitro studies. These results underscore the relevance of the 3D bioprinted models we produced as tools to investigate AD, enabling advances in future studies involving the molecular mechanisms of the disease and potential therapeutic targets.
Globally, it is estimated that the most common form of dementia in society, Alzheimer’s disease (AD), will affect around 75 million people by 2030 and over 130 million by 2050. The primary pathophysiological mechanisms of AD include the extracellular formation of amyloid plaques (A) and intracellular neurofibrillary tangles. Both alterations cause extreme neurotoxicity, resulting in synaptic damage and increased oxidative stress, accompanied by a local inflammatory response followed by neuronal death. Neuroinflammation also inhibits neurogenesis, contributing to accelerated neural stem cell senescence, memory loss, and cognitive impairment. In vivo and in vitro models are essential for understanding neurodegenerative pathologies, including AD. The development of new biomaterials and bioprinting technology has recently contributed to the proposal of more complex in vitro models. Combining cells with biocompatible materials minimizes the use of experimental animals and allows for the in vitro simulation of key pathogenic events of diseases. Thus, bioprinting and three-dimensional (3D) culture represent alternative approaches to mimic the 3D microenvironment of brain tissue and study cell-cell and cell-extracellular microenvironment interactions. The objective of this project was to produce an in vitro model mimicking the AD microenvironment using 3D bioprinting with bioink containing A oligomers (AβOs) and neurospheres derived from neural stem cells extracted from neurogenic zones of the brains of wild-type mice (C57bl/6) and transgenic AD models, including APPswe/PSEN1dE9 (APP/PS1) and 5xFAD. Biomaterials were developed, characterized, and standardized for use in 3D bioprinting. The biological parameters evaluated included cell differentiation within neurospheres, oxidative stress, and cell death. The 3D constructs produced using the protocol developed during this project are promising in vitro models for studying AD. The biomaterial composition we used provided favorable conditions for bioprinting, maintaining the constructs' organization and integrity highlighting their suitability for cell culture. Neurospheres bioprinted in the control group increased and showed results comparable to those observed in suspension culture, indicating that the bioprinting process was not detrimental to the cells. Moreover, adding AβOs to the bioink allowed us to reproduce key AD characteristics, such as reduced cell viability, induction of oxidative stress, and differentiation of neural stem cells (NSCs), reinforcing the model's potential for studying pathological mechanisms. Comparative analyses of 3D cultures containing neurospheres from APP/PS1 and 5xFAD transgenic AD mouse models demonstrate the model’s potential for in vitro studies. These results underscore the relevance of the 3D bioprinted models we produced as tools to investigate AD, enabling advances in future studies involving the molecular mechanisms of the disease and potential therapeutic targets.
Descrição
Citação
FERREIRA, Natalia Dall Agnol. Bioimpressão 3D de neuroesferas murinas em microambiente contendo oligômero beta amiloide para modelar a doença de alzheimer. 2024. 64 f. Trabalho de Conclusão de Curso (Graduação em Biomedicina) - Escola Paulista de Medicina, Universidade Federal de São Paulo (UNIFESP). São Paulo, 2024.
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3. Saúde e bem-estar