Thermal lamination and laser cut (TLLC) method for enclosed Micro-fluidic paper analytical devices (μPADs) by controlled ablation

Cancer stem cells (CSCs) are most likely the main cause of lung cancer formation, metastasis, drug resistance, and genetic heterogeneity. Three-dimensional (3D) ex vivo cell culture models can facilitate stemness improvement and CSC enrichment. Considering the critical role of extracellular matrix (ECM) on CSC properties, the present study developed a thermo-responsive hydrogel using the porcine decellularized lung for 3D cell culture, and the cell-laden hydrogel culturing model was used to explore the CSC characteristics and potential utilization in CSC-specific drug evaluation. Results showed that the lung dECM hydrogel (LEH) was composed of the main ECM components and displayed excellent cellular compatibility. In addition, lung cancer cells 3D cultured in LEH displayed the overexpression of metastasis-related genes and enhanced migration properties, as compared with those in two-dimensional (2D) conditions. Notably, the CSC features, including the expression level of stemness-associated genes, colony formation capability, drug resistance, and the proportion of cancer stem-like cells (CD133+), were also enhanced in 3D cells. Furthermore, the attenuation effect of epigallocatechin gallate (EGCG) on CSC properties in the 3D model was observed, confirming the potential practicability of the 3D culture on CSC-targeted drug screening. Overall, our results suggest that the fabricated LEH is an effective and facile platform for 3D cell culture and CSC-specific drug evaluation.

Macrophage-based drug delivery systems, such as macrophage-liposome conjugates (Mϕ-Lip), leverage the natural tumor-homing ability of macrophages and offer a potential solution for overcoming biological barriers and delivering chemotherapy drugs to challenging tumor regions. However, reliable platforms to assess the tumor-targeting efficiency, penetration capabilities, and therapeutic effectiveness of drug-laden macrophages remain largely unavailable. In this study, we developed a three-dimensional (3D) cell culture platform that mimics the structural and biological complexity of invivo tumors, enabling real-time observation and analysis of Mϕ-Lip as they migrate, penetrate, and exert anti-tumor effects. Beyond evaluating the delivery process, this work focuses on the rational design and optimization of dosage regimens for co-delivering cisplatin (CDDP) and paclitaxel (Taxol) using Mϕ-Lip. Experimental results demonstrated that the drugs encapsulated within the liposomes influenced the invasive behavior of Mϕ-Lip, which in turn impacted their tumor-killing efficiency. Using this 3D cell culture platform, we identified optimal dosage regimens for co-delivering combination chemotherapy drugs through the Mϕ-Lip. This newly developed approach provides a reliable and versatile tool not only for evaluating but also for fine-tuning cell-based drug delivery strategies. It holds significant promise for advancing targeted chemotherapy strategies and improving therapeutic outcomes for solid tumors.

Dynamic bond crosslinked poly(γ-glutamic acid)/Salecan derived hydrogel as a platform for 3D cell culture

SIS-ECM BIOMATERIAL DECREASES HUMAN CARDIAC MYOFIBROBLAST ACTIVATION AND ATTENUATES LOCAL MATRIX REMODELING

As the complex structure of nervous tissue cannot be mimicked in two-dimensional (2D) cultures, the development of three-dimensional (3D) neuronal cell culture platforms is a topical issue in the field of neuroscience and neural tissue engineering. Computer-assisted laser-based fabrication techniques such as direct laser writing by two-photon polymerization (2PP-DLW) offer a versatile tool to fabricate 3D cell culture platforms with highly ordered geometries in the size scale of natural 3D cell environments. In this study, we present the design and 2PP-DLW fabrication process of a novel 3D neuronal cell culture platform based on tubular microtowers. The platform facilitates efficient long-term 3D culturing of human neuronal cells and supports neurite orientation and 3D network formation. Microtower designs both with or without intraluminal guidance cues and/or openings in the tower wall are designed and successfully fabricated from Ormocomp. Three of the microtower designs are chosen for the final culture platform: a design with openings in the wall and intralumial guidance cues (webs and pillars), a design with openings but without intraluminal structures, and a plain cylinder design. The proposed culture platform offers a promising concept for future 3D cultures in the field of neuroscience.

Detection and quantification of cell viability and growth in two-dimensional (2D) and three-dimensional (3D) cell cultures commonly involve harvesting of cells and therefore requires a parallel set-up of several replicates for time-lapse or dose–response studies. Thus, developing a non-invasive and touch-free detection of cell growth in longitudinal studies of 3D tumor spheroid cultures or of stem cell regeneration remains a major unmet need. Since surface acoustic waves (SAWs) permit mass loading-based biosensing and have been touted due to their many advantages including low cost, small size and ease of assembly, we examined the potential of SAW-biosensing to detect and quantify cell growth. Herein, we demonstrate that a shear horizontal-surface acoustic waves (SH-SAW) device comprising two pairs of resonators consisting of interdigital transducers and reflecting fingers can be used to quantify mass loading by the cells in suspension as well as within a 3D cell culture platform. A 3D COMSOL model was built to simulate the mass loading response of increasing concentrations of cells in suspension in the polydimethylsiloxane (PDMS) well in order to predict the characteristics and optimize the design of the SH-SAW biosensor. The simulated relative frequency shift from the two oscillatory circuit systems (one of which functions as control) were found to be concordant to experimental data generated with RAW264.7 macrophage and A549 cancer cells. In addition, results showed that SAW measurements per se did not affect viability of cells. Further, SH-SAW biosensing was applied to A549 cells cultured on a 3D electrospun nanofiber scaffold that generate tumor spheroids (tumoroids) and the results showed the device's ability to detect changes in tumor spheroid growth over the course of eight days. Taken together, these results demonstrate the use of SH-SAW device for detection and quantification of cell growth changes over time in 2D suspension cultures and in 3D cell culture models, which may have potential applications in both longitudinal 3D cell cultures in cancer biology and in regenerative medicine.

BackgroundAlthough rare, uveal melanoma (UM) is a life-threatening malignancy. Understanding its biology is necessary to improve disease outcome. Three-dimensional (3D) in vitro culture methods have emerged as tools that incorporate physical and spatial cues that better mimic tumor biology and in turn deliver more predictive preclinical data. Herein, we comprehensively characterize UM cells under different 3D culture settings as a suitable model to study tumor cell behavior and therapeutic intervention.MethodsSix UM cell lines were tested in two-dimensional (2D) and 3D-culture conditions. For 3D cultures, we used anchorage-dependent (AD) methods where cells were embedded or seeded on top of basement membrane extracts and anchorage-free (AF) methods where cells were seeded on agarose pre-coated plates, ultra-low attachment plates, and on hanging drops, with or without methylcellulose. Cultures were analyzed for multicellular tumor structures (MCTs) development by phase contrast and confocal imaging, and cell wellbeing was assessed based on viability, membrane integrity, vitality, apoptotic features, and DNA synthesis. Vascular endothelial growth factor (VEGF) production was evaluated under hypoxic conditions for cell function analysis.ResultsUM cells cultured following anchorage-free methods developed MCTs shaped as spheres. Regardless of their sizes and degree of compaction, these spheres displayed an outer ring of viable and proliferating cells, and a core with less proliferating and apoptotic cells. In contrast, UM cells maintained under anchorage-dependent conditions established several morphological adaptations. Some remained isolated and rounded, formed multi-size irregular aggregates, or adopted a 2D-like flat appearance. These cells invariably conserved their metabolic activity and conserved melanocytic markers (i.e., expression of Melan A/Mart-1 and HMB45). Notably, under hypoxia, cells maintained under 3D conditions secrete more VEGF compared to cells cultured under 2D conditions.ConclusionsUnder an anchorage-free environment, UM cells form sphere-like MCTs that acquire attributes reminiscent of abnormal vascularized solid tumors. UM cells behavior in anchorage-dependent manner exposed diverse cells populations in response to cues from an enriched extracellular matrix proteins (ECM) environment, highlighting the plasticity of UM cells. This study provides a 3D cell culture platform that is more predictive of the biology of UM. The integration of such platforms to explore mechanisms of ECM-mediated tumor resistance, metastatic abilities, and to test novel therapeutics (i.e., anti-angiogenics and immunomodulators) would benefit UM care.

The conventional two-dimensional (2D) in vitro cell culture system has been a staple in biological research for decades. However, due to numerous limitations, this strategy is being replaced by 3D cell culture, representing a significant advancement in cell culture techniques. The 3D culture has garnered substantial attention across various scientific fields due to its ability to replicate in vivo-like physiological conditions. In the same spirit, mimicking in vivo scenarios is crucial for assessing the adverse effects of materials, in particularly carbon-based nanomaterials. This review highlights recent advancements in cell culture techniques and various innovative 3D cell culturing strategies, and concurrently addresses the current status and limitations of cytotoxicological studies related to carbon-based nanomaterials. The application of 3D cell cultures in toxicological studies of carbon-based nanomaterials is discussed with an emphasis on the potential of these advanced techniques. Additionally, the review delves into the remaining challenges associated with strategies for assessing the toxicity of carbon nanomaterials in 3D cell culture platforms and explores future perspectives in this evolving field.

Electrospun PBLG/PLA nanofiber membrane for constructing in vitro 3D model of melanoma

3D cell culture platforms have emerged as a setting that resembles in vivo environments replacing the traditional 2D platforms. Over the recent years, an extensive effort has been made on the development of more physiologically relevant 3D cell culture platforms. Extracellular matrix-based materials have been reported as a bioactive and biocompatible support for cell culture. For example, human plasma derivatives have been extensively used in cell culture. Despite all the promising results, in most cases these types of materials have poor mechanical properties and poor stability in vitro. Here plasma-based hydrogels with increased stability are proposed. Platelet lysates are modified by addition of methacryloyl groups (PLMA) that polymerize in controlled geometries upon UV light exposure. The hydrogels could also generate porous scaffolds after lyophilization. The results show that PLMA materials have increased mechanical properties that can be easily adjusted by changing PLMA concentration or modification degree. Cells readily adhere, proliferate, and migrate, exhibiting high viability when encapsulated in PLMA hydrogels. The innovation potential of PLMA materials is based on the fact that it is a complete xeno-free solution for human cell culture, thus an effective alternative to the current gold standards for 3D cell culture based on animal products.

Enabling high throughput drug discovery in 3D cell cultures through a novel bioprinting workflow.

The healing process of diabetic foot ulcers (DFUs) presents a slow pattern with an increased risk of infections, ischemia, and thrombosis correlated with high levels of reactive oxygen species production. Vascular injury is one of the factors contributing to the difficulty of wound healing in diabetic patients. Although the understanding of the pathophysiology of DFUs has significantly increased in recent years, associated treatments still have a high level of failure, leading to high morbidity rates, mortality, and amputations. Three-dimensional (3D) cell culture platforms offer a new approach to investigating and treating these wounds, as they can reproduce one or more physiological systems in a relevant microenvironment. This systematic review describes the advancements, challenges, and future implications of advanced 3D culture models in vascularization, encompassing pathophysiological understanding, treatment, and prognostic perspectives. We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. This work was registered under PROSPERO protocol number CRD42022336473. The eligibility criteria addressed studies related to chronic DFUs that analyzed vascularization during the healing process. The selected study designs involved 3D cultures and organ-on-a- chip (OoC) platforms, utilizing either primary or secondary human cell lines. Studies published more than 10 years ago or using only animal cells in 2D culture environments were excluded. The search was conducted in PubMed, LILACS, Embase, MEDLINE, IEEE, the BSV regional portal, ScienceDirect, Scopus, CINAHL, EBSCO, and Web of Science. A total of 2,539 relevant studies were identified as of June 1, 2025. After screening, only seven met the inclusion criteria. These studies reflect a growing interest in using hydrogel scaffolds and microfluidic systems to replicate diabetic skin environments; however, the field remains in an early stage of development. OoC platforms, in particular, stand out for their ability to recreate dynamic, tissue-like conditions and vascular function. While some promising attempts have been made to combine hydrogels with these technologies, the evidence is still limited and inconclusive for chronic wound modeling. This review underscores both the potential and the urgent need for more robust and translational research in this area—especially toward building personalized, clinically relevant models to support future drug testing and therapeutic innovation.

Extracellular vesicles (EVs) play a critical role in intercellular communication and hold great promise as diagnostic biomarkers and therapeutic agents. Due to the limited availability of patient samples, in vitro cell culture models have become indispensable tools for generating EVs under controlled conditions and investigating their biological roles. While conventional 2D cultures are widely used, they lack the complexity of native tissues. In contrast, 3D culture platforms better mimic in vivo conditions and may influence EV secretion dynamics and characteristics. However, there is a lack of research directly comparing these various 2D and 3D platforms for EV production. In this study, we temporarily compared 2D culture with three 3D platforms composed of distinct biomaterials: ultralow attachment (ULA) plates with a nonadherent surface, collagen-coated plates with a biologically active matrix, and AlgiMatrix plates with porous alginate sponges. Cell growth and EV production were evaluated over multiple time points using the human mammary epithelial cell (HMEC) as a model, including assessments of cell morphology, EV yield, size distribution, and morphology. The results showed that both ULA and collagen-based platforms effectively produced smaller and more uniform EVs compared to the 2D platform, with yields exceeding those observed in 2D. In contrast, the AlgiMatrix system was unsuitable for size-based EV quantification due to contamination from scaffold-related materials. These results demonstrate distinct EV production shaped by the physical and biochemical features of each culture platform, highlighting the importance of biomaterial selection and time-course analysis when optimizing EV production for downstream applications, such as diagnostics and therapeutic development.

The demands of tissue engineering have driven a tremendous amount of research effort in 3D tissue culture technology and, more recently, in 3D printing. The need to use 3D tissue culture techniques more broadly in all of cell biology is well-recognized, but the transition to 3D has been impeded by the convenience, effectiveness, and ubiquity of 2D culture materials, assays, and protocols, as well as the lack of 3D counterparts of these tools. Interestingly, progress and discoveries in 3D bioprinting research may provide the technical support needed to grow the practice of 3D culture. Here we investigate an integrated approach for 3D printing multicellular structures while using the same platform for 3D cell culture, experimentation, and assay development. We employ a liquid-like solid (LLS) material made from packed granular-scale microgels, which locally and temporarily fluidizes under the focused application of stress and spontaneously solidifies after the applied stress is removed. These rheological properties enable 3D printing of multicellular structures as well as the growth and expansion of cellular structures or dispersed cells. The transport properties of LLS allow molecular diffusion for the delivery of nutrients or small molecules for fluorescence-based assays. Here, we measure viability of 11 different cell types in the LLS medium, we 3D print numerous structures using several of these cell types, and we explore the transport properties in molecular time-release assays.

Precision medicine holds the promise of designing patient-specific therapies to improve therapeutic efficiency. However, the scarcity of tumor and biopsy tissue is a limiting factor in the development of diagnostic assays. Cells isolated from these tissues could be used to overcome these issues, while serving as the basis for assays to diagnose and guide treatment. It is critical that the in vitro culture of these cells be performed in three-dimensional (3D) environments that can better replicate the native tumor microenvironment. However, currently available 3D cell culture platforms, like Matrigel, suffer from technical limitations in reproducibility and handling that make the development of such assays difficult. Towards that end, this study isolates cells from human prostate cancer (PC) and renal cell carcinoma (RCC) tumor biopsies and patient-derived xenografts (PDX) and prints them into spheroids using magnetic 3D bioprinting. The core principle of magnetic 3D bioprinting is the magnetization of cells and their aggregation using mild magnetic forces. Once aggregated, these cells form spheroids that mimic native tumor environments in extracellular matrix and cell-cell and cell-ECM interactions. This technique can be used to actively magnetize cells and generate spheroids from a scarce cell source, while overcoming the limitations of other 3D cell culture platforms. In this study, we demonstrated our ability to print spheroids from cells isolated from human tumor biopsies and PDX. Isolation techniques ranging from simple mincing and filtration to enzymatic digestion were employed. Next, these cells were magnetized by incubation with a biocompatible magnetic nanoparticle assembly, NanoShuttle. Once magnetized, these cells were printed into spheroids of varying sizes, from 1,000-20,000 cells, in 384-well plates. These cells were cultured for days, after which viability was measured using CellTiter-Glo. Our preliminary studies demonstrated our ability to isolate cells and print them into spheroids. Isolation was best with either mincing and filtration alone or collagenase II (400 U/mL) digestion for 1 h. These cells were then successfully magnetized and printed into spheroids, which remained viable after 72 h. Spheroids of 10,000-20,000 cells were the most successful, and further optimization is needed to reduce the size needed for viable spheroids to take full advantage of scarce resources such as tumor biopsies. We also demonstrated the ability to assay compound toxicity, showing a dose-dependent toxicity on spheroids derived from PDX tumors. In all, we demonstrated our ability to isolate cells from human tumor biopsies and PDX models and print them into spheroids with high throughput. These preliminary results will serve as a platform for the further development of precision medicine assays to optimize PC and RCC treatment. Citation Format: Hubert Tseng, Jacob A. Gage, Pujan K. Desai, Reynolds Brobey, Sheri Skinner, Mehdi Dehghani, Kevin P. Rosenblatt, Wenliang Li, Robert J. Amato, Glauco R. Souza. Development of spheroids derived from tumor biopsies and patient-derived xenografts using magnetic 3D bioprinting. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4251.