Cancer is intrinsically complex, comprising both heterogeneous cellular composition and extracellular matrix. In vitro cancer research models have been widely used in the past to model and study cancer. Although two-dimensional (2D) cell culture models have traditionally been used for cancer research, they have many limitations, such as the disturbance of interactions between cellular and extracellular environments and changes in cell morphology, polarity, division mechanism, differentiation and cell motion. Moreover, 2D cell models are usually monotypic. This implies that 2D tumor models are ineffective at accurately recapitulating complex aspects of tumor cell growth, as well as their radiation responses. Over the past decade there has been significant uptake of three-dimensional (3D) in vitro models by cancer researchers, highlighting a complementary model for studies of radiation effects on tumors, especially in conjunction with chemotherapy. The introduction of 3D cell culture approaches aims to model in vivo tissue interactions with radiation by positioning itself halfway between 2D cell and animal models, and thus opening up new possibilities in the study of radiation response mechanisms of healthy and tumor tissues.

/pdf/3d-cell-models-in-radiobiology-improving-the-predictive-176ojd4m.pdf

3D Cell Models in Radiobiology: Improving the Predictive Value of In Vitro Research

Advances in cancer biology over the past decades have revealed that metabolic adaptation of cancer cells is an essential aspect of tumorigenesis. However, recent insights into tumour metabolism in vivo have revealed dissimilarities with results obtained in vitro. This is partly due to the reductionism of in vitro cancer models that struggle to reproduce the complexity of tumour tissues. This review describes some of the discrepancies in cancer cell metabolism between in vitro and in vivo conditions, and presents current methodological approaches and tools used to bridge the gap with the clinically relevant microenvironment. As such, these approaches should generate new knowledge that could be more effectively translated into therapeutic opportunities.

Modelling cancer metabolism in vitro: current improvements and future challenges.

Over the past decade, the development of three-dimensional (3D) models has increased exponentially, facilitating the unravelling of fundamental and essential cellular mechanisms by which cells communicate with each other, assemble into tissues and organs and respond to biochemical and biophysical stimuli under both physiological and pathological conditions. This section presents a concise overview of the most recent updates on the significant contribution of different types of 3D cell cultures including spheroids, organoids and organ-on-chip and bio-printed tissues in advancing our understanding of cellular and molecular mechanisms. The case studies presented include the 3D cultures of breast cancer (BC), endometriosis, the liver microenvironment and infections. In BC, the establishment of 3D culture models has permitted the visualization of the role of cancer-associated fibroblasts in the delivery of exosomes, as well as the significance of the physical properties of the extracellular matrix in promoting cell proliferation and invasion. This approach has also become a valuable tool in gaining insight into general and specific mechanisms of drug resistance. Given the considerable heterogeneity of endometriosis, 3D models offer a more accurate representation of the in vivo microenvironment, thereby facilitating the identification and translation of novel targeted therapeutic strategies. The advantages provided by 3D models of the hepatic environment, in conjunction with the high throughput characterizing various platforms, have enabled the elucidation of complex molecular mechanisms underlying various threatening hepatic diseases. A limited number of 3D models for gut and skin infections have been developed. However, a more profound comprehension of the spatial and temporal interactions between microbes, the host and their environment may facilitate the advancement of in vitro, ex vivo and in vivo disease models. Additionally, it may pave the way for the development of novel therapeutic approaches in diverse research fields. The interested reader will also find concluding remarks on the challenges and prospects of using 3D cell cultures for discovering cellular and molecular mechanisms in the research areas covered in this review. 

Growing Role of 3D In Vitro Cell Cultures in the Study of Cellular and Molecular Mechanisms: Short Focus on Breast Cancer, Endometriosis, Liver and Infectious Diseases

Neural stem cell (NSC)-based therapies are at the forefront of regenerative medicine strategies for various neural defects and injuries such as stroke, traumatic brain injury, and spinal cord injury. For several clinical applications, NSC therapies require biocompatible scaffolds to support cell survival and to direct differentiation. Here, we investigate decellularized plant tissue as a novel scaffold for three-dimensional (3D), in vitro culture of NSCs. Plant cellulose scaffolds were shown to support the attachment and proliferation of adult rat hippocampal neural stem cells (NSCs). Further, NSCs differentiated on the cellulose scaffold had significant increases in their expression of neuron-specific beta-III tubulin and glial fibrillary acidic protein compared to 2D culture on a polystyrene plate, indicating that the scaffold may enhance the differentiation of NSCs towards astrocytic and neuronal lineages. Our findings suggest that plant-derived cellulose scaffolds have the potential to be used in neural tissue engineering and can be harnessed to direct the differentiation of NSCs. 

Plant Cellulose as a Substrate for 3D Neural Stem Cell Culture

Chondrocytes are the primary cells responsible for maintaining cartilage integrity and function. Their role in cartilage homeostasis and response to inflammation is crucial for understanding the progression and potential therapeutic interventions for various cartilage-related disorders. Developing an accessible and cost-effective model to generate viable chondrocytes and to assess their response to different bioactive compounds can significantly advance our knowledge of cartilage biology and contribute to the discovery of novel therapeutic approaches.We developed a novel, streamlined protocol for generating chondrocytes from bone marrow-derived mesenchymal stem cells (BMSCs) in a 3D culture system that offers significant implications for the study of cartilage biology and the discovery of potential therapeutic interventions for cartilage-related and associated disorders.We developed a streamlined protocol for generating chondrocytes from BMSCs in a 3D culture system using an "in-tube" culture approach. This simple pellet-based 3D culture system allows for cell aggregation and spheroid formation, facilitating cell-cell and cell-extracellular matrix interactions that better mimic the in vivo cellular environment compared with 2D monolayer cultures. A proinflammatory chondrocyte model was created by treating the chondrocytes with lipopolysaccharide and was subsequently used to evaluate the anti-inflammatory effects of vitamin D, curcumin, and resveratrol.The established protocol successfully generated a large quantity of viable chondrocytes, characterized by alcian blue and toluidine blue staining, and demonstrated versatility in assessing the anti-inflammatory effects of various bioactive compounds. The chondrocytes exhibited reduced inflammation, as evidenced by the decreased tumor necrosis factor-α levels, in response to vitamin D, curcumin, and resveratrol treatment.Our novel protocol offers an accessible and cost-effective approach for generating chondrocytes from BMSCs and for evaluating potential therapeutic leads in the context of inflammatory chondrocyte-related diseases. Although our approach has several advantages, further investigation is required to address its limitations, such as the potential differences between chondrocytes generated using our protocol and those derived from other established methods, and to refine the model for broader applicability and clinical translation. 

pdf/efficient-generation-of-chondrocytes-from-bone-marrow-186y8gu6pw.pdf

Efficient Generation of Chondrocytes From Bone Marrow–Derived Mesenchymal Stem Cells in a 3D Culture System: Protocol for a Practical Model for Assessing Anti-Inflammatory Therapies

From growing cells in spheroids to arranging them on complex engineered scaffolds, three-dimensional cell culture protocols are rapidly expanding and diversifying. While these systems may often improve the physiological relevance of cell culture models, they come with technical challenges, as many of the analytical methods used to characterize traditional two-dimensional (2D) cells must be modified or replaced to be effective. Here we review the advantages and limitations of quantification methods based either on biochemical measurements or microscopy imaging. We focus on the most basic of parameters that one may want to measure, the number of cells. Precise determination of this number is essential for many analytical techniques where measured quantities are only meaningful when normalized to the number of cells (e.g. cytochrome p450 enzyme activity). Thus, accurate measurement of cell number is often a prerequisite to allowing comparisons across different conditions (culturing conditions or drug and treatment screening) or between cells in different spatial states. We note that this issue is often neglected in the literature with little or no information given regarding how normalization was performed, we highlight the pitfalls and complications of quantification and call for more accurate reporting to improve reproducibility.

Current strategies with implementation of three-dimensional cell culture: the challenge of quantification

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Current strategies with implementation of three-dimensional cell culture: the challenge of quantification