• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Doctoral Thesis
DOI
https://doi.org/10.11606/T.60.2020.tde-21092021-055958
Document
Author
Full name
Robson Luis Ferraz do Amaral
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2020
Supervisor
Committee
Antonietto, Kamilla Swiech (President)
Moraes, Ângela Maria
Cominetti, Márcia Regina
Engler, Silvya Stuchi Maria
Title in Portuguese
Análise comparativa de diferentes sistemas de cultivo tridimensionais para estabelecimento de um modelo in vitro de câncer de bexiga para ensaios de eficácia terapêutica
Keywords in Portuguese
Câncer de bexiga
Cultivos 3D
Esferoides tumorais
Modelo in vitro
Abstract in Portuguese
Diferentes técnicas de cultivos tridimensionais (3D) foram avaliadas a fim de estabelecer um modelo de câncer de bexiga com capacidade de recapitular in vitro características do tumor in vivo. Na primeira parte, utilizando a linhagem celular de câncer de bexiga RT4, compararam-se sistemas de cultivo 3D livres de suporte, utilizando as técnicas de flutuação forçada (Placas ULA, Ultra low attachment) e gota suspensa (Placas HD, hanging drop), e sistemas de cultivo dependentes de suporte, utilizando microcarregadores e hidrogéis (Matrigel, Alginato e HydroMatrix), quanto à formação de esferoides RT4. Estes foram caracterizados quanto aos aspectos morfológicos, de crescimento celular e metabolismo. Somente os cultivos utilizando as técnicas ULA, HD e os suportes Matrigel (50% v/v) e HydroMatrix (0,25% v/v) foram capazes de gerar esferoides com parâmetros de forma adequados (solidez > 0,95; esfericidade ~ 0,90; arredondamento entre 0,7 e 0,9). Nos cultivos ULA e HD, foi gerado um único esferoide por poço, com diâmetros entre 200 e 600 μm. O crescimento celular foi reduzido e, consequentemente, a atividade metabólica menos intensa nos esferoides quando comparados aos cultivos 2D. Os cultivos em Matrigel e HydroMatrix, por sua vez, geraram vários esferoides por poço, com diâmetros que não ultrapassaram 200 μm. Os esferoides em Matrigel apresentaram crescimento celular e metabolismo superiores aos observados nos cultivos 2D. Foram selecionados os esferoides RT4 gerados em ULA e em Matrigel para a avaliação da sensibilidade à Doxorrubicina (DOX) e da expressão de genes comumente superexpressos em tumores sólidos de bexiga. Os esferoides RT4 gerados em ULA foram menos sensíveis ao tratamento com DOX (IC50 8,62 ± 1,53 μM) do que os cultivos 2D (IC50 2,35 ± 0,39 μM) e Matrigel (IC50 3,31 ± 1,28 μM). Nos esferoides RT4 em ULA, houve maior expressão dos genes ALDH1A1 e H-RAS, dois dos principais genes envolvidos nos mecanismos de resistência e reincidência de tumores de bexiga. Já nos cultivos em Matrigel, quase todos os genes analisados foram mais expressos (HIF-1α, IFIT5, MDR-1, IL3, ALDH1A1 e H-RAS). Na segunda parte do projeto, a estratégia de cultivo em ULA com 5% Matrigel foi utilizada com sucesso para a geração de esferoides com células primárias de tumor de bexiga humano (BL0293 e BL0808) derivados de modelos PDX (patient-derived xenografts). Os perfis de sensibilidade dos esferoides BL0293 e BL0808 à Cisplatina e à Gemcitabina foram semelhantes aos obtidos previamente nos ensaios in vivo com os modelos PDX BL0293 e BL0808. De forma geral, foi possível concluir que os cultivos 3D realizados utilizando a técnica de gota suspensa em placas ULA e em Matrigel foram capazes de recapitular in vitro características do tumor in vivo, podendo ser considerados como promissores modelos 3D de câncer de bexiga. O presente trabalho traz de forma inédita uma análise comparativa entre os esferoides de tumor de bexiga gerados com diferentes técnicas de cultivo 3D em relação a morfologia e a capacidade em recapitular algumas característica do tumor in vivo.
Title in English
Comparative analysis of different three-dimensional culture systems for the establishment of an in vitro bladder cancer model for therapeutic-efficacy assays
Keywords in English
3D cell culture
Bladder cancer
In vitro models
Tumor spheroids
Abstract in English
Different three-dimensional (3D) cell cultures techniques were evaluated in order to establish a bladder cancer model with the ability to recapitulate in vitro the tumor features observed in vivo. Using the bladder cancer cell line RT4, the first part of the project focused on comparing the scaffold-free 3D cell culture systems, employing forced floating (Ultra-low attachment plates, ULA) and hanging drop (HD plates) techniques, and scaffold-dependent systems, employing microcarriers and hydrogels (Matrigel, Alginate and HydroMatrix), to generate RT4 spheroids. These were characterized regarding morphological parameters, cell growth and metabolism. Only 3D cell culture using the forced floating and hanging drop techniques, and Matrigel (50% v/v) and HydroMatrix (0.25% v/v) scaffolds, were able to generate spheroids with suitable parameters (solidity>0.95; sphericity~0.90; rounding between 0.70- 0.90). In the ULA and HD cell cultures, a single spheroid was generated per well, with diameters ranging between 200 and 600 μm. The cell growth and, consequently, the metabolism observed in these spheroids were lower than in 2D cultures. The Matrigel and HydroMatrix cultures, on the other hand, generated several spheroids per well, with diameters that did not exceed 200 μm. The spheroids in Matrigel showed cell growth and metabolism higher than those observed in 2D cultures. RT4 spheroids generated in ULA plates and Matrigel were selected to assess the Doxorubicin (DOX) sensitivity and the expression of commonly overexpressed genes in solid bladder tumors. ULA plates' spheroids were less sensitive to DOX treatment (IC50 8.62 ± 1.53 μM) than 2D (IC50 2.35 ± 0.39 μM) and Matrigel (IC50 3.31 ± 1.28 μM) cultures. Moreover, it was observed a higher expression of ALDH1A1 and HRAS genes in RT4 spheroids generated in ULA, two of the main genes involved in the mechanisms of resistance and recurrence of bladder tumors. Almost all the analyzed genes (HIF-1α, IFIT5, MDR-1, IL3, ALDH1A1 and HRAS) were more expressed in Matrigel cultures. In the second part of the project, the strategy based on the use of ULA cultures with 5% of Matrigel (v/v) was successfully applied to generate spheroids using primary human bladder tumor cells (BL0293 and BL0808) derived from PDX models (patient-derived xenografts). The sensitivity profiles of the BL0293 and BL0808 spheroids to Cisplatin and Gemcitabine were similar to those obtained previously in the in vivo tests using PDX models BL0293 and BL0808. In general, it was possible to conclude that both 3D cultures generated using the forced floating technique (ULA plates) and Matrigel scaffold were able to recapitulate in vitro some characteristics of the solid tumor in vivo and can be considered as promising 3D models of bladder cancer. The present work shows an unprecedented comparative analysis between the bladder tumor spheroids generated with different 3D cell culture techniques regarding the morphology and the ability to recap some features of the tumor in vivo.
 
WARNING - Viewing this document is conditioned on your acceptance of the following terms of use:
This document is only for private use for research and teaching activities. Reproduction for commercial use is forbidden. This rights cover the whole data about this document as well as its contents. Any uses or copies of this document in whole or in part must include the author's name.
Publishing Date
2021-09-22
 
WARNING: Learn what derived works are clicking here.
All rights of the thesis/dissertation are from the authors
CeTI-SC/STI
Digital Library of Theses and Dissertations of USP. Copyright © 2001-2024. All rights reserved.