Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Hydrogel/microcarrier cell scaffolds for rapid expansion of satellite cells from large yellow croakers: Differential analysis between 2D and 3D cell culture.

Cell culture meat is based on the scaled-up expansion of seed cells. The biological differences between seed cells from large yellow croakers in the two-dimensional (2D) and three-dimensional (3D) culture systems have not been explored. Here, satellite cells (SCs) from large yellow croakers (Larimichthys crocea) were grown on cell climbing slices, hydrogels, and microcarriers for five days to analyze the biological differences of SCs on different cell scaffolds. The results exhibited that SCs had different cell morphologies in 2D and 3D cultures. Cell adhesion receptors (Itgb1andsdc4) and adhesion spot markervclof the 3D cultures were markedly expressed. Furthermore, myogenic decision markers (Pax7andmyod) were significantly enhanced. However, the expression of myogenic differentiation marker (desmin) was significantly increased in the microcarrier group. Combined with the transcriptome data, this suggests that cell adhesion of SCs in 3D culture was related to the integrin signaling pathway. In contrast, the slight spontaneous differentiation of SCs on microcarriers was associated with rapid cell proliferation. This study is the first to report the biological differences between SCs in 2D and 3D cultures, providing new perspectives for the rapid expansion of cell culture meat-seeded cells and the development of customized scaffolds.

Generation of 3D Fibroblast-Derived Extracellular Matrix and Analysis of Tumor Cell-Matrix Interactions and Signaling.

Fibroblasts are the major producers of the extracellular matrix and regulate its organization. Aberrant signaling in diseases such as fibrosis and cancer can impact the deposition of the matrix proteins, which can in turn act as an adhesion scaffold and signaling reservoir promoting disease progression. To study the composition and organization of the extracellular matrix as well as its interactions with (tumor) cells, this protocol describes the generation and analysis of 3D fibroblast-derived matrices and the investigation of (tumor) cells seeded onto the 3D scaffolds by immunofluorescent imaging and cell adhesion, colony formation, migration, and invasion/transmigration assays.

Advances in the application of extracellular vesicles derived from three-dimensional culture of stem cells.

Stem cells (SCs) have been used therapeutically for decades, yet their applications are limited by factors such as the risk of immune rejection and potential tumorigenicity. Extracellular vesicles (EVs), a key paracrine component of stem cell potency, overcome the drawbacks of stem cell applications as a cell-free therapeutic agent and play an important role in treating various diseases. However, EVs derived from two-dimensional (2D) planar culture of SCs have low yield and face challenges in large-scale production, which hinders the clinical translation of EVs. Three-dimensional (3D) culture, given its ability to more realistically simulate the in vivo environment, can not only expand SCs in large quantities, but also improve the yield and activity of EVs, changing the content of EVs and improving their therapeutic effects. In this review, we briefly describe the advantages of EVs and EV-related clinical applications, provide an overview of 3D cell culture, and finally focus on specific applications and future perspectives of EVs derived from 3D culture of different SCs.

A Pillar/Perfusion Plate Enhances Cell Growth, Reproducibility, Throughput, and User Friendliness in Dynamic 3D Cell Culture.

Static three-dimensional (3D) cell culture has been demonstrated in ultralow attachment well plates, hanging droplet plates, and microtiter well plates with hydrogels or magnetic nanoparticles. Although it is simple, reproducible, and relatively inexpensive, thus potentially used for high-throughput screening, statically cultured 3D cells often suffer from a necrotic core due to limited nutrient and oxygen diffusion and waste removal and have a limited in vivo-like tissue structure. Here, we overcome these challenges by developing a pillar/perfusion plate platform and demonstrating high-throughput, dynamic 3D cell culture. Cell spheroids were loaded on the pillar plate with hydrogel by simple sandwiching and encapsulation and cultured dynamically in the perfusion plate on a digital rocker. Unlike traditional microfluidic devices, fast flow velocity was maintained within perfusion wells and the pillar plate was separated from the perfusion plate for cell-based assays. It was compatible with common lab equipment and allowed cell culture, testing, staining, and imaging in situ. The pillar/perfusion plate enhanced cell growth by rapid diffusion, reproducibility, assay throughput, and user friendliness in a dynamic 3D cell culture.

Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Open-Top Patterned Hydrogel-Laden 3D Glioma Cell Cultures for Creation of Dynamic Chemotactic Gradients to Direct Cell Migration.

The laminar flow profiles in microfluidic systems coupled to rapid diffusion at flow streamlines have been widely utilized to create well-controlled chemical gradients in cell cultures for spatially directing cell migration. However, within hydrogel-based closed microfluidic systems of limited depth (≤0.1 mm), the biomechanical cues for the cell culture are dominated by cell interactions with channel surfaces rather than with the hydrogel microenvironment. Also, leaching of poly(dimethylsiloxane) (PDMS) constituents in closed systems and the adsorption of small molecules to PDMS alter chemotactic profiles. To address these limitations, we present the patterning and integration of a PDMS-free open fluidic system, wherein the cell-laden hydrogel directly adjoins longitudinal channels that are designed to create chemotactic gradients across the 3D culture width, while maintaining uniformity across its ∼1 mm depth to enhance cell-biomaterial interactions. This hydrogel-based open fluidic system is assessed for its ability to direct migration of U87 glioma cells using a hybrid hydrogel that includes hyaluronic acid (HA) to mimic the brain tumor microenvironment and gelatin methacrylate (GelMA) to offer the adhesion motifs for promoting cell migration. Chemotactic gradients to induce cell migration across the hydrogel width are assessed using the chemokine CXCL12, and its inhibition by AMD3100 is validated. This open-top hydrogel-based fluidic system to deliver chemoattractant cues over square-centimeter-scale areas and millimeter-scale depths can potentially serve as a robust screening platform to assess emerging glioma models and chemotherapeutic agents to eradicate them.

Colon cancer inhibitory properties of Caulerpa lentillifera polysaccharide and its molecular mechanisms based on three-dimensional cell culture model.

Caulerpa lentillifera is rich in polysaccharides, and its polysaccharides show a significant effect in different biological activities including anti-cancer activity. As an edible algae-derived polysaccharide, exploring the role of colon cancer can better develop the application from a dietary therapy perspective. However, more in-depth studies of C. lentillifera polysaccharide on anti-colon cancer activity and mechanism are needed. In this study, we found that Caulerpa lentillifera polysaccharides (CLP) showed potential anti-colon cancer effect on human colon cancer cell HT29 in monolayer (IC50 = 1.954 mg/mL) and spheroid (IC50 = 0.402 mg/mL). Transcriptomics and metabolomics analyses revealed that CLP had an inhibitory effect on HT29 3D spheroid cells by activating aminoacyl-tRNA biosynthesis as well as arginine and proline metabolism pathways. Furthermore, the anti-colon cancer effects of CLP were confirmed through other human colon cancer cell HCT116 and LoVo in monolayer cells (IC50 = 1.890 mg/mL and 1.437 mg/mL, respectively) and 3D spheroid cells (IC50 = 0.344 mg/mL and 0.975 mg/mL, respectively), and three patient-derived organoids with IC50 values of 6.333-8.780 mg/mL. This study provided basic data for the potential application of CLP in adjuvant therapeutic food for colon cancer on multiple levels, while further investigation of detailed mechanism in vivo was still required.

Characterization of TGFß1-induced tendon-like structure in the scaffold-free three-dimensional tendon cell culture system.

The biological mechanisms regulating tenocyte differentiation and morphological maturation have not been well-established, partly due to the lack of reliable in vitro systems that produce highly aligned collagenous tissues. In this study, we developed a scaffold-free, three-dimensional (3D) tendon culture system using mouse tendon cells in a differentially adherent growth channel. Transforming Growth Factor-ß (TGFß) signaling is involved in various biological processes in the tendon, regulating tendon cell fate, recruitment and maintenance of tenocytes, and matrix organization. This known function of TGFß signaling in tendon prompted us to utilize TGFß1 to induce tendon-like structures in 3D tendon constructs. TGFß1 treatment promoted a tendon-like structure in the peripheral layer of the constructs characterized by increased thickness with a gradual decrease in cell density and highly aligned collagen matrix. TGFß1 also enhanced cell proliferation, matrix production, and morphological maturation of cells in the peripheral layer compared to vehicle treatment. TGFß1 treatment also induced early tenogenic differentiation and resulted in sufficient mechanical integrity, allowing biomechanical testing. The current study suggests that this scaffold-free 3D tendon cell culture system could be an in vitro platform to investigate underlying biological mechanisms that regulate tenogenic cell differentiation and matrix organization.

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture.

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel® SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel®Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel® Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels® with laminin. Laminin was found to require negatively charged PeptiGel® Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

The effects of microgravity on stem cells and the new insights it brings to tissue engineering and regenerative medicine.

Conventional two-dimensional (2D) cell culture techniques may undergo modifications in the future, as life scientists have widely acknowledged the ability of three-dimensional (3D) in vitro culture systems to accurately simulate in vivo biology. In recent years, researchers have discovered that microgravity devices can address many challenges associated with 3D cell culture. Stem cells, being pluripotent cells, are regarded as a promising resource for regenerative medicine. Recent studies have demonstrated that 3D culture in microgravity devices can effectively guide stem cells towards differentiation and facilitate the formation of functional tissue, thereby exhibiting advantages within the field of tissue engineering and regenerative medicine. Furthermore, We delineate the impact of microgravity on the biological behavior of various types of stem cells, while elucidating the underlying mechanisms governing these alterations. These findings offer exciting prospects for diverse applications.

Advancements in culture technology of adipose-derived stromal/stem cells: implications for diabetes and its complications.

Stem cell-based therapies exhibit considerable promise in the treatment of diabetes and its complications. Extensive research has been dedicated to elucidate the characteristics and potential applications of adipose-derived stromal/stem cells (ASCs). Three-dimensional (3D) culture, characterized by rapid advancements, holds promise for efficacious treatment of diabetes and its complications. Notably, 3D cultured ASCs manifest enhanced cellular properties and functions compared to traditional monolayer-culture. In this review, the factors influencing the biological functions of ASCs during culture are summarized. Additionally, the effects of 3D cultured techniques on cellular properties compared to two-dimensional culture is described. Furthermore, the therapeutic potential of 3D cultured ASCs in diabetes and its complications are discussed to provide insights for future research.

Recent Advances in 3D Cultures.

Methods and protocols for creating complex 3D cell culture systems have been rapidly advancing in the past decade from the perspective of biomaterials [...].

Methodology and Characterization of a 3D Bone Organoid Model Derived from Murine Cells.

Here, we report on the development of a cost-effective, well-characterized three-dimensional (3D) model of bone homeostasis derived from commonly available stocks of immortalized murine cell lines and laboratory reagents. This 3D murine-cell-derived bone organoid model (3D-mcBOM) is adaptable to a range of contexts and can be used in conjunction with surrogates of osteoblast and osteoclast function to study cellular and molecular mechanisms that affect bone homeostasis in vitro or to augment in vivo models of physiology or disease. The 3D-mcBOM was established using a pre-osteoblast murine cell line, which was seeded into a hydrogel extracellular matrix (ECM) and differentiated into functional osteoblasts (OBs). The OBs mineralized the hydrogel ECM, leading to the deposition and consolidation of hydroxyapatite into bone-like organoids. Fourier-transform infrared (FTIR) spectroscopy confirmed that the mineralized matrix formed in the 3D-mcBOM was bone. The histological staining of 3D-mcBOM samples indicated a consistent rate of ECM mineralization. Type I collagen C-telopeptide (CTX1) analysis was used to evaluate the dynamics of OC differentiation and activity. Reliable 3D models of bone formation and homeostasis align with current ethical trends to reduce the use of animal models. This functional model of bone homeostasis provides a cost-effective model system using immortalized cell lines and easily procured supplemental compounds, which can be assessed by measuring surrogates of OB and OC function to study the effects of various stimuli in future experimental evaluations of bone homeostasis.

Three-Dimensional Cultivation a Valuable Tool for Modelling Canine Mammary Gland Tumour Behaviour In Vitro.

Cell cultivation has been one of the most popular methods in research for decades. Currently, scientists routinely use two-dimensional (2D) and three-dimensional (3D) cell cultures of commercially available cell lines and primary cultures to study cellular behaviour, responses to stimuli, and interactions with their environment in a controlled laboratory setting. In recent years, 3D cultivation has gained more attention in modern biomedical research, mainly due to its numerous advantages compared to 2D cultures. One of the main goals where 3D culture models are used is the investigation of tumour diseases, in both animals and humans. The ability to simulate the tumour microenvironment and design 3D masses allows us to monitor all the processes that take place in tumour tissue created not only from cell lines but directly from the patient's tumour cells. One of the tumour types for which 3D culture methods are often used in research is the canine mammary gland tumour (CMT). The clinically similar profile of the CMT and breast tumours in humans makes the CMT a suitable model for studying the issue not only in animals but also in women.

State of the Art in 3D Culture Models Applied to Thyroid Cancer.

Thyroid cancer (TC) is the prevalent endocrine tumor with a rising incidence, particularly in higher-income countries, leading to an increased interest in its management and treatment. While overall, survival rates for TC are usually favorable, advanced cases, especially with metastasis and specific histotypes, pose challenges with poorer outcomes, advocating the need of systemic treatments. Targeted therapies have shown efficacy in both preclinical models and clinical trials but face issues of resistance, since they usually induce partial and transient response. These resistance phenomena are currently only partially addressed by traditional preclinical models. This review explores the limitations of traditional preclinical models and emphasizes the potential of three-dimensional (3D) models, such as transwell assays, spheroids, organoids, and organ-on-chip technology in providing a more comprehensive understanding of TC pathogenesis and treatment responses. We reviewed their use in the TC field, highlighting how they can produce new interesting insights. Finally, the advent of organ-on-chip technology is currently revolutionizing preclinical research, offering dynamic, multi-cellular systems that replicate the complexity of human organs and cancer-host interactions.

Three-Dimensional Cell Culture of Adipose-Derived Stem Cells in a Hydrogel with Photobiomodulation Augmentation.

Adipose-derived stem cells (ADSCs), possessing multipotent mesenchymal characteristics akin to stem cells, are frequently employed in regenerative medicine due to their capacity for a diverse range of cell differentiation and their ability to enhance migration, proliferation, and mitigate inflammation. However, ADSCs often face challenges in survival and engraftment within wounds, primarily due to unfavorable inflammatory conditions. To address this issue, hydrogels have been developed to sustain ADSC viability in wounds and expedite the wound healing process. Here, we aimed to assess the synergistic impact of photobiomodulation (PBM) on ADSC proliferation and cytotoxicity within a 3D cell culture framework. Immortalized ADSCs were seeded into 10 µL hydrogels at a density of 2.5 x 103 cells and subjected to irradiation using 525 nm and 825 nm diodes at fluencies of 5 J/cm2 and 10 J/cm2. Morphological changes, cytotoxicity, and proliferation were evaluated at 24 h and 10 days post-PBM exposure. The ADSCs exhibited a rounded morphology and were dispersed throughout the gel as individual cells or spheroid aggregates. Importantly, both PBM and 3D culture framework displayed no cytotoxic effects on the cells, while PBM significantly enhanced the proliferation rates of ADSCs. In conclusion, this study demonstrates the use of hydrogel as a suitable 3D environment for ADSC culture and introduces PBM as a significant augmentation strategy, particularly addressing the slow proliferation rates associated with 3D cell culture.

Development of a novel high-throughput culture system for hypoxic 3D hydrogel cell culture.

Animal models lack physiologic relevance to the human system which results in low clinical translation of results derived from animal testing. Besides spheroids or organoids, hydrogel-based 3D in vitro models are used to mimic the in vivo situation increasing the relevance while reducing animal testing. However, to establish hydrogel-based 3D models in applications such as drug development or personalized medicine, high-throughput culture systems are required. Furthermore, the integration of oxygen-reduced (hypoxic) conditions has become increasingly important to establish more physiologic culture models. Therefore, we developed a platform technology for the high-throughput generation of miniaturized hydrogels for 3D cell culture. The Oli-Up system is based on the shape of a well-plate and allows for the parallel culture of 48 hydrogel samples, each with a volume of 15 µl. As a proof-of-concept, we established a 3D culture of gelatin-methacryloyl (GelMA)-encapsulated mesenchymal stem/stromal cells (MSCs). We used a hypoxia reporter cell line to establish a defined oxygen-reduced environment to precisely trigger cellular responses characteristic of hypoxia in MSCs. In detail, the expression of hypoxia response element (HRE) increased dependent on the oxygen concentration and cell density. Furthermore, MSCs displayed an altered glucose metabolism and increased VEGF secretion upon oxygen-reduction. In conclusion, the Oli-Up system is a platform technology for the high-throughput culture of hydrogel-based 3D models in a defined oxygen environment. As it is amenable for automation, it holds the potential for high-throughput screening applications such as drug development and testing in more physiologic 3D in vitro tissue models.

Establishment and validation of an efficient method for the 3D culture of osteoclasts in vitro.

INTRODUCTION: Osteoclasts (OCs) play a crucial role in maintaining bone health. Changes in OC activity are linked to different bone diseases, making them an intriguing focus for research. However, most studies on OCs have relied on 2D cultures, limiting our understanding of their behavior. Yet, there's a lack of knowledge regarding platforms that effectively support osteoclast formation in 3D cultures. METHODS: In our investigation, we explored the capacity of collagen and GelMA hydrogels to facilitate osteoclast development in 3D culture settings. We assessed the osteoclast development by using different hydrogels and cell seeding strategies and optimizing cell seeding density and cytokine concentration. The osteoclast development in 3D cultures was further validated by biochemical assays and immunochemical staining. RESULTS: Our findings revealed that 0.3 % (w/v) collagen was conducive to osteoclast formation in both 2D and 3D cultures, demonstrated by increased multinucleation and higher TRAP activity compared to 0.6 % collagen and 5 % to 10 % (w/v) GelMA hydrogels. Additionally, we devised a "sandwich" technique using collagen substrates and augmented the initial macrophage seeding density and doubling cytokine concentrations, significantly enhancing the efficiency of OC culture in 3D conditions. Notably, we validated osteoclasts derived from macrophages in our 3D cultures express key osteoclast markers like cathepsin K and TRAP. CONCLUSIONS: To conclude, our study contributes to establishing an effective method for cultivating osteoclasts in 3D environments in vitro. This innovative approach not only promises a more physiologically relevant platform to study osteoclast behavior during bone remodeling but also holds potential for applications in bone tissue engineering. CLINICAL SIGNIFICANCE: This study introduces an efficient method for cultivating osteoclasts in 3D environments in vitro. It offers a more physiologically relevant platform to investigate osteoclast behavior and holds promise to advance research in bone biology and regenerative dentistry.

Protocol for high throughput 3D drug screening of patient derived melanoma and renal cell carcinoma.

High Throughput Screening (HTS) with 3D cell models is possible thanks to the recent progress and development in 3D cell culture technologies. Results from multiple studies have demonstrated different drug responses between 2D and 3D cell culture. It is now widely accepted that 3D cell models more accurately represent the physiologic conditions of tumors over 2D cell models. However, there is still a need for more accurate tests that are scalable and better imitate the complex conditions in living tissues. Here, we describe ultrahigh throughput 3D methods of drug response profiling in patient derived primary tumors including melanoma as well as renal cell carcinoma that were tested against the NCI oncologic set of FDA approved drugs. We also tested their autologous patient derived cancer associated fibroblasts, varied the in-vitro conditions using matrix vs matrix free methods and completed this in both 3D vs 2D rendered cancer cells. The result indicates a heterologous response to the drugs based on their genetic background, but not on their maintenance condition. Here, we present the methods and supporting results of the HTS efforts using these 3D of organoids derived from patients. This demonstrated the possibility of using patient derived 3D cells for HTS and expands on our screening capabilities for testing other types of cancer using clinically approved anti-cancer agents to find drugs for potential off label use.