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Spheroids

Spheroids

Table of Contents

Definition

A spheroid is a three-dimensional, multicellular aggregate in which suspension cells self-assemble into a compact, roughly spherical body when cultured under conditions that prevent adhesion to a flat substrate1. The phenomenon was observed in cell culture as early as the 1950s, and was defined as a spheroid in the early 1970s, when Sutherland and colleagues reported that cultured hamster lung cells arranged themselves into near-spherical aggregates and proposed these structures as in vitro models of nodular carcinomas2 3.

Structurally, a spheroid recreates features of living tissue that monolayers cannot: extensive cell-cell junctions, cell-extracellular matrix interactive networks, and the production of endogenous extracellular matrix and growth factors at levels higher than those achieved in cell suspension or sheet culture4. Because interior cells depend entirely on diffusion, radial gradients of oxygen, nutrients, and metabolites emerge and produce three concentric zones: an outer proliferating rim, an intermediate quiescent layer, and a hypoxic or necrotic core4. Aggregates smaller than approximately 200 micrometers remain predominantly normoxic and proliferative, characteristic zonation appears at 200 to 300 micrometers, and necrotic cores develop in aggregates approaching 500 micrometers in diameter5. Larger spheroids thereby reproduce the microenvironment of avascular tumors and micrometastases4.

Purpose

The primary value proposition of the spheroid is that it restores a third dimension to cell culture, thereby positioning itself between the oversimplified physiology of two-dimensional monolayers and the complexity of living tissue. The widespread reliance on two-dimensional monolayer assays has been challenged by a decline in the discovery of novel medications, which has renewed interest in spheroid culture as a more appropriate model of human physiology and disease6.

Its principal advantage over legacy models is human relevance: spheroids can be constructed from human primary, patient-derived, or induced pluripotent stem cell-derived cells. Primary human hepatocyte spheroids, for example, retain morphology, viability, and hepatocyte-specific functions for culture periods of at least five weeks, an endurance unattainable in conventional monolayers7.

Spheroids also reduce dependence on animal testing. The FDA Modernization Act 2.0, signed into law on December 29, 2022, permits cell-based assays, organoids, and organ-on-chip systems as alternatives to animal testing in drug development, establishing a regulatory context in which spheroid models are directly relevant8.

Methodology

Spheroid construction rests on a single unifying principle: deny the cells a flat adhesive surface so that gravity and cellular cohesion drive self-assembly into a rounded aggregate9.

Primary formation techniques include:

  • Hanging drop culture, in which cells concentrate at the apex of an inverted droplet and aggregate under gravity; 384-well hanging drop arrays render the method compatible with existing high-throughput screening instruments10.
  • Ultra-low attachment plates, round-bottomed vessels coated with non-adherent or bioinert surfaces that force suspended cells to interact with one another and form a single aggregate per well; the format is compatible with automated liquid handling9.
  • Spinner flasks and bioreactors, which sustain bulk production of large spheroid numbers through continuous gentle agitation11.
  • Pellet culture, in which centrifugation compacts cells into a disc-shaped aggregate.
  • Microfluidic biochips, which trap cells behind U-shaped barriers to enforce uniform size, shape, and density, thereby improving assay reproducibility12 13.

Substrates and matrices include agarose, collagen, basement membrane extracts eg Matrigel, and synthetic hydrogels, which modulate aggregate cohesion and permit embedded culture11. Defined media are chosen according to application: chemically defined conditions support long-term hepatic spheroid culture14, while serum-free media supplemented with mitogens such as epidermal growth factor and fibroblast growth factor sustain neural progenitor sphere assays15. Co-culture with fibroblasts and immune cells is routinely applied to reproduce multicellular microenvironments, and the presence of these additional cell types measurably alters compound potency in dose-response testing16.

Standard tools comprise commercial spheroid microplates, 3D-optimized viability reagents eg CellTiter-Glo 3D, high-content and confocal imaging, and dedicated spheroid analysis software16. Automated platforms that standardize seeding, media exchange, and dosing have been shown to enhance assay robustness and precision relative to manual workflows17.

Performance

Empirically, spheroids outperform legacy cultures on temporal stability. Primary human hepatocyte spheroids remain phenotypically stable for at least five weeks while retaining hepatocyte-specific functions, and their sensitivity to chronic drug exposure permits detection of toxicity that short-lived monolayers miss7 14. A multicenter study conducted across six independent laboratories confirmed that primary human hepatocyte spheroid cultures deliver reproducible long-term viability and function, and supported the spheroid configuration over two-dimensional sandwich culture for long-term toxicity applications18.

Reproducibility metrics depend strongly on size control. Physiological zonation thresholds of approximately 200, 300, and 500 micrometers define the experimental window within which oxygenation and viability can be predicted5. Microfluidic trapping and optimized surface chemistry demonstrably reduce variability in spheroid size, form, and density, which in turn improves the precision of drug-dose response assessment13.

Current technical boundaries are well characterized:

  • Spheroids face reproducibility and data interpretation issues that limit their utility, particularly in co-culture configurations19.
  • Heterogeneous protocols across laboratories have impeded standardization, and reviews of specific disease applications continue to identify methodological gaps20.
  • The absence of vasculature means that oxygen and nutrient delivery are constrained by diffusion, and oxygen consumption rates must be estimated to predict the onset of hypoxia and necrosis in larger aggregates21.
  • Imaging and quantitative analysis remain more demanding than for two-dimensional assays because most instruments and protocols were designed for flat cultures.

Applications

Primary research and industrial applications include:

  • Oncology drug screening. Multicellular tumor spheroids reproduce the gradients and avascular microenvironment of solid tumors and are the dominant three-dimensional format for in vitro therapeutic screening, including high-throughput testing in 384-well formats10 4 22.
  • Radiation research. From their origin, spheroids have served to assess the effects of nutrition and oxygenation on growth and to determine growth fraction after treatment with drugs or radiation3.
  • Drug-induced liver injury prediction. Long-term stable hepatic spheroids in chemically defined conditions detect hepatotoxicity under chronic and repeated-dose exposure regimes that monolayers cannot sustain7 14.
  • Immuno-oncology. Spheroid microarrays support high-throughput, high-content quantification of natural killer cell-mediated cytotoxicity and combination immunotherapy testing23.
  • Personalized medicine. Patient-derived cancer spheroids are in expanding use for drug screening, with systematic reviews cataloguing seeding methodologies across cancer types24.

Standardized contexts of use are established in pharmaceutical discovery and preclinical screening, where commercial platforms eg Corning spheroid microplates and InSphero Akura 96- and 384-well systems, together with patient-derived tumor models, support routine deployment16 25. Adoption is broad in industrial research and development, and automation continues to raise assay robustness17. Regulatory readiness is partial: spheroid assays are accepted components of the alternative-methods toolkit under the FDA Modernization Act 2.0, yet full protocol standardization and standalone regulatory substitution remain works in progress8 20.

Variations

  • Multicellular Tumor Spheroids: Aggregates derived from tumor cell lines or patient tumors that reproduce tumor gradients and the avascular microenvironment for drug response and radiation studies4 5.
  • Hepatic Spheroids: Primary human hepatocyte aggregates that retain liver-specific function for at least five weeks, enabling long-term metabolism and drug-induced liver injury studies7.
  • Neurospheres: Free-floating clonal aggregates of neural stem and progenitor cells first reported by Reynolds and Weiss, propagated in serum-free medium supplemented with epidermal and fibroblast growth factors15 26.
  • Mammospheres: A non-adherent, non-differentiating assay that propagates human mammary stem and progenitor cells, developed by Dontu and colleagues and later adapted for cancer stem cell research27.
  • Cardiac Spheroids: Human induced pluripotent stem cell-derived cardiomyocyte aggregates and microtissues applied to in vitro drug-induced cardiotoxicity assessment28.
  • Mesenchymal Stem Cell Spheroids: Aggregates of human mesenchymal stem cells that confer therapeutic advantages, including enhanced secretory activity, and provide in vitro glimpses of the in vivo state for regenerative medicine29.

  1. Białkowska K, Komorowski P, Bryszewska M, Miłowska K. Spheroids as a Type of Three-Dimensional Cell Cultures: Examples of Methods of Preparation and the Most Important Application, International Journal of Molecular Sciences, 2020;21(17):6225.
    A review contrasting two-dimensional monolayer culture, in which cells are deprived of cell-cell and cell-extracellular matrix interactions, with spheroid three-dimensional culture, and surveying the principal methods of spheroid preparation, approaches to quantitative estimation of spheroid properties, and the most important applications in biomedical research. ↩︎

  2. CELLINK. What is a spheroid? And why they are important, CELLINK Blog, accessed 2026-10-15.
    An accessible overview of spheroid history, noting that spherical cell aggregates were observed in culture as early as the 1950s and that the phenomenon was defined as a spheroid in the early 1970s after Sutherland and colleagues observed hamster V79 lung cells self-arranging into near-perfect spheres in suspension, and explaining why the format is central to modern three-dimensional cell culture. ↩︎

  3. Sutherland RM, McCredie JA, Inch WR. Growth of Multicell Spheroids in Tissue Culture as a Model of Nodular Carcinomas, JNCI: Journal of the National Cancer Institute, 1971;46(1):113-120. doi: 10.1093/jnci/46.1.113.
    The foundational paper of the field, in which the authors grew multicell spheroids as in vitro models of nodular carcinomas, showed that the aggregates could be disaggregated by trypsinization and assayed for colony formation, and proposed the model for assessing the effects of nutrition and oxygenation on growth and for determining growth fraction after treatment with drugs or radiation. ↩︎ ↩︎

  4. Weiswald LB, Bellet D, Dangles-Marie V. Spherical cancer models in tumor biology, Neoplasia, 2015;17(1):1-15. doi: 10.1016/j.neo.2014.12.004.
    A review of spherical cancer models describing how spheroids reconstitute cell-cell and cell-matrix interactions lost in monolayer culture, how aggregates organize into concentric compartments of proliferating, quiescent, and necrotic cells under diffusion-limited oxygen and nutrient supply, and how this architecture mirrors the microenvironment of avascular tumor nodules. ↩︎ ↩︎ ↩︎ ↩︎ ↩︎

  5. Däster S, Amatruda N, Calabrese D, et al. Induction of hypoxia and necrosis in multicellular tumor spheroids is associated with resistance to chemotherapy treatment, Oncotarget, 2017;8(2):1725-1736.
    A study on oxygenation gradients in multicellular tumor spheroids reporting that aggregates below approximately 200 micrometers are mostly normoxic and proliferative, that zonation into proliferative, quiescent, and hypoxic compartments emerges at approximately 200 to 300 micrometers, and that necrotic cores develop in spheroids approaching 500 micrometers, with hypoxia and necrosis linked to resistance to chemotherapy. ↩︎ ↩︎ ↩︎

  6. Mittler F, Obeïd P, Rulina AV, et al. High-Content Monitoring of Drug Effects in a 3D Spheroid Model, Frontiers in Oncology, 2017;7:293.
    An application paper arguing that a decline in the discovery of novel medications challenges the widespread use of two-dimensional monolayer assays in drug discovery, that the resulting need for more appropriate models of human physiology and disease has renewed interest in spheroid culture, and demonstrating high-content monitoring of drug effects in three-dimensional spheroids. ↩︎

  7. Bell CC, Hendriks DF, Moro SM, et al. Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease, Scientific Reports, 2016;6:25187.
    A characterization study showing that primary human hepatocyte spheroids remain phenotypically stable and retain morphology, viability, and hepatocyte-specific functions for at least five weeks in culture, that cellular adenosine triphosphate content remains constant over this period, and that chronic drug exposure reveals toxicity sensitivity that short-lived monolayer assays fail to detect. ↩︎ ↩︎ ↩︎ ↩︎

  8. Zushin PJH, Mukherjee S, et al. FDA Modernization Act 2.0: transitioning beyond animal models with human cells, organoids, and AI/ML-based approaches, Journal of Clinical Investigation, 2023;133(21):e175824.
    A commentary on the FDA Modernization Act 2.0, signed into law on December 29, 2022, which permits specified alternatives to animal testing in drug development, including cell-based assays built on human induced pluripotent stem cells, organoids, and organs-on-chips, thereby establishing the regulatory context for the adoption of non-animal models. ↩︎ ↩︎

  9. ibidi. Generation and Cultivation of 3D Cell Models, ibidi, accessed 2026-10-15.
    A technical guide to three-dimensional model generation describing the hanging drop technique, in which an inverted droplet concentrates cells at its lowest point so that gravity and cellular cohesion drive aggregation into a compact sphere, and the bioinert and ultra-low attachment surfaces that prevent vessel adhesion and force suspended cells to interact with one another instead. ↩︎ ↩︎

  10. Tung YC, Hsiao AY, Allen SG, et al. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array, Analyst, 2011;136(3):473-478. doi: 10.1039/C0AN00609B.
    A methods paper describing a 384-well format hanging drop culture plate that renders spheroid formation, maintenance, and subsequent drug testing as straightforward to perform and as adaptable to existing high-throughput screening instruments as conventional two-dimensional cultures. ↩︎ ↩︎

  11. Lee KH. Recent Advances in Multicellular Tumor Spheroid Generation for Drug Screening, Biosensors, 2021;11(11):445.
    A review of the technologies developed to generate and incubate multicellular tumor spheroids, covering the importance of the extracellular matrix microenvironment for cell-cell and cell-matrix interactions, the better mimicry of solid tumors achieved by multicellular spheroids in cytotoxicity assessment, and advanced materials, microwell and microfluidic designs, and biosensing techniques for drug screening platforms. ↩︎ ↩︎

  12. Barisam M, Saidi MS, Kashaninejad N, Nguyen NT. Prediction of Necrotic Core and Hypoxic Zone of Multicellular Spheroids in a Microbioreactor with a U-Shaped Barrier, Micromachines, 2018;9(3):94.
    A finite element simulation study of a U-shaped barrier microbioreactor for spheroid culture that systematically examines oxygen and glucose concentration distributions as a function of spheroid diameter, inlet concentration, and medium flow rate, and identifies the necrotic core and hypoxic or quiescent zones that arise within aggregates of different sizes. ↩︎

  13. Azizipour N, Avazpour R, Weber MH, Sawan M, Ajji A, Rosenzweig DH. Uniform Tumor Spheroids on Surface-Optimized Microfluidic Biochips for Reproducible Drug Screening and Personalized Medicine, Micromachines, 2022;13(4):587.
    A study addressing the limited controllability of spheroid size, form, and density in conventional culture methods, which reduces the reproducibility and precision of bioassay results and drug-dose response assessment, and demonstrating surface-optimized microfluidic biochips that generate uniform tumor spheroids for reproducible drug screening and personalized medicine. ↩︎ ↩︎

  14. Vorrink SU, Zhou Y, Ingelman-Sundberg M, Lauschke VM. Prediction of Drug-Induced Hepatotoxicity Using Long-Term Stable Primary Hepatic 3D Spheroid Cultures in Chemically Defined Conditions, Toxicological Sciences, 2018;163(2):655-665. doi: 10.1093/toxsci/kfy058.
    A toxicity study demonstrating that primary hepatic spheroids in chemically defined conditions remain stable over multi-week culture, support repeated-dose and chronic exposure regimes that monolayers cannot sustain, and predict drug-induced hepatotoxicity, with cross-species comparisons of spheroids generated from mouse, rat, and non-human primate hepatocytes. ↩︎ ↩︎ ↩︎

  15. Richards LJ, Kilpatrick TJ, Bartlett PF. De novo generation of neuronal cells from the adult mouse brain, Proceedings of the National Academy of Sciences, 1992;89(18):8591-8595.
    One of the two foundational neurosphere studies, reporting that cells isolated from the adult mouse striatum proliferate in serum-free medium supplemented with growth factors such as basic fibroblast growth factor and that the resulting free-floating spheres generate neurons and astrocytes de novo, refuting the belief that mammalian central nervous system neurogenesis ends shortly after birth. ↩︎ ↩︎

  16. Bergeron A, Gitschier H. Co-culturing and Assaying Spheroids in the Corning Spheroid Microplate, Corning Application Note CLS-AN-390, accessed 2026-10-15.
    An application note demonstrating spheroid co-culture and assay workflows in 384-well spheroid microplates, including dose-response curves of chemotherapeutic compounds applied to mono-, co-, and tri-culture spheroids with the CellTiter-Glo 3D cell viability assay, and showing that the presence of fibroblasts and immune cells affects the potency of several compounds. ↩︎ ↩︎ ↩︎

  17. Hellmold D, Ziemianowicz D, Ellermann F, et al. Automation of 3D liver spheroid generation and acetaminophen dose-response on the MO:BOT enhances assay robustness and precision, Scientific Reports, 2026.
    An engineering study presenting MO:BOT, a modular benchtop platform that standardizes and automates critical steps of three-dimensional cell culture, including accurate cell seeding, precise media exchange, and dosing, situated within the regulatory momentum of the FDA Modernization Act 2.0, and demonstrating that automation enhances the robustness and precision of liver spheroid assays and acetaminophen dose-response testing. ↩︎ ↩︎

  18. Bell CC, Dankers ACA, Lauschke VM, et al. Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study, Toxicological Sciences, 2018;162(2):655-666. doi: 10.1093/toxsci/kfx289.
    A multicenter comparison in which hepatocytes from three donors were cultured in two-dimensional sandwich and three-dimensional spheroid configurations at six independent laboratories, finding that viability remains stable in both formats for up to two weeks in culture and supporting the spheroid configuration over two-dimensional sandwich culture for long-term toxicity applications. ↩︎

  19. Yadav C, Evtushenko AS, Bistrović Popov A, et al. A 3D spheroid model for assessing nanocarrier-based drug delivery to solid tumors, npj Biomedical Innovations, 2025;1:41.
    A methods study acknowledging that spheroids face reproducibility and data interpretation issues that limit their utility, and describing a simple and reproducible co-culture spheroid model compatible with high-throughput screening for the study of nanocarrier-based drug delivery in pancreatic ductal adenocarcinoma. ↩︎

  20. Castro LM, Paiva L, Lobo I, et al. Modeling 3D Glioblastoma: A review of spheroid culture techniques and standardization challenges, Methods, 2026. doi: 10.1016/j.ymeth.2026.05.013.
    A review of spheroid culture techniques applied to glioblastoma, arguing that traditional two-dimensional cultures fall short of replicating the complex tumor microenvironment, comparatively evaluating the major spheroid formation methodologies, identifying the lack of standardization as a critical barrier to translation, and outlining a framework for advancing standardized protocols. ↩︎ ↩︎

  21. Grimes DR, Kelly C, Bloch K, Partridge M. A method for estimating the oxygen consumption rate in multicellular tumour spheroids, Journal of the Royal Society Interface, 2014;11(92):20131124.
    A theoretical and experimental study motivated by the limited spatial resolution of clinical hypoxia imaging, which presents a method for estimating oxygen consumption rates in multicellular tumour spheroids from growth data and uses the estimates to predict oxygen gradients and the onset of proliferating and hypoxic regions within aggregates. ↩︎

  22. Zanoni M, Piccinini F, Arienti C, et al. 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained, Scientific Reports, 2016;6:19103.
    A study demonstrating that spheroid tumor models composed of cells in different proliferative and metabolic states hold value for developing new anticancer strategies, and noting that reproducibility of data originating from three-dimensional model experiments is insufficiently documented in the literature, which the authors address through a systematic approach. ↩︎

  23. Gopal S, Kwon SJ, Ku B, et al. 3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity, Communications Biology, 2021;4:893.
    A methods study presenting a spheroid microarray platform for analyzing natural killer cell-mediated cytotoxicity and combination therapies against established cancer cell lines, reported to mimic the tumor microenvironment and to support high-throughput, high-content immuno-oncology profiling. ↩︎

  24. Lonkwic K, Zajdel R, Kaczka K. Unlocking the Potential of Spheroids in Personalized Medicine: A Systematic Review of Seeding Methodologies, International Journal of Molecular Sciences, 2025;26(13):6478.
    A systematic review of patient-derived cancer spheroids for drug screening, extracting data on cancer type, culture and seeding methods, spheroid characteristics, and therapeutic responses under defined inclusion criteria, and arguing that three-dimensional spheroid models offer more physiologically relevant alternatives to two-dimensional cultures for personalized medicine. ↩︎

  25. InSphero. 3D In Vitro Oncology and Immunology, InSphero, accessed 2026-10-15.
    A commercial platform description of three-dimensional in vitro oncology and immunology services, supporting robust high-throughput drug screening in Akura 96- and 384-well plate formats and reporting that patient-derived tumor spheroids retain the architecture and complexity of the original tumor tissue. ↩︎

  26. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system, Science, 1992;255(5052):1707-1710. doi: 10.1126/science.255.5052.1707.
    The original neurosphere study reporting that cells isolated from the adult mouse striatum can be induced to proliferate in serum-free medium and generate both neurons and astrocytes, overturning the belief that mammalian central nervous system neurogenesis ends shortly after birth and establishing the free-floating sphere assay. ↩︎

  27. Dontu G, Abdallah WM, Foley JM, et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells, Genes and Development, 2003;17(10):1253-1270. doi: 10.1101/gad.1061803.
    The paper that developed the mammosphere assay, in which cells derived from reduction mammoplasties are seeded in non-adherent, non-differentiating culture conditions, and the cells capable of surviving and proliferating form discrete spheres that propagate as mammary stem and progenitor cells, providing the template for subsequent tumorsphere assays in cancer stem cell research. ↩︎

  28. Takeda M, Miyagawa S, Fukushima S, et al. Development of In Vitro Drug-Induced Cardiotoxicity Assay by Using Three-Dimensional Cardiac Tissues Derived from Human Induced Pluripotent Stem Cells, Tissue Engineering Part C: Methods, 2018;24(1):56-67. doi: 10.1089/ten.tec.2017.0247.
    A methods study developing an in vitro drug-induced cardiotoxicity assay based on three-dimensional cardiac tissues derived from human induced pluripotent stem cell-derived cardiomyocytes, on the premise that single cardiomyocytes are limited in their ability to mimic native tissue architecture and function. ↩︎

  29. Yen BL, Hsieh CC, Hsu PJ, Chang CC, Wang LT, Yen ML. Three-Dimensional Spheroid Culture of Human Mesenchymal Stem Cells: Offering Therapeutic Advantages and In Vitro Glimpses of the In Vivo State, Stem Cells Translational Medicine, 2023;12(5):235-244.
    A review of mesenchymal stem cell spheroid culture noting that these cells naturally reside in three-dimensional tissues in vivo despite the plastic-adherence criterion used to define them, and describing the therapeutic advantages of the aggregate format, including enhanced functional activity, alongside its value as an in vitro window onto the in vivo state. ↩︎