Characterization of in vitro 3D cultures

Abstract

SummaryOver the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient‐derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient‐specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high‐throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole‐image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular‐like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11‐day period. The parental tumours were analysed by next‐generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS‐mutated parental tumours showed a significantly higher growth rate compared to their wild‐type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell‐derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine

Inhibition of O-GlcNAcylation Reduces Cell Viability and Autophagy and Increases Sensitivity to Chemotherapeutic Temozolomide in Glioblastoma

“…In addition, we cultivated U87MG in a 3D culture system. This type of culture simulates tumor characteristics, such as senescence, hypoxia, and anti-apoptotic behavior [73][74][75]. In this work, the treatment of spheroids with TMZ only reduced the sizes of the spheroids after one week of treatment.…”

Fibroblasts and Endothelial Cells in Three-Dimensional Models: A New Tool for Addressing the Pathogenesis of Systemic Sclerosis as a Prototype of Fibrotic Vasculopathies

“…These limitations can be partially overcome, by the employment of three-dimensional (3D) in vitro models [ 1 , 2 , 9 , 10 ]. These 3D models can closely mimic the in vivo environment of native tissues [ 11 , 12 ]. Recent advances in cell biology, microfabrication techniques, and tissue engineering have made it possible to create different methods of assembling 3D cell cultures [ 1 , 2 ].…”

“…The bioreactor cell culture chambers operate in a dynamic culture environment and can be used independently or inter-connectedly in modular multi-compartmental platforms simulating a multi-organ system. Despite the complexity of the starting setting, the use of bioreactors in 3D cultures allows the study of cell functions, interactions, and crosstalk between different tissues/organs under flow conditions, reproducing the mechanisms of in vivo perfusion [ 2 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Fibrotic vasculopathies are characterized by vascular damage together with the deposition of fibrotic tissue in organs and inflammation.…”