Search Results (15)

Tissue engineering research for neurological applications has demonstrated that biomaterial-based structural bridges present a promising approach for promoting regeneration. This is particularly relevant for penetrating traumatic brain injuries, where the clinical prognosis is typically poor, with no available regeneration-enhancing therapies. Specifically, repurposing clinically approved biomaterials offers many advantages (reduced approval time and achieving commercial scaleup for clinical applications), highlighting the need for detailed screening of potential neuromaterials. A major challenge in experimental testing is the limited availability of neuromimetic, technically accessible, cost-effective, and humane models of neurological injury for efficient biomaterial testing in injury-simulated environments. Three dimensional (3D) organotypic brain slices bridge the gap between live animal models and simplified co-cultures and are a versatile tool for studies on neural development, neurodegenerative disease and in drug testing. Despite this, their utility for investigation of neural cell responses to biomaterial implantation is poorly investigated. We demonstrate that murine brain organotypic slices can be used to develop a model of penetrating traumatic brain injury, wherein a surgical-grade biomaterial scaffold can be implanted into the lesion cavity. Critically, the model allowed for examination of key cellular responses involved in CNS injury pathology/biomaterial handling: astrogliosis, microglial activation and axonal sprouting. The approach offers a technically simple and versatile methodology to study biomaterial interventions as a regenerative therapy for neurological injuries. Full article

Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor–stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME. Full article

Biocompatibility testing of materials is carried out in 2D cell cultures or animal models despite serious limitations. 3D skin equivalents are advanced in vitro models for human skin. Silicone has been shown to be noncytotoxic but capable of eliciting an immune response. Our aim was to (1) establish a 3D skin equivalent to (2) assess the proinflammatory properties of silicone. We developed a coculture of keratinocytes and fibroblasts resulting in a 3D skin equivalent with an implant using samples from a breast implant. Samples with and without the silicone implant were studied histologically and immunohistochemically in comparison to native human skin samples. Cytotoxicity was assessed via LDH-assay, and cytokine response was assessed via ELISA. Histologically, our 3D skin equivalents had a four-layered epidermal and a dermal component. The presence of tight junctions was demonstrated in immunofluorescence. The only difference in 3D skin equivalents with implants was an epidermal thinning. Implanting the silicone samples did not cause more cell death, however, an inflammatory cytokine response was triggered. We were able to establish an organotypical 3D skin equivalent with an implant, which can be utilised for studies on biocompatibility of materials. This first integration of silicone into a 3D skin equivalent confirmed previous findings on silicone being non-cell-toxic but capable of exerting a proinflammatory effect. Full article

Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen is Staphylococcus aureus (S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects of S. aureus infection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model with S. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1β) proved host–pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected with S. aureus resulting in the formation of biofilm, including host–pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema. Full article

Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments. Full article

The invasion of skin tissue by Staphylococcus aureus is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant Staphylococcus aureus (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains. Full article

The vancomycin-resistance associated sensor/regulator, VraSR two-component regulatory-system (VraSR), regulates virulence and the response of Staphylococcus aureus (SA) to environmental stress. To investigate the role of VraSR in SA skin and soft tissue infections (SSTI), we inactivated the VraSR of a clinical CA-MRSA ST30 strain by insertional mutation in vraR gene using the TargeTron-Gene Knockout System. We constructed an organotypic keratinocyte fibroblast co-culture (3D-skin model) and a humanized mouse as SSTI infection models. In the 3D-skin model, inactivation of VraSR in the strains ST30 and USA300 showed 1-log reduction in adhesion and internalization (p < 0.001) compared to the respective wildtype. The mutant strains of ST30 (p < 0.05) and USA300-LAC (p < 0.001) also exhibited reduced apoptosis. The wildtype ST30 infection in the humanized mouse model demonstrated increased skin lesion size and bacterial burden compared to BALB/c mice (p < 0.01). The response of the humanized mouse towards the MRSA infection exhibited human similarity indicating that the humanized mouse SSTI model is more suitable for evaluating the role of virulence determinants. Inactivation of VraSR in ST30 strain resulted in decreased skin lesion size in the humanized mouse SSTI model (p < 0.05) and reduction in apoptotic index (p < 0.01) when compared with the wildtype. Our results reveal that inactivating the VraSR system may be a potent anti-virulence approach to control MRSA infection. Full article

To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization. Full article

Decellularized skeletal muscle (dSkM) constructs have received much attention in recent years due to the versatility of their applications in vitro. In search of adequate in vitro models of the skeletal muscle tissue, the dSkM offers great advantages in terms of the preservation of native-tissue complexity, including three-dimensional organization, the presence of residual signaling molecules within the construct, and their myogenic and neurotrophic abilities. Here, we attempted to develop a 3D model of neuromuscular tissue. To do so, we repopulated rat dSkM with human primary myogenic cells along with murine fibroblasts and we coupled them with organotypic rat spinal cord samples. Such culture conditions not only maintained multiple cell type viability in a long-term experimental setup, but also resulted in functionally active construct capable of contraction. In addition, we have developed a customized culture system which enabled easy access, imaging, and analysis of in vitro engineered co-cultures. This work demonstrates the ability of dSkM to support the development of a contractile 3D in vitro model of neuromuscular tissue fit for long-term experimental evaluations. Full article

Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer. Full article

(1) Background: Despite progress in surgery and radio-chemotherapy of glioblastoma (GB), the prognosis remains very poor. GB cells exhibit a preference for hypoxia to maintain their tumor-forming capacity. Enhancing oxidative phosphorylation—known as the anti-Warburg effect—with cyclic AMP activators has been demonstrated to drive GB cells from proliferation to differentiation thereby reducing tumor growth in a cell culture approach. Here we re-evaluate this treatment in a more clinically relevant model. (2) Methods: The effect of treatment with dibutyryl cyclic AMP (dbcAMP, 1 mM) and the cAMP activator forskolin (50µM) was assessed in a GB cell line (U87GFP+, 10⁴ cells) co-cultured with mouse organotypic brain slices providing architecture and biochemical properties of normal brain tissue. Cell viability was determined by propidium-iodide, and gross metabolic effects were excluded in the extracellular medium. Tumor growth was quantified in terms of area, volume, and invasion at the start of culture, 48 h, 7 days, and 14 days after treatment. (3) Results: The tumor area was significantly reduced following dbcAMP or forskolin treatment (F_2,249 = 5.968, p = 0.0029). 3D volumetric quantification utilizing two-photon fluorescence microscopy revealed that the treated tumors maintained a spheric shape while the untreated controls exhibited the GB typical invasive growth pattern. (4) Conclusions: Our data demonstrate that treatment with a cAMP analog/activator reduces GB growth and invasion. Full article

Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16^INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16^INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response. Full article

Despite the well-known role of chronic human papillomavirus (HPV) infections in causing tumors (i.e., all cervical cancers and other human malignancies from the mucosal squamous epithelia, including anogenital and oropharyngeal cavity), its persistence is not sufficient for cancer development. Other co-factors contribute to the carcinogenesis process. Recently, the critical role of the underlying stroma during the HPV life cycle and HPV-induced disease have been investigated. The tumor stroma is a key component of the tumor microenvironment (TME), which is a specialized entity. The TME is dynamic, interactive, and constantly changing—able to trigger, support, and drive tumor initiation, progression, and metastasis. In previous years, in vitro organotypic raft cultures and in vivo genetically engineered mouse models have provided researchers with important information on the interactions between HPVs and the epithelium. Further development for an in-depth understanding of the interaction between HPV-infected tissue and the surrounding microenvironment is strongly required. In this review, we critically describe the HPV-related cancers modeled in vitro from the simplified ‘raft culture’ to complex three-dimensional (3D) organotypic models, focusing on HPV-associated cervical cancer disease platforms. In addition, we review the latest knowledge in the field of in vitro culture systems of HPV-associated malignancies of other mucosal squamous epithelia (anogenital and oropharynx), as well as rare cutaneous non-melanoma associated cancer. Full article

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH₂, denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice. Full article

Colorectal cancer is the third most common cancer worldwide, and the fourth leading cause of malignancy-related mortality. This highlights the need to understand the processes driving this disease in order to develop new treatments and improve patient outcomes. A potential therapeutic target is the increased stiffness of the tumour microenvironment, which is linked to aggressive cancer cell behaviour by enhancing biomechanical signalling. In this study, we used an siRNA-based approach to investigate the contribution of the protein cross-linking enzyme transglutaminase-2 (TG2) to matrix remodelling and biomechanical properties of the tumour microenvironment. TG2 inhibited cancer cell growth in organotypic 3D fibroblast/SW480 co-culture models, and biomechanical analysis demonstrated that colorectal cancer cells induced fibroblast-mediated stiffness which was inhibited by silencing TG2. These biomechanical changes were associated with observed alterations to collagen fibre structure, notably fibre thickness. Our in vitro findings of collagen composition changes were also seen with imaging biopsied tissues from patients with colorectal cancer, with TG2 correlating positively with thicker collagen fibres, and associating with poor outcome as determined by disease recurrence post-surgery and overall survival. In conclusion, this study demonstrates a role for TG2 in the stromal response to invading tumour, leading to tissue stiffening and poor outcome in patients. Full article