Three-dimensional Culture Model Research Articles

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

Patient-Derived Organoids: A Game-Changer in Personalized Cancer Medicine.

Research on cancer therapies has benefited from predictive tools capable of simulating treatment response and other disease characteristics in a personalized manner, in particular three-dimensional cell culture models. Such models include tumor-derived spheroids, multicellular spheroids including organotypic multicellular spheroids, and tumor-derived organoids. Additionally, organoids can be grown from various cancer cell types, such as pluripotent stem cells and induced pluripotent stem cells, progenitor cells, and adult stem cells. Although patient-derived xenografts and genetically engineered mouse models replicate human disease in vivo, organoids are less expensive, less labor intensive, and less time-consuming, all-important aspects in high-throughput settings. Like in vivo models, organoids mimic the three-dimensional structure, cellular heterogeneity, and functions of primary tissues, with the advantage of representing the normal oxygen conditions of patient organs. In this review, we summarize the use of organoids in disease modeling, drug discovery, toxicity testing, and precision oncology. We also summarize the current clinical trials using organoids.

3D Scaffold-Based Culture System Enhances Preclinical Evaluation of Natural Killer Cell Therapy in A549 Lung Cancer Cells.

Cell-based immunotherapies have emerged as promising cancer treatment modalities, demonstrating remarkable clinical efficacy. As interest in applying immune cell-based therapies to solid tumors has gained momentum, experimental models that enable long-term monitoring and mimic clinical administration are increasingly necessary. This study explores the potential of scaffold-based cell culture technologies, specifically three-dimensional (3D) extracellular matrix (ECM)-like frameworks, as promising solutions. These frameworks facilitate unhindered immune cell growth and enable continuous cancer cell culture. The three-dimensional (3D) cell culture model was developed using tailored scaffolds for natural killer (NK) cell culture. Within this framework, A549 lung cancer cells were cocultured with NK cells, allowing real-time monitoring for up to 28 days. The expression of critical markers associated with anticancer drug resistance and epithelial-mesenchymal transition (EMT) was evaluated in cancer cells within this 3D culture context. Compared to conventional 2D monolayer cultures, this 3D scaffold-based culture revealed that solid tumor cells, specifically A549 cells, exhibited heightened resistance to anticancer drugs. Additionally, the 3D culture environment upregulated the expression of EMT markers namely vimentin, N-cadherin, and fibronectin, while NK and zEGFR-CAR-NK cells displayed anticancer effects. In the two-dimensional (2D) coculture, only zEGFR-CAR-NK cells exhibited such effects in the 3D coculture system, highlighting an intriguing inconsistency with the 2D culture model, further confirmed by in vivo experiments. This in vitro 3D cell culture model reliably predicts outcomes in NK immunotherapy experiments. Thus, it represents a valuable tool for investigating drug resistance mechanisms and assessing the efficacy of immune cell-based therapies. By bridging the gap between in vitro and in vivo investigations, this model effectively translates potential treatments into animal models and facilitates rigorous preclinical evaluations.

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p>0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

The Use of Patient-Derived Organoids in the Study of Molecular Metabolic Adaptation in Breast Cancer

Around 13% of women will likely develop breast cancer during their lifetime. Advances in cancer metabolism research have identified a range of metabolic reprogramming events, such as altered glucose and amino acid uptake, increased reliance on glycolysis, and interactions with the tumor microenvironment (TME), all of which present new opportunities for targeted therapies. However, studying these metabolic networks is challenging in traditional 2D cell cultures, which often fail to replicate the three-dimensional architecture and dynamic interactions of real tumors. To address this, organoid models have emerged as powerful tools. Tumor organoids are 3D cultures, often derived from patient tissue, that more accurately mimic the structural and functional properties of actual tumor tissues in vivo, offering a more realistic model for investigating cancer metabolism. This review explores the unique metabolic adaptations of breast cancer and discusses how organoid models can provide deeper insights into these processes. We evaluate the most advanced tools for studying cancer metabolism in three-dimensional culture models, including optical metabolic imaging (OMI), matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), and recent advances in conventional techniques applied to 3D cultures. Finally, we explore the progress made in identifying and targeting potential therapeutic targets in breast cancer metabolism.

Abstract C172: Diversifying translational research models to address outcomes disparities in lung cancer

Abstract Introduction: Racial disparities in lung cancer remains a persistent and challenging problem, with African Americans experiencing the highest mortality of any race or ethnic group. Emerging evidence suggests that multiple biological factors affect the development and progression of lung cancer. Three-dimensional organoid culture models have become important instruments to use for biomarker analysis and drug sensitivity screening. Despite this strong rationale and potential, there is a lack of representative preclinical models from African American patients with lung cancer. Furthermore, there are no studies evaluating the ability to create organoids successfully using samples from different races. As such, our goal was to establish a diverse biobank of organoid models and determine the rate of successful development based on race and other factors. Methods: Patients with lung cancer from a large urban medical center were recruited to participate in the study from 2021 to 2024. Specimens were collected from surgical pulmonary resection or endobronchial biopsy. Each tissue specimen was divided into 3 pieces and allocated for genomic, transcriptomic and organoid development. Once collected, specimens allocated for organoid development were processed within 8 hours. Cells were isolated from tissues by mechanical and enzymatic dissociation and plated in Matrigel drops overlayed with optimized organoid media. Patient demographics, tumor characteristics and method of specimen collection were recorded. Results: A total of 29 patients consented to the study and had evaluable specimens collected. Mean age was 67 years. Fifty-five percent of patients (16/29) were female. White patients were 55 percent of cohort (16/29), while Black patients made up 45% (13/29). Approximately 62 (18/29) percent of patients were diagnosed with adenocarcinoma, followed by squamous cell carcinoma in 28 percent (8/29) of patients. Tumor stage was 55%, 28%, 3% and 14% for stages I, II, III and IV respectively. Overall, there was a successful organoid development rate of 69 percent (20/29). There was a strong trend toward higher success rates in Black patients compared to White patients (85% vs. 56%, p = 0.10). There were no significant differences in successful organoid development based on gender (p = 0.43), histology (p = 0.18) or tumor stage (p = 0.84). Conclusions: Establishment of a racially diverse set of organoid models will be an invaluable tool for biomarker discovery and for screening novel therapeutic agents. Black patients appear to have similar to improved propensity for successful organoid development. Future efforts should be focused on creating diverse biobanks to strengthen precision oncology efforts for Black patients, collectively helping to reduce racial outcomes disparities in patients with lung cancer. Citation Format: Amanda Pilling, Hollis Hutchings, Avi Cohen, Shirish Gadgeel, Ikenna Okereke. Diversifying translational research models to address outcomes disparities in lung cancer [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C172.

Development of Tissue-Engineered Model of Fibrotic Scarring after Spinal Cord Injury to Study Astrocyte Activation and Neurite Outgrowth In Vitro.

Traumatic spinal cord injuries (SCI) are debilitating injuries affecting twenty-seven million people worldwide and cause functional impairments. Despite decades of research and medical advancements, current treatment options for SCI remain limited, in part due to the complex pathophysiology of spinal cord lesions including cellular transformation and extracellular matrix (ECM) remodeling. Recent studies have increased focus on fibrotic scarring after SCI, and yet much remains unclear about the impact of fibrotic scarring on SCI lesion progression. Here, using collagen and decellularized spinal cord-based composite hydrogels, a three-dimensional (3D) cell culture model mimicking the fibrous core of spinal cord lesions was implemented to investigate its influence on the surrounding astrocytes. To mimic the fibrotic milieu, collagen fibril thickness was tuned using previously established temperature-controlled casting methods. In our platforms, astrocytes in fibro-mimetic hydrogels exhibited increased levels of activation markers such as glial fibrillary acidic protein and N-cadherin. Furthermore, astrocytes in fibro-mimetic hydrogels deposited more fibronectin and laminin, further hinting that astrocytes may also contribute to fibrotic scarring. These markers were decreased when Rho-ROCK and integrin β1 were inhibited via pharmacological inhibitors. Mechanistic analysis of Yes-associated protein reveals that blocking integrin β1 prevents mechanosensing of astrocytes, contributing to altered phenotypes in variable culture conditions. In the presence of these inhibitors, astrocytes increased the secretion of brain-derived neurotrophic factor, and a greater degree of dorsal root ganglia neurite infiltration into the underlying hydrogels was observed. Altogether, this study presents a novel tissue-engineered platform to study fibrotic scarring after SCI and may be a useful platform to advance our understanding of SCI lesion aggravation.

A Facile, Transfection-Free Approach to siRNA Delivery in In Vitro 3D Spheroid Models.

Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two-dimensional (2D) cell culture fails to faithfully model the complexity found in vivo, and novel drug candidates that show promising results in 2D models often do not translate to the clinic. More recently, three-dimensional (3D) cell culture models have gained popularity owing to their greater physiological relevance to in vivo biology. In particular, 3D spheroid models are becoming widely used due to their ability to mimic solid tumors, both in architecture and gradation of nutrients distributed from the outer, proliferative layers into the inner, quiescent layers of cells. Similar to in vivo tumors, cell lines grown in 3D spheroid models tend to be more resistant to antitumor drug treatments than their 2D cultured counterparts, though distinct signaling pathways and gene targets conferring this resistance have yet to be fully explored. RNA interference (RNAi) is an effective tool to elucidate gene function and discover novel druggable targets in 2D models; however, only a few studies have successfully performed RNAi in complex 3D models to date. Here, we demonstrate efficient RNAi-mediated knockdown using "transfection-free" Dharmacon Accell siRNAs in three spheroid culture models, in the presence or absence of the extracellular matrix. This methodology has the potential to be scaled up for complex arrayed screening experiments, which may aid in the identification of novel druggable targets with greater clinical relevance than those identified in 2D experiments. © 2024 Dharmacon, Inc. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 3D spheroids in matrix-free ULA plates Alternate Protocol 1: Generation of Matrigel matrix-embedded 3D spheroids Alternate Protocol 2: Generation of GrowDex hydrogel-embedded 3D spheroids Basic Protocol 2: Delivery of siRNA and collection of matrix-free 3D spheroids Alternate Protocol 3: Delivery of siRNA and collection of matrix-embedded spheroids Basic Protocol 3: RNA and protein extraction from spheroids for characterization of gene knockdown.

380. GROWTH CHARACTERISTICS OF ESOPHAGEAL PATIENT DERIVED ORGANOIDS REFLECT CLINICOPATHOLOGIC VARIABLES

Abstract Background Esophageal patient derived organoids (PDOs) are three-dimensional cell culture models derived from esophageal cancer tissue. We have previously shown that PDOs recapitulate the genetic and morphologic characteristics of the primary tumour, and that PDOs may predict patient responses to neoadjuvant regimens and potentially allow personalisation perioperative therapies. Nevertheless, up to 30% of tissue samples fail to establish organoids in vivo, although the reasons for this are unclear. This study interrogates the clinical characteristics of biopsies that establish or fail to establish PDOs. Methods Esophageal adenocarcinomas from locally advanced (LA) or metastatic (M) disease were biopsied at initial endoscopy, following surgical resection, or image-guided biopsy of metastatic disease (n=75 total). Following a standardised protocol, cells were dissociated and seeded for organoid culture. Organoids are considered ‘established’ after passage 4. Samples were discarded if there was obvious contamination (n=9) or if there were no obvious tumour cells (n=6). Clinicopathologic variables for successfully established or unsuccessfully established were compared for both LA and M disease. Statistical analysis was performed to interrogate characteristics associated with successfully establishing organoids. Results There were significant differences between successful and unsuccessful organoids. Organoids were more likely to be established from endoscopic biopsies (p=0.002). Samples pre-treated with neoadjuvant chemoradiation or chemotherapy were less likely to grow (p=0.02). Samples from poorly differentiated tumours were less likely to grow compared with well and moderately differentiated tumours (p=0.05). Tumour stage, lymphovascular invasion, perineural invasion, MSI status and HER-2 status were not associated with growth characteristics. Conclusion Growth characteristics of esophageal adenocarcinoma PDOs correlate with clinicopathologic characteristics. Samples from patients who have undergone chemotherapy or chemoradiation, and poorly differentiated tumours are less likely to establish in vitro models. Understanding the altered in vitro growth patterns is key to translating use of organoids into clinical practice.

The use of hyaluronic acid in a 3D biomimetic scaffold supports spheroid formation and the culture of cancer stem cells

Three-dimensional (3D) bioprinting culture models capable of reproducing the pathological architecture of diseases are increasingly advancing. In this study, 3D scaffolds were created using extrusion-based bioprinting method with alginate, gelatin, and hyaluronic acid to investigate the effects of hyaluronic acid on the physical properties of the bioscaffold as well as on the formation of liver cancer spheroids. Conformational analysis, rheological characterization, and swelling-degradation tests were performed to characterize the scaffolds. After generating spheroids from hepatocellular carcinoma cells on the 3D scaffolds, cell viability and proliferation assays were performed. Flow cytometry and immunofluorescence microscopy were used into examine the expression of albumin, CD44, and E-cadherin to demonstrate functional capability and maturation levels of the spheroid-forming cells. The results show that hyaluronic acid in the scaffolds correlates with spheroid formation and provides high survival rates. It is also associated with an increase in CD44 expression and a decrease in E-cadherin, while there is no significant change in the albumin expression in the cells. Overall, the findings demonstrate that hyaluronic acid in a 3D hydrogel scaffold supports spheroid formation and may induce stemness. We present a promising 3D scaffold model for enhancing liver cancer spheroid formation and mimicking solid tumors. This model also has the potential for further studies to examine stem cell properties in 3D models.

Influence of inflammation on the expression of microRNA-140 in extracellular vesicles from 2D and 3D culture models of synovial-membrane-derived stem cells.

In osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an in vitro inflammatory environment. Four experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1β and TNFα. The culture media samples were collected at 24h, 72h, and 120h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level. Encapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72h and in the 2D-OA group at 120h (p < 0.001). However, the 3D-OA culture showed higher expression of the mRNA Adamts5 in the EVs at 120h. The responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.

Using Spheroids as Building Blocks Towards 3D Bioprinting of Tumor Microenvironment.

Cancer still ranks as a leading cause of mortality worldwide. Although considerable efforts have been dedicated to anticancer therapeutics, progress is still slow, partially due to the absence of robust prediction models. Multicellular tumor spheroids, as a major three-dimensional (3D) culture model exhibiting features of avascular tumors, gained great popularity in pathophysiological studies and high throughput drug screening. However, limited control over cellular and structural organization is still the key challenge in achieving in vivo like tissue microenvironment. 3D bioprinting has made great strides toward tissue/organ mimicry, due to its outstanding spatial control through combining both cells and materials, scalability, and reproducibility. Prospectively, harnessing the power from both 3D bioprinting and multicellular spheroids would likely generate more faithful tumor models and advance our understanding on the mechanism of tumor progression. In this review, the emerging concept on using spheroids as a building block in 3D bioprinting for tumor modeling is illustrated. We begin by describing the context of the tumor microenvironment, followed by an introduction of various methodologies for tumor spheroid formation, with their specific merits and drawbacks. Thereafter, we present an overview of existing 3D printed tumor models using spheroids as a focus. We provide a compilation of the contemporary literature sources and summarize the overall advancements in technology and possibilities of using spheroids as building blocks in 3D printed tissue modeling, with a particular emphasis on tumor models. Future outlooks about the wonderous advancements of integrated 3D spheroidal printing conclude this review.

Polydopamine Nanoparticle-Based Combined Chemotherapy and Photothermal Therapy for the Treatment of Liver Cancer.

Polydopamine nanoparticles (PDA NPs) are proposed as an anti-cancer tool against hepatocellular carcinoma through the combination of near-infrared (NIR)-mediated hyperthermia and loading with a chemotherapeutic drug, sorafenib (SRF). Cell membranes isolated from a liver cancer cell line (HepG2) have been exploited for the coating of the nanoparticles (thus obtaining CM-SRF-PDA NPs), to promote homotypic targeting toward cancer cells. The selective targeting ability and the combined photothermal and chemotherapeutic activity of the CM-SRF-PDA NPs following NIR irradiation have been evaluated on cell cultures in static and dynamic conditions, besides three-dimensional culture models. Eventually, the therapeutic effectiveness of the proposed approach has also been tested ex ovo on HepG2 spheroid-grafted quail embryos. This comprehensive investigation, supported by proteomic analysis, showed the effectiveness of the proposed nanoplatform and strongly suggests further pre-clinical testing in the treatment of liver cancer.

Fabrication of heterocellular spheroids with controllable core-shell structure using inertial focusing effect for scaffold-free 3D cell culture models

Three-dimensional (3D) cell culture models capable of emulating the biological functions of natural tissues are pivotal in tissue engineering and regenerative medicine. Despite progress, the fabrication ofin vitroheterocellular models that mimic the intricate structures of natural tissues remains a significant challenge. In this study, we introduce a novel, scaffold-free approach leveraging the inertial focusing effect in rotating hanging droplets for the reliable production of heterocellular spheroids with controllable core-shell structures. Our method offers precise control over the core-shell spheroid's size and geometry by adjusting the cell suspension density and droplet morphology. We successfully applied this technique to create hair follicle organoids, integrating dermal papilla cells within the core and epidermal cells in the shell, thereby achieving markedly enhanced hair inducibility compared to mixed-structure models. Furthermore, we have developed melanoma tumor spheroids that accurately mimic the dynamic interactions between tumor and stromal cells, showing increased invasion capabilities and altered expressions of cellular adhesion molecules and proteolytic enzymes. These findings underscore the critical role of cellular spatial organization in replicating tissue functionalityin vitro. Our method represents a significant advancement towards generating heterocellular spheroids with well-defined architectures, offering broad implications for biological research and applications in tissue engineering.

A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Impact of Cyclic Strain on Elastin Synthesis in a 3D Human Myometrial Culture Model.

The synthesis and assembly of mature, organized elastic fibers remains a limitation to the clinical use of many engineered tissue replacements. There is a critical need for a more in-depth understanding of elastogenesis regulation for the advancement of methods to induce and guide production of elastic matrix structures in engineered tissues that meet the structural and functional requirements of native tissue. The dramatic increase in elastic fibers through normal pregnancy has led us to explore the potential role of mechanical stretch in combination with pregnancy levels of the steroid hormones 17β-estradiol and progesterone on elastic fiber production by human uterine myometrial smooth muscle cells in a three-dimensional (3D) culture model. Opposed to a single strain regimen, we sought to better understand how the amplitude and frequency parameters of cyclic strain influence elastic fiber production in these myometrial tissue constructs (MTC). Mechanical stretch was applied to MTC at a range of strain amplitudes (5%, 10%, and 15% at 0.5 Hz frequency) and frequencies (0.1 Hz, 0.5 Hz, 1 Hz, and constant 0 Hz at 10% amplitude), with and without pregnancy-level hormones, for 6 days. MTC were assessed for cell proliferation, matrix elastin protein content, and expression of the main elastic fiber genes, tropoelastin (ELN) and fibrillin-1 (FBN1). Significant increases in elastin protein and ELN and FBN1 mRNA were produced from samples subjected to a 0.5 Hz, 10% strain regimen, as well as samples stretched at higher amplitude (15%, 0.5 Hz) and higher frequency (1 Hz, 10%); however, no significant effects because of third-trimester mimetic hormone treatment were determined. These results establish that a minimum level of strain is required to stimulate the synthesis of elastic fiber components in our culture model and show this response can be similarly enhanced by increasing either the amplitude or frequency parameter of applied strain. Further, our results demonstrate strain alone is sufficient to stimulate elastic fiber production and suggest hormones may not be a significant factor in regulating elastin synthesis. This 3D culture model will provide a useful tool to further investigate mechanisms underlying pregnancy-induced de novo elastic fiber synthesis and assembly by uterine smooth muscle cells.

Targeting tumor-associated macrophages with mannosylated nanotherapeutics delivering TLR7/8 agonist enhances cancer immunotherapy

Tumor-associated macrophages (TAMs) constitute 50–80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic-co-glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from “cold tumor” into “hot tumor”, with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy.

Variations in fluid chemical potential induce fibroblast mechano-response in 3D hydrogels

Mechanical deformation of skin creates variations in fluid chemical potential, leading to local changes in hydrostatic and osmotic pressure, whose effects on mechanobiology remain poorly understood. To study these effects, we investigate the specific influences of hydrostatic and osmotic pressure on primary human dermal fibroblasts in three-dimensional hydrogel culture models. Cyclic hydrostatic pressure and hyperosmotic stress enhanced the percentage of cells expressing the proliferation marker Ki67 in both collagen and PEG-based hydrogels. Osmotic pressure also activated the p38 MAPK stress response pathway and increased the expression of the osmoresponsive genes PRSS35 and NFAT5. When cells were cultured in two-dimension (2D), no change in proliferation was observed with either hydrostatic or osmotic pressure. Furthermore, basal, and osmotic pressure-induced expression of osmoresponsive genes differed in 2D culture versus 3D hydrogels, highlighting the role of dimensionality in skin cell mechanotransduction and stressing the importance of 3D tissue-like models that better replicate in vivo conditions. Overall, these results indicate that fluid chemical potential changes affect dermal fibroblast mechanobiology, which has implications for skin function and for tissue regeneration strategies.

The application of organoids in colorectal diseases.

Intestinal organoids are a three-dimensional cell culture model derived from colon or pluripotent stem cells. Intestinal organoids constructed in vitro strongly mimic the colon epithelium in cell composition, tissue architecture, and specific functions, replicating the colon epithelium in an in vitro culture environment. As an emerging biomedical technology, organoid technology has unique advantages over traditional two-dimensional culture in preserving parental gene expression and mutation, cell function, and biological characteristics. It has shown great potential in the research and treatment of colorectal diseases. Organoid technology has been widely applied in research on colorectal topics, including intestinal tumors, inflammatory bowel disease, infectious diarrhea, and intestinal injury regeneration. This review focuses on the application of organoid technology in colorectal diseases, including the basic principles and preparation methods of organoids, and explores the pathogenesis of and personalized treatment plans for various colorectal diseases to provide a valuable reference for organoid technology development and application.

Selective nitration of Hsp90 acts as a metabolic switch promoting tumor cell proliferation

Tumors develop in an oxidative environment characterized by peroxynitrite production and downstream protein tyrosine (Y) nitration. We showed that tyrosine nitration supports schwannoma cell proliferation and regulates cell metabolism in the inheritable tumor disorder NF2-related Schwannomatosis (NF2-SWN). Here, we identified the chaperone Heat shock protein 90 (Hsp90) as the first nitrated protein that acts as a metabolic switch to promote schwannoma cell proliferation. Doubling the endogenous levels of nitrated Hsp90 in schwannoma cells or supplementing nitrated Hsp90 into normal Schwann cells increased their proliferation. Metabolically, nitration on either Y33 or Y56 conferred Hsp90 distinct functions; nitration at Y33 (Hsp90NY33) down-regulated mitochondrial oxidative phosphorylation, while nitration at Y56 (Hsp90NY56) increased glycolysis by activating the purinergic receptor P2X7 in both schwannoma and normal Schwann cells. Hsp90NY33 and Hsp90NY56 showed differential subcellular and spatial distribution corresponding with their metabolic and proliferative functions in schwannoma three-dimensional cell culture models. Collectively, these results underscore the role of tyrosine nitration as a post-translational modification regulating critical cellular processes. Nitrated proteins, particularly nitrated Hsp90, emerge as a novel category of tumor-directed therapeutic targets.