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

Abstract

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.

Keywords: 3D cell cultures; 3D in vitro model; bacterial infections; breast cancer; cellular and molecular mechanism; endometriosis; hepatic environment.

Figure 1

(a) In 2D adherent cultures, cells grow as a monolayer on a flat surface, allowing unrestricted access to a similar number of nutrients and growth factors in the culture medium, resulting in homogeneous growth and proliferation. Cell–cell interactions and the extracellular environment are absent. The 3D model recapitulates the characteristics of the tumour microenvironment. Adequate cell–cell and extracellular environment interactions are allowed. A variable availability of oxygen, nutrients, metabolites and signalling molecules is established (adapted from [82] under the terms and conditions of the Creative Commons Attribution (CC-BY) license (CC-BY 4.0)). (b–d) Schematic representation of different culture conditions (b), expression of CCL5 receptors (CCR1 and CCR5) in mono- and co-culture spheroids of ASCs and MDA-MB-231 or MCF-7 compared to indirect and direct 2D cultures (c,d). * indicates statistically significant differences (p < 0.05) between culture systems; Δ indicates statistically significant differences (p < 0.05) to corresponding monocultures (adapted from [56] under the terms of the CC-BY 4.0 publishing license). (e,f) Images of HCC1954 spheroids stiffened by different concentrations of ribose (0.50 and 200 mM). (e) Fixed samples show the distribution of ERK (green) and F-actin (magenta), with counterstained nuclei in blue. Scale bars, 20 μm. (f) Spheroids embedded in a 3D collagen structure show the localization of YAP (green); nuclei are stained blue. Scale bars, 20 μm (adapted from [57] under the terms of the CC-BY 4.0 publishing license). (g) Box representation of doxorubicin effects in the MDA-MB-231 cell line cultured within the 3D biomimetic collagen scaffold, indicating the most significantly altered pathways implicated in DOX resistance (green = up-regulation; red = down-regulation) (adapted from [64] under the terms of the CC-BY 4.0 publishing license).

Figure 2

3D culture models of the human endometrium. (a) Available in vitro experimental systems used in endometriotic studies, which reflect the multifactorial nature of the endometriotic lesion (adapted from [83] under the terms of a CC-BY 4.0 publishing license). (b,c) After 7 days of the 3D culture, EEC12Z and EEC16 both form dense, smooth and symmetrical spheroids. (b) Phase contrast and H&E images, (c) Cytokeratin expression is increased in 3D models versus that in 2D models and (d) Expression of genes relevant in endometriosis in EEC16 and EEC12Z after being cultures in 3D for 7 days. * p > 0.05. (adapted from [86] under the terms of a CC-BY 4.0 publishing license). (e) Spheroids’ shape and dimension characterization: bright-field images (Scale bars 250 µm), Cell Tracker staining (Red: St-T1b; green: 12Z. Scale bar 200 µm) and relative quantitative analysis. ** p < 0.01; *** p < 0.001, **** p < 0.0001; (adapted from ref. [87] under the terms of a CC-BY 4.0 publishing license). (f) Representative images showing a 3D cell culture model system for endometriosis based on a slice from a full-thickness human endometrium (adapted from ref. [90] under the terms of a CC-BY 4.0 publishing license).

Figure 3

Beneficial impact of growing hepatic cells within a three-dimensional environment. (a) When cell lines are cultured in a 3D environment, the systematic up-regulation of in vivo-like functions has been observed. Scheme created with BioRender.com. (b) The increase in albumin secretion, as well as the up-regulation of relevant cytochromes, were observed for HepG2 cells grown within a 3D-bioprinted hydrogel matrix, the scale bars are all equal to 200 μm (adapted from [101] under the terms of a CC-BY 4.0 publishing license). (c) 3D culture environments positively impact both the establishment and the maintenance of physiological-like hepatic functions in primary hepatocytes, as well as in iPSC-derived hepatocytes. Scheme created with BioRender.com. (d) 3D cultures have also shown their potential in guiding the differentiation of iPSC-derived hepatocytes, as the coculture of the iPSC spheroid and primary hepatocytes spheroid led to the establishment of multiple physiological-like hepatic functions. Statistical significance is expressed as ns: p > 0.05, **: 0.01 ≤ p < 0.001, ***: p ≤ 0.001 (adapted from [115] under the terms of a CC-BY 4.0 publishing license).

Figure 4

Infection in 3D culture models. (a) Human intestinal enteroids (HIE) monolayers were infected with wild-type (WT) Shigella and the avirulent CSF100 strain. The panel shows host gene expression (as log₂-fold change) after three hours of Shigella infection (adapted from [131] with permission from Copyright American Society for Microbiology-License number 1494825-1). Statistical significance is expressed as p < 0.05, *. (b,c) Salmonella infection in LSMMG and in control culture conditions (adapted from [132] under the terms of a CC-BY 4.0 publishing license). The analyzed strains were the wild-type (WT) and the mutant delta-hfq strains. (b) Bacterial genes that were up- and down-regulated, in red and blue, respectively, were associated with Salmonella Pathogenic Islands (SPI) 1, 2, motility and chemotaxis. The expression was reported as the mean log₂ fold change. (c) Plots illustrating the up- (red dots) and down-regulation (blue dots) of genes expressed by host cells infected at 24 h post-infection (hpi) by WT (left panel) and delta-hfq (right panel) strains in LSMMG conditions. Expression reported as the logFC (logged fold change) as a function of the FDR (false discovery rate) < 0.05. (d) Label of a section of a skin 3D model after 48 h of infection with MRSA bacterial strains (ST8, ST30, ST59, ST22, ST45, ST239). Each line composed of i, ii, iii and iv represents a bacterial strain. The white dashed line marks the dermal epidermal barrier between the stratum basale and the collagen gel containing fibroblasts. Specifically, (i) shows HaCaT keratinocytes nuclei, at the strata basale and spinosum (yellow line), marked in blue with Hoechst stain. (ii) shows MRSA bacteria labeled with an anti-S. aureus antibody and Alexa Fluor^® 568 conjugated secondary antibody. Those indicated by yellow arrows are in the collagen gel. (iii) shows the Click-iT^® TUNEL Alexa Fluor^® 488 cell for the detection of damaged DNA. Finally, (iv) is an overlay where bacteria and apoptosis/DNA damage are co-localized in keratinocytes in the stratum spinosum. The yellow circles in (iv) depict the model’s skin being exfoliated. Scale Bar (i–iv) of 50 µm (adapted from [133] under the terms of a CC-BY 4.0 publishing license).