A Comparative Study of 3D Culture Methods in Lung Cancer Research: Finding the Optimal Path for Spheroid Formation

Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative of the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods allow cells to grow in structures more resembling the in vivo environment. Cells can develop cell-cell and cell-ECM interactions in 3D. The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and subsequently incubated at 37°C to allow the formation of a cell-seeded hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. The RAFT™ Absorbers gently remove abundant medium, thus compacting the cell/collagen hydrogel. Additional epithelial or endothelial cells may be added as overlays on top to study co-cultures or more complex cultures. Another commonly used 3D cell culture model is spheroids. Spheroids are compact aggregates of cells that are generated without the addition of exogenous ECM – e.g. in so-called ultra-low attachment (ULA) plates or by the hanging-drop method. While 3D cultures more accurately mimic the in vivo cell environment, it might be difficult to analyze cells in 3D due to the dense, tissue-like structure of these cultures compared to 2D cell monolayers. This presentation explains how to measure cell viability in both RAFT™ 3D cell cultures and in spheroid cultures with the ViaLight™ Plus Cell Proliferation and Cytotoxicity BioAssay. The ViaLight™ Assay is based on the bioluminescent detection of cellular ATP as a measure of cell viability. Only minor modifications of the standard two-step protocol that is used for 2D cell cultures are required for using the assay for 96-well and 24-well RAFT™ 3D cell cultures as well as for spheroid cultures in 96-well ULA plates. Two different cell types were used in this study: Normal Human Dermal Fibroblasts (NHDFneo) and the colon cancer cell line HCT-116. Both cell types efficiently form spheroids in ULA plates. Also in RAFT™ 3D cultures HCT-116 cells build compact aggregate-like structures, whereas NHDFneo grow in as individual cells interspersed in the collagen scaffold. By elongating the first step of the Vialight™ Assay, the lysis step, from 10 minutes to 30 minutes, a linear performance of the assay for cell numbers of up to 96,000 cells in the 96-well format and 480,000 cells in the 24-well format could be obtained for RAFT™ cultures. For spheroid cultures an extension of the lysis time to 60 minutes was required to obtain efficient lysis of spheroids with a diameter of up to 400μm, as confirmed by CalceinAM and propidium iodide staining. In summary this presentation shows that analyzing cells in 3D cultures can easily and routinely be done by slightly adjusting standard 2D cell culture assays like the ViaLight™ Assay. Citation Format: Stefanie Buesch, John Langer, Sabine Schaepermeier, Lubna Hussain, Jeffrey Bergeron, Volker Vogel, Jenny Schroeder. Analyzing cell viability in 3-D tissue models with the ViaLight™ plus cell proliferation and cytotoxicity bioassay. [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 607.

ObjectiveThree-dimensional (3D) culture systems offer a more physiologically relevant environment than conventional two-dimensional (2D) cultures, particularly for studying tumor biology. Here, we systematically compared two widely used 3D platforms—poly(2-hydroxyethyl methacrylate) (Poly-HEMA, PH)-coating and ultra-low attachment (ULA) plates—to evaluate their impact on pancreatic cancer (PCa) cell behavior. We assessed spheroid morphology, chemotherapeutic response, invasion potential, and adhesion molecule expression in two PCa cell lines (PANC-1 and SU.86.86).ResultsSpheroid morphology differed markedly between PH and ULA platforms, with ULA generally promoting larger and more cohesive spheroids. Gemcitabine resistance was the highest in SU.86.86 spheroids on ULA plates. Matrigel invasion assays revealed enhanced single-cell migration in SU.86.86 spheroids grown on PH, whereas ULA spheroids exhibited broader matrix degradation and collective invasion. Moreover, gene and protein expression levels of key adhesion molecules, including E-Cadherin, N-Cadherin, and integrins, varied between platforms in both cells. These findings demonstrate that 3D culture systems distinctly influence PCa cell characteristics and highlight the necessity of selecting appropriate culture environment for studying tumor biology and drug response. Further mechanistic studies are warranted to uncover how different 3D environments shape tumor cell behavior and therapy resistance.

Culturing cells in three-dimensions (3D) has become a well established approach as it is more representative of the in vivo environment than traditional two-dimensional (2D) cultures. Allowing cells to interact with each other in a spheroid creates a micro-environment which mimics in vivo tissue and is a better model for examining the effect of drugs in cancer. Developing uniform spheroids becomes especially important as it forms the basis for robust and reliable assays. S-BIO PrimeSurface® cultureware are ultra low attachment (ULA) dishes and plates that promote scaffold free, self assembly spheroid formation. The plates are pre-coated with a proprietary hydrophilic polymer that enables spontaneous spheroid formation of uniform size. PrimeSurface 96 and 384 ULA plates have good optical clarity making them highly suitable for brightfield and fluorescent imaging. Imaging technologies such as the BioTek Cytation™ 5 allows researchers to study not only spheroid proliferation through brightfield imaging, but also phenotypic events such as hypoxia, apoptosis, or necrosis induction through the use of fluorescent probes and fluorescence imaging. Incorporation of z-stacking and projection techniques in the Gen5™ Microplate Reader and Imager Software create in-focus images of spheroidal cells, allowing accurate, robust, and repeatable determination of the effect of test molecules or conditions. In this app note, we present data generated with BioTek Cytation 5 using PrimeSurface ULA plates to develop simple robust spheroid assays for brightfield and fluorescence imaging.

Various three-dimensional (3D) culture methods have been introduced to overcome the limitations of in vitro culture and mimic in vivo conditions. This study aimed to evaluate two microsphere-forming culture methods and a monolayer culture method. We evaluated cell morphology, viability, osteo-, adipo-, and chondrogenic differentiation potential of dental pulp stem cells (DPSCs) cultured in 3D culture plates: ultra-low attachment (ULA) and U-bottomed StemFit 3D (SF) plates, and a two-dimensional (2D) monolayer plate. RNA sequencing (RNA-seq) revealed differentially expressed gene (DEG) profiles of the DPSCs. In contrast to an increasing pattern in the 2D group, cell viability in 3D groups (ULA and SF) showed a decreasing pattern; however, high multilineage differentiation was observed in both the 3D groups. RNA-seq showed significantly overexpressed gene ontology categories including angiogenesis, cell migration, differentiation, and proliferation in the 3D groups. Hierarchical clustering analysis revealed a similar DEG regulation pattern between the 3D groups; however, a comparatively different DEG was observed between the 2D and 3D groups. Taken together, this study shows that DPSCs cultured in microsphere-forming plates present superior multilineage differentiation capacities and demonstrate higher DEG expression in regeneration-related gene categories compared to that in DPSCs cultured in a conventional monolayer plate.

Background and Aims Three-dimensional (3D) culture techniques represent a significant evolution from traditional monolayer culture methods for cell biology and regenerative medicine studies. These cell models provide a more accurate representation of human tissues, allowing a higher reliable simulation of cell-cell and cell-extracellular matrix (ECM) interactions and of the biological processes occurring in both physiological and pathological conditions. Different cell types can be combined into hybrid and multicellular 3D structures, to study how they interact and distribute. Urine derived renal epithelial cells (URECs) are a heterogeneous cells population voided in urine from the upper urinary tract, and particularly from proximal tubule; these cells are rarely detected in healthy subjects, while their number increases in presence of acute kidney damage and after kidney transplant as a response to ischemia reperfusion events and the increased cell turnover. In particular, we previously described URECs derived from kidney transplanted patients, focusing on their phenotype and their immunomodulatory properties in traditional monolayer culture [1, 2]. Here, we aimed to establish a 3D model combining URECs derived from kidney transplanted patients with Wharton's jelly mesenchymal stromal cells (WJ-MSCs), to evaluate structure, cell distribution and viability of the newly generated spheroids. For a functional characterization of the 3D model, the immunomodulatory properties were analysed by coculturing spheroids with peripheral blood mononuclear cells (PBMCs); the proliferation of CD4 and CD8 T cells, as well as the percentage of Treg cells and Granzyme B positive cells were evaluated after coculture. Method URECs and WJ-MSCs were combined to obtain spheroids by seeding cells in ultra low attachment (ULA) plates, allowing cell aggregation in 24 h Different condition and ratio were tested: URECs or WJ-MSCs only, URECs:WJ-MSCs 1:1 and 2:1 ratio. Spheroids formation and cell distribution were analysed using Incucyte, and by staining URECs and WJ-MSCs with different fluorochromes. The viability was assessed by Live/Dead assay at different time point. Spheroids were then cultured with PBMCs, and after 72 h immune cells were analysed by flow cytometry, comparing the results with activated PBMCs set as control. Results Stable spheroids were obtained combining URECs and WJ-MSCs (Fig. 1a and b), with a higher viability compared to WJ-MSCs spheres, while URECs alone could not self-aggregate (Fig. 1a, b and d). URECs were mostly located in the core of the 3D model, while WJ-MSCs were in the outer layer (Fig. 1c). During coculture with activated PBMCs (Fig. 2a), hybrid spheroids in both 1:1 and 1:2 ratio, significantly reduced CD4 and CD8 T cell proliferation (Fig. 2b), while WJ-MSCs only spheroids do not affect the proliferation rate, compared to activated PBMCs cultured without spheroids. For what concerns specific T cell subsets, hybrid spheroids reduced the percentage of Granzyme B producing cells, while increasing the CD4+CD25+FoxP3+ regulatory T cell subset (Fig. 2c). Conclusion The date obtained in our study indicate a long term stability and viability of UREC:WJ-MSCs spheroids, with a specific cell distribution into the 3D structure. Moreover, these 3D models showed promising immunomodulatory properties during coculture with PBMCs, which need to be further assessed by evaluating their effect on other immune cell subset including B cell and monocytes. These findings may provide the background for studies testing unknown effects of this new hybrid 3D cell model for cell therapy and regenerative medicine applications.

Three-dimensional cell cultures may better mimic avascular tumors. Yet, they still lack characterization and standardization. Therefore, this study aimed to (a) generate multicellular aggregates (MCAs) of four breast cell lines: MCF7, MDA-MB-231, and SKBR3 (tumoral) and MCF12A (non-tumoral) using ultra-low attachment (ULA) plates, (b) detail the methodology used for their formation and analysis, providing technical tips, and (c) characterize the MCAs using morphometry, qualitative cytology (at light and electron microscopy), and quantitative immunocytochemistry (ICC) analysis. Each cell line generated uniform MCAs with structural differences among cell lines: MCF7 and MDA-MB-231 MCAs showed an ellipsoid/discoid shape and compact structure, while MCF12A and SKBR3 MCAs were loose, more flattened, and presented bigger areas. MCF7 MCAs revealed glandular breast differentiation features. ICC showed a random distribution of the proliferating and apoptotic cells throughout the MCAs, not fitting in the traditional spheroid model. ICC for cytokeratin, vimentin, and E-cadherin showed different results according to the cell lines. Estrogen (ER) and progesterone (PR) receptors were positive only in MCF7 and human epidermal growth factor receptor 2 (HER-2) in SKBR3. The presented characterization of the MCAs in non-exposed conditions provided a good baseline to evaluate the cytotoxic effects of potential anticancer compounds.

Different three-dimensional (3D) cell cultures techniques were evaluated in order to establish a bladder cancer model with the ability to recapitulate in vitro the tumor features observed in vivo. Using the bladder cancer cell line RT4, the first part of the project focused on comparing the scaffold-free 3D cell culture systems, employing forced floating (Ultra-low attachment plates, ULA) and hanging drop (HD plates) techniques, and scaffold-dependent systems, employing microcarriers and hydrogels (Matrigel, Alginate and HydroMatrix), to generate RT4 spheroids. These were characterized regarding morphological parameters, cell growth and metabolism. Only 3D cell culture using the forced floating and hanging drop techniques, and Matrigel (50% v/v) and HydroMatrix (0.25% v/v) scaffolds, were able to generate spheroids with suitable parameters (solidity>0.95; sphericity~0.90; rounding between 0.70-0.90). In the ULA and HD cell cultures, a single spheroid was generated per well, with diameters ranging between 200 and 600 m. The cell growth and, consequently, the metabolism observed in these spheroids were lower than in 2D cultures. The Matrigel and HydroMatrix cultures, on the other hand, generated several spheroids per well, with diameters that did not exceed 200 m. The spheroids in Matrigel showed cell growth and metabolism higher than those observed in 2D cultures. RT4 spheroids generated in ULA plates and Matrigel were selected to assess the Doxorubicin (DOX) sensitivity and the expression of commonly overexpressed genes in solid bladder tumors. ULA plates' spheroids were less sensitive to DOX treatment (IC 50 8.62 1.53 M) than 2D (IC 50 2.35 0.39 M) and Matrigel (IC 50 3.31 1.28 M) cultures. Moreover, it was observed a higher expression of ALDH1A1 and HRAS genes in RT4 spheroids generated in ULA, two of the main genes involved in the mechanisms of resistance and recurrence of bladder tumors. Almost all the analyzed genes (HIF-1, IFIT5, MDR-1, IL3, ALDH1A1 and HRAS) were more expressed in Matrigel cultures. In the second part of the project, the strategy based on the use of ULA cultures with 5% of Matrigel (v/v) was successfully applied to generate spheroids using primary human bladder tumor cells (BL0293 and BL0808) derived from PDX models (patient-derived xenografts). The sensitivity profiles of the BL0293 and BL0808 spheroids to Cisplatin and Gemcitabine were similar to those obtained previously in the in vivo tests using PDX models BL0293 and BL0808. In general, it was possible to conclude that both 3D cultures generated using the forced floating technique (ULA plates) and Matrigel scaffold were able to recapitulate in vitro some characteristics of the solid tumor in vivo and can be considered as promising 3D models of bladder cancer. The present work shows an unprecedented comparative analysis between the bladder tumor spheroids generated with different 3D cell culture techniques regarding the morphology and the ability to recap some features of the tumor in vivo.

The acquired drug chemoresistance represents the main challenge of the ovarian cancer treatment. In addition, the absence of an adequate in vitro model able to reproduce the native tumor environment can contribute to the poor success rate of pre-clinical studies of new compounds. Three-dimensional (3D) culture models have been recently used for drug screening purposes due to their ability to reproduce the main characteristics of in vivo solid tumors. Here we describe the establishment and characterization of 3D ovarian cancer spheroids using different adenocarcinoma tumor cell lines (SKOV-3 and OVCAR-3 cells) in two different 3D scaffold-free methods: forced-floating in ultra-low attachment (ULA) plates and hanging drop (HD). Spheroids were evaluated in both 3D cultures in order to establish the best condition to perform the drug response analysis with Paclitaxel, a common drug used to treat ovarian cancer. SKOV-3 and OVCAR-3 spheroids with the desired characteristics (roundness close to 1.0 and diameter in the 200-500 μm range) were obtained using both methods after addition of the methylcellulose (MC) in the culture medium (0.25% and 0.5%, w/v). We also observed the presence of microvilli on the surface of the spheroids, higher presence of apoptotic cells and higher drug resistance, when compared with 2D cultures. The 3D cultures obtained seem to provide more reliable results in terms of drug response than those provided by 2D monolayer culture. The forced floating method was considered more suitable and straightforward to generate ovarian cancer spheroids for drug screening/cytotoxicity assays.

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.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammatory disease associated with epithelial dysfunction and impaired mucosal barrier integrity. Dupilumab, an IL-4 receptor alpha antagonist, has shown clinical efficacy, but its cellular effects on nasal epithelium remain poorly understood. Advanced in vitro models such as 3D spheroid cultures may provide insight into epithelial organization under treatment. We conducted a preliminary study using nasal epithelial cells obtained from three patient groups: CRSwNP treated with Dupilumab for 16 weeks (n = 3), untreated CRSwNP (n = 3), and turbinate hypertrophy controls (n = 3). Cells were isolated by enzymatic digestion and cultured in ultra-low attachment plates using sphere-promoting medium to assess spheroid formation. Observations were performed using phase-contrast microscopy. Due to the limited sample size, data were analyzed qualitatively without statistical testing. Control cells formed compact spheroids, while untreated CRSwNP cells failed to generate structured spheroids, showing only aggregates. Cells from Dupilumab-treated patients produced well-organized spheroids, suggesting improved epithelial organization. Occasional surface movement was observed but not quantitatively assessed. No molecular or ultrastructural assays were performed to confirm mechanistic hypotheses. Our preliminary findings indicate that Dupilumab treatment may be associated with improved epithelial organization in CRSwNP, as shown by spheroid formation in 3D culture. However, these observations are preliminary and based on a small cross-sectional cohort. Future studies should include longitudinal sampling, functional assays, and molecular analyses to confirm mechanisms and validate these results.

Generating 3D tumor spheroid in vitro has been a straightforward method not only to replicate in vivo like cytoarchitecture and also to study complex interactions to the 3D milieu. Although various approaches have been developed to date, most of the in vitro techniques were hampered by the limitations like small-volume, non-uniformity, irregular sphericity, short-term culture, labor-intensive and studies’ complexities. Here, we have developed very intuitive methods to generate 3D tumor spheroid models and scaffold-based 3D culture models in a single well of universal plate for long-term cultivation. Experimental Procedures: The prototype of 3D printed chamber was designed by Sketchup software (Trimble) and printed at Illinois MakerLab (http://makerlab.illinois.edu) for this study. The human colon cancer cell (HCT116), obtained from American Type Culture Collection (ATCC), were cultured in DMEM medium containing 10% FBS and stored in a humidified incubator of 5% CO 2 at 37 °C. Five universal 96 well plates (Corning, Thermo Scientific, CytoOne, Advangene, and Genesee Scientific), Elisa 96 well strip (Corning), Black 96-well plate (Thermo scientific) were used without any modification (Not a type of ultra-low attachment (ULA) plate). The prototype designs are flexible to be modified and will be utilized to develop a product beyond conceptual frameworks in the market. Results: Recently, microfabricated approaches have been intensively studied for micro 3D tumor models (< 300 µm in diameter) implemented with high-throughput analysis. However, the micro tumor model incubated in a short-term might not effectively develop tumor heterogeneity such as a non-perfusion core, diffusion gradient and increased interstitial pressure. We intentionally generated a tumor spheroid model incubated over 1 month with appropriate media replacement to supply nutrients. The spheroid in the system grew over 1.5mm of diameter with an excellent sphericity (> 0.95). Besides, our methodology can perform various in-situ spheroid assays in basic cancer research and also generate scaffold-free/based 3D tumor culture models in the system. To replicate in vivo like cytoarchitecture more physiologically relevant, decellularized tissue scaffold-based 3D culture was conceptually demonstrated. Conclusions: We developed a simple and effective process that generates 3D tumor culture models in millimeter size and allows long-term incubation to develop local heterogeneity. Importantly, this approach does not require additional hidden costs and any specialized instruments. We believe the present strategy satisfies all essential criteria (i.e., easiness, accessibility, reproducibility, effectiveness, and applicability) to appeal to general researchers compared to other available methods by far. Citation Format: Yoon Jeong, Ashley Tin, Joseph Irudayaraj. Long-term three-dimensional (3D) tumor culture models using a universal well-plate platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6029.

Three-dimensional (3D) spheroid models better replicate the in vivo tumor microenvironment than conventional two-dimensional (2D) cultures, making them valuable tools for preclinical drug screening in oral squamous cell carcinoma (OSCC). Scaffold-free spheroids were generated from CAL 27 and OECM-1 OSCC cell lines using three approaches: methylcellulose suspension, agarose-coated plates, and ultra-low attachment (ULA) plates. Spheroid morphology and size uniformity were assessed microscopically. Functional characterization included viability assays and histological evaluation for necrosis. Molecular profiling of cancer hallmark genes was performed using qRT-PCR. Drug response assays for cisplatin and doxorubicin were compared between 2D and 3D cultures. Compact, uniform spheroids (~ 150-200μm) formed within 72h in both cell lines, with ULA plates producing the highest reproducibility and structural integrity. Histological analysis revealed central necrosis with viable peripheral cell layers. Gene expression analysis confirmed the modulation of hallmark cancer-associated genes in spheroid associated with hypoxia, angiogenesis, stress-response, adhesion and EMT-related pathways. Drug assays demonstrated significantly higher resistance to cisplatin and doxorubicin in 3D spheroids than in 2D monolayers (p < 0.05). ULA-based 3D spheroids provide a reproducible, structurally stable, and scalable model that closely mimics OSCC tumor biology and chemoresistance. These findings support their application as a robust platform for high-throughput drug screening and translational cancer research. The online version contains supplementary material available at 10.1007/s44164-025-00099-2.

3D cell culture technologies have recently shown very valuable promise for applications in regenerative medicine, but the most common 3D culture methods for mesenchymal stem cells still have limitations for clinical application, mainly due to the slowdown of inner cell proliferation and increase in cell death rate. We previously developed a new 3D culture of adipose-derived mesenchymal stem cells (ASCs) based on its self-feeder layer, which solves the two issues of ASC 3D cell culture on ultra-low attachment (ULA) surface. In this study, we compared the 3D spheroids formed on the self-feeder layer (SLF-3D ASCs) with the spheroids formed by using ULA plates (ULA-3D ASCs). We discovered that the cells of SLF-3D spheroids still have a greater proliferation ability than ULA-3D ASCs, and the volume of these spheroids increases rather than shrinks, with more viable cells in 3D spheroids compared with the ULA-3D ASCs. Furthermore, it was discovered that the SLF-3D ASCs are likely to exhibit the abovementioned unique properties due to change in the expression level of ECM-related genes, like COL3A1, MMP3, HAS1, and FN1. These results indicate that the SLF-3D spheroid is a promising way forward for clinical application.

Developing accurate in vitro models that replicate the in vivo tumor environment is essential for advancing cancer research and therapeutic development. Traditional 2D cell cultures often fail to capture the complex structural and functional heterogeneity of tumors, limiting the translational relevance of findings. In contrast, 3D culture systems, such as spheroids, provide a more physiologically relevant context by replicating key aspects of the tumor microenvironment. This study aimed to compare the metabolism of three intrahepatic cholangiocarcinoma cell lines in 2D and 3D cultures to identify metabolic shifts associated with spheroid formation. Cells were cultured in 2D on adhesion plates and in 3D using ultra-low attachment plates. Metabolic exchange rates were measured using NMR, and intracellular metabolites were analyzed using LC-MS. Significant metabolic differences were observed between 2D and 3D cultures, with notable changes in central carbon and glutathione metabolism in 3D spheroids. The results suggest that 3D cultures, which more closely mimic the in vivo environment, may offer a more accurate platform for cancer research and drug testing.

Background: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. Methods: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. Results: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. Conclusions: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.