Open research questions in 3D Printing in Biomedical Research

What the literature leaves open

• Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Itoh, M. et al. Development of an immunodeficient pig model allowing long-term accommodation of artificial human vascular tubes. Nat. Commun. 10, 2244 (2019). 70. Shiwarski, D. J. et al. 3D bioprinting of collagen-based high-resolution internally perfusable scaffolds for engineering fully biologic tissue systems. Sci. Adv. 11, eadu5905 (2025). 48. Arai, K. et al. Fabrication of scaffold-free tubular cardiac constructs 71. Sexton, Z. A. et al. Rapid model-guided design of organ-scale using a bio-3D printer. PLoS One 13, e0209162 (2018). 49. Moldovan, L. et al. iPSC-derived vascular cell spheroids as building blocks for scaffold-free biofabrication. Biotechnol. J. 12, 1700444 (2017). 50. Oh, H. et al. Engineering volumetric tissue analogs via assembly of endothelial cell-covered spheroidal microtissues. Adv. Healthc. Mater. 15, e02418 (2026). 51. Neves, S. C. et al. A hybrid construct with tailored 3D structure for directing pre-vascularization in engineered tissues. Mater. Today Bio. 29, 101291 (2024). synthetic vasculature for biomanufacturing. Science 388, 1198–1204 (2025). 72. Endo, Y. et al. Bioartificial pulsatile cuffs fabricated from human induced pluripotent stem cell-derived cardiomyocytes using a prevascularization technique. npj Regen. Med. 7, 22 (2022). 73. Wimmer, R. A. et al. Human blood vessel organoids as a model of diabetic vasculopathy. Nature 565, 505–510 (2019). 74. Yoshizato, H. et al. A scaffold-free cartilage construct fabricated using a bio 3D printer accelerates critical-size bone defect regeneration. J. Orthop. Transl. 57, 101033 (2026). 52. Kim, C. H. et al. Mesenchymal stem cells improve wound healing 75. Hong, Y. et al. A strongly adhesive hemostatic hydrogel for the repair in vivo via early activation of matrix metalloproteinase-9 and vascular endothelial growth factor. J. Korean Med. Sci. 26, 726–733 (2011). 53. De Moor, L. et al. High-throughput fabrication of vascularized spheroids for bioprinting. Biofabrication 10, 035009 (2018). 54. Hsu, T. W. et al. Transplantation of 3D MSC/HUVEC spheroids with neuroprotective and proangiogenic potentials ameliorates ischemic stroke brain injury. Biomaterials 272, 120765 (2021). 55. Lin, S., He, X. & He, Y. Co-culture of ASCs/EPCs and dermal extracellular matrix hydrogel enhances the repair of full-thickness skin wound by promoting angiogenesis. Stem. Cell Res. Ther. 12, 129 (2021). 56. Ayan, B. et al. Aspiration-assisted bioprinting for precise positioning of biologics. Sci. Adv. 6, eaaw5111 (2020). 57. Aguilar, I. N. et al. Scaffold-free bioprinting of mesenchymal stem cells with the Regenova printer: optimization of printing parameters. Bioprinting 15, e00048 (2019). 58. Kim, M. H. & Ozbolat, I. T. Aspiration-assisted bioprinting of spheroids. Nat. Protoc. 21, 1325–1373 (2026). 59. Singh, S. K. et al. Critical role of three-dimensional tumorsphere size on experimental outcome. Biotechniques 69, 333–338 (2020). 60. Vilinski-Mazur, K. et al.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Data availability The data presented in this study are included within the article. Data shown in Figure 5D were generated by the authors in previous experiments. 23. Son, J. et al. Bioprinting of pre-vascularized constructs for enhanced in vivo neo-vascularization. Biofabrication 15, 034101 (2023). 24. Melhem, M. R. et al.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• behavior without physical scaffold interference. For a clearer comparison, the summarized in Table 118,24,26,28,37–39,42,45,61–66.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Fig. 6 | Design of spheroid composition and construct structure. A a–b Skewering multicellular spheroids onto the Kenzan and fabrication of a scaffold-free vascular graft. c Distribution of CD31-positive cells within the construct. d Anastomosis with the rat aorta and Doppler ultrasonography showing vessel patency on day 5. Adapted under the Creative Commons CC BY license from35. B Spheroids composed of three cell types, including hepatocytes (Heps), HUVECs, and MSCs. Adapted under the Creative Commons CC BY license from39. C a Spheroids composed of three cell types, including human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), HUVECs, and normal human dermal fibroblasts (NHDFs). b Histological and immunohistochemical staining of cell-specific markers. Adapted under the Creative Commons CC BY license from38. D a Design of porous liver-like tissue using micro-gaps. b Changes in the construct during tissue culture. c Histological and immunostaining of the gaps between spheroids and the distribution of HUVECs, indicating the formation of endothelial networks within the cellular construct. Adapted under the Creative Commons CC BY license from39. structures were observed via immunohistochemical staining like (Fig. 6A, a–c)35. The cellular constructs were first cultured on microneedles to allow spheroid fusion, followed by further maturation after removal from the needle array. These constructs were then transplanted into nude rats, resulting in grafted vessels that remained patent and exhibited excellent short-term biocompatibility (Fig. 6A, d). However, the tensile strength was only approximately half that of native vessels, and elastic fibers were not observed within the reconstructed tissue. In a subsequent study, Itoh et al. transplanted human dermal fibroblast-based tubular constructs into immunodeficient minipigs, where the grafts underwent in vivo remodeling into vascular constructs through interactions with host cells47. Another study suggested that the inclusion of mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) with hepatocytes allowed the expansion of spheroid size to 500 μm, exceeding the typical size limitation of hepatocyte spheroids (100–150 μm) imposed by hypoxia (Fig. 6B)39. Notably, a cell-sorting phenomenon occurred, in which hepatocytes migrated to the surface, whereas MSCs and HUVECs localized to the core. The authors proposed that this core localization may mitigate hypoxic stress in hepatocytes and may support vascularization through the secretion of growth factors, such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor. These findings are consistent with other studies demonstrating that organ-specific cells migrate to the periphery, whereas stromal cells and ECs form the core to support spheroid stability and maintenance of organ-specific cells (Fig. 6C)38,48. These results suggest that formed within multicellular fragments bioassembled microvascular spheroids may contribute to enhanced core viability, thereby demonstrating their potential for constructing large-scale vascularized tissues48,49. A porous structural design may be applied to promote angiogenic induction. Yanagi et al. utilized “micro-gaps” within tubular structures to facilitate the outward migration of vascular cells, thereby forming interstructures within the printed constructs connected microvascular (Fig. 6D)39. These results are particularly interesting because, although the differential adhesion hypothesis suggests that highly adhesive cells localize to the core and less adhesive cells to the periphery, the opposite distribution was observed, with ECs positioned internally within the multicellular spheroids. However, in structures incorporating micro-gaps, culture medium flow through these gaps generates localized shear stress, which may induce cellular rearrangement and drive ECs toward the spheroid periphery. In a similar study, Oh et al. demonstrated the formation of microvascular networks using a core–shell bioprinting strategy, in which organ-specific cells and ECs were precisely compartmentalized50. Under dynamic perfusion culture conditions, the printed microtissues underwent fusion and evolved into a unified construct. While the core–shell approach achieved vascularization through artificial compartmentalization of cell types and controlled perfusion, the Kenzan method relied on the predesigned porosity of the constructs to facilitate medium flow, thus enabling spontaneous endothelial redistribution and network formation. Despite these different fabrication approaches, both strategies underscore the importance of porous structural design coupled with dynamic culture conditions in promoting functional vascularization.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Fig. 4 | Components of a bio-3D printer. a Bio-3D printer hardware. Adapted under the Creative Commons CC BY license from42. b Control unit. Fig. 5 | Fabrication of cellular constructs using a bio-3D printer. A a Schematic diagram of tubular tissue conversion into a patch. b Cellular patch. Reproduced with permission from43. Copyright 2018, Elsevier. B a Design data of a trachea-like tube model. b Multi-layered trachea-like construct. Reproduced with permission from44. Copyright 2019, John Wiley and Sons. C a Design data of an articular surface. b Articular surface construct. Adapted under the Creative Commons CC BY license from45. D a Design data of a loop-type structure. b Loop-type construct. preclinical studies showed that the bio-3D conduit promoted superior nerve regeneration, as evidenced by increased numbers of myelinated axons, enhanced Schwann cell expression, and consistent nerve bridging compared with conventional silicone conduits46. These findings suggest a promising therapeutic strategy for neurological diseases. Despite this potential, the technical complexity and high operational cost of bio-3D printers may limit their widespread adoption compared with other bioprinting technologies. In particular, research on the construction of functional microvascular structures within artificial tissues remains limited. Nevertheless, this Perspective aims to outline potential strategies and future directions for scaffold-free microvascular construction by optimizing key parameters, such as spheroid composition and structural design, based on existing studies of vascularized tissues. Design of spheroids and structures for construction of microvascularized tissues One strategy involves fabricating multicellular spheroids by combining ECs with organ-specific cells to engineer target constructs35,38,39. Itoh et al.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Fig. 3 | Light-based bioprinting. A Schematic of the SLA process and printing of cell-laden gel patches. Reproduced with permission from24. Copyright 2017, American Chemical Society. B Schematic of the DLP process and fabrication of vascular structures. Adapted under the Creative Commons CC BY license from27. C a Microchannels filled with FITC-dextran (top) and fabrication of vascular units incorporating RFP-HUVECs and supportive cells (bottom). b Endothelial sprouting after 5 days (left) and intertwined spiral channels composed of GFP-ADSCs and RFP-HUVECs (right). Adapted under the Creative Commons CC BY license from28. source of endothelial sprouting within hydrogels. Additionally, predefined microchannel structures were endothelialized by seeding endothelial spheroids to form vessel-like linings, resulting in spatially separated endo- thelial and supporting-cell compartments (GFP-ADSCs), thereby estab- lishing a microvascular barrier structure. These results demonstrate that 2PP enables the precise fabrication of complex microvascular architectures on microfluidic chips and highlight its potential for organ-on-a-chip applications.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Fig. 2 | Extrusion-based bioprinting. A Fabrication of an EC-laden tubular construct using a coaxial nozzle and insertion of a polycaprolactone frame to improve the mechanical properties of the structure. Adapted under CC BY-NC 4.0 from17. B Fabrication of biomimetic blood vessels using a combination of SWIFT and a coaxial printhead. Scale bars: 10 mm (top) and 50 µm (bottom). Reproduced with permission from20, Copyright 2024, John Wiley and Sons. C Induction of angiogenic sprouting through the fabrication of endothelialized and fibroblast-laden channels. Scale bars: 200 µm (left) and 500 µm (right). Reproduced with permission from22, Copyright 2021, John Wiley and Sons. Furthermore, micropores ( ~ 25 μm) created within the collagen scaffold following the removal of gelatin microparticles contributed to endothelial migration and angiogenesis. Similarly, the sacrificial writing into functional tissue (SWIFT) method developed by Skylar-Scott et al. utilizes sacrificial printing within high-density cell spheroid baths19. Upon removal of the sacrificial filaments, perfusable microchannels are created within the functional tissue matrix. In another study, the feasibility of fabricating hierarchical vascular networks using a coaxial sacrificial ink printing strategy combined with SWIFT was demonstrated (Fig. 2B)20. However, the size of the printed vessels remains limited to a few hundred micrometers. Fang et al. developed the sequential printing in a reversible ink template technique, which employs a microgel-based biphasic system that serves as both a support bath and a cell-laden bioink, thus enabling the fabrication of complex 3D architectures and engineered microvascular networks21. Notably, this technique allows the integration of bulk tissue constructs with hierarchical vascular networks within a single printing process. Although embedded bioprinting technologies can stably fabricate complex 3D geometries without structural collapse by utilizing support media, they still share the fundamental limitation that resolution is constrained by the internal diameter of the extrusion nozzle, similar to conventional extrusion-based approaches. Therefore, regardless of the presence or absence of support media, realizing complex microscale structures remains a significant technical challenge for all nozzle-based bioprinting methods. In particular, although extrusion-based bioprinting can rapidly generate structures on the scale of hundreds of micrometers, comparable to arterioles, it still faces substantial challenges in replicating capillary-scale architectures ranging from 6 to 14 μm. An alternative strategy involves leveraging the intrinsic ability of ECs to undergo sprouting morphogenesis to form microvascular networks. For example, Son et al. induced angiogenesis by positioning fibroblast-lined channels adjacent to endothelialized channels, thereby leveraging angiogenic gradients to promote endothelial sprouting (Fig. 2C)22.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Fig. 1 | Classification and size ranges of blood vessels. Created in BioRender. Yu, J. (2026) https://BioRender.com/32t44q0. in which collagen and fibronectin are deposited by vascular resident cells8,9. Structurally, arteries are characterized by thicker walls and circular lumina to withstand high hemodynamic pressure, whereas veins typically exhibit thinner walls and elliptical luminal cross-sections. Arterioles, the smallest arteries, measure 10–300 μm in diameter and are composed of three distinct layers10,11. Similar to arteries, the tunica intima is composed of a single layer of ECs supported by a basement membrane, providing a thromboresistant surface. The tunica media, composed of a dense layer of SMCs arranged concentrically with elastic fibers, regulates blood flow and blood pressure. Finally, the tunica externa, a perivascular connective tissue layer, contributes to mechanical stabilization. Capillaries, measuring 6–14 μm in diameter, consist of a single layer of ECs and a basement membrane10,12. Pericytes surrounding this layer regulate vascular stability, barrier function, and maturation through interactions with ECs13. This structure, characterized by low blood flow and a large surface area, is optimized for material exchange. Venules are divided into postcapillary and muscular venules and range in size from 10 to 200 μm10,12. While muscular venules possess a very thin tunica media composed of one or two layers of SMCs and are surrounded by an adventitia consisting of connective tissue with few elastic fibers, postcapillary venules lack both tunica media and adventitia12. Similar to veins, some larger venules also possess valves to prevent backflow14. From an engineering perspective, reproducing vascular networks involves more than simply creating tubular structures. It is essential to ensure robust barrier function through junctions between ECs and maintain biological function under shear stress generated by blood flow. These complex intercellular interactions and the precise control of the microenvironment remain key challenges in building microvascular networks within artificial tissues. However, current bioprinting approaches primarily focus on the geometric formation of microchannels and therefore still face limitations in fully recapitulating these biological functions. Nevertheless, precise structural control is considered a key requirement for the formation of functional vasculature. Accordingly, the following section examines various bioprinting approaches, including extrusion-based and light-based techniques, for fabricating microscale vascular structures. Scaffold-based techniques for engineering microvascularized tissues Extrusion-based bioprinting Extrusion-based bioprinting, which mechanically deposits bioinks onto a substrate, is an accessible technology that offers considerable material flexibility. To fabricate perfusable and endothelialized vascularized constructs, extrusion-based bioprinting utilizes multi-nozzle or coaxial printing heads in combination with sacrificial inks such as Pluronic F-127 or polyethylene glycol to create tubular structures15,16. Gu et al. used extrusionbased bioprinting equipped with a coaxial nozzle to fabricate a perfusable vascular construct with an approximate diameter of 900 µm17. ECs were encapsulated within methacrylate gelatin (GelMA) bioinks, whereas gelatin served as a sacrificial component that was subsequently removed to generate a hollow channel. To enhance the overall mechanical stability of the construct, polycaprolactone was used as a supporting structure (Fig. 2A). In general, conventional extrusion-based bioprinting often struggles to maintain structural stability because low-viscosity hydrogels tend to collapse under their own weight, thereby requiring additional support structures to fabricate complex 3D constructs with high structural fidelity. Embedded bioprinting based on conventional extrusion printing has been developed to address these challenges. This approach utilizes a support bath that rapidly stabilizes printed filaments, providing the buoyancy and mechanical support necessary to prevent the sagging or collapse of thin hydrogels owing to gravity. This environment effectively provides the “time and space” required for stable crosslinking. For instance, the freeform reversible embedding of suspended hydrogels (FRESH) technique, which utilizes an optimized slurry of gelatin microparticles, enables the extrusion of filaments with diameters as small as 20 μm18. Using the FRESH method, vascular structures measuring approximately 100 μm could be fabricated.

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Challenges and opportunities in generating microvasculature using bioprinting techniques https://doi.org/10.1038/s44385-026-00100-x Junjie Yu1, Daiki Murata1, Manabu Itoh2 & Koichi…

  Challenges and opportunities in generating microvasculature using bioprinting techniques · 2026 · DOI

• Addi- tionally, a better characterization of PEG4MAL con- centrations between 2% and 5% remains warranted, as well as the use of other small molecules, other than RGD, that may induce desirable changes in mechanical, fluid, and adhesion properties of PEG4MAL.

  Microfluidic encapsulation of the human gut microbiota—a tool for research and beyond · 2026 · DOI

• This phase focused exclusively on design and technical devel- opment; clinical usability, user comfort, psychosexual acceptability, and long-term durability under real-world conditions remain to be tested. Environmental factors such as room temperature or mucosal moisture may influence colour-transition thresholds and should be investigated fur- ther during pilot studies.

  Design and Development of a Silicone Vaginal Dilator with Thermochromic Properties for Quantifiable Biofeedback in Gynecologic Rehabilitation · 2026 · DOI

• Four convergent trends are likely to reshape OoC technology over the next decade. Integration with artificial intelligence and machine learning—both for analysis of multi-modal sensor streams and for model-based extrapolation of chip data to whole-body pharmacokinetics—will transform OoC platforms into closed-loop autonomous experimentation systems. Combination with single-cell and spatial omics technologies will yield unprecedented mechanistic resolution of drug effects within chip tissues. Convergence with patient-derived iPSCs and CRISPR-edited isogenic controls will enable truly personalized precision-medicine platforms for routine clinical decision support. Finally, body-on-a-chip platforms integrating six or more organs with physiologically scaled fluid volumes are expected to enter regulatory submissions for first-in-human dose selection within five years, potentially redefining the boundary between preclinical and clinical drug development [41,42].

  From mice to microchips: Organ-on-a-chip technology as a paradigm shift in drug discovery, disease modeling, and personalized medicine · 2026 · DOI

• Brain organoid models have substantially advanced the study of human brain development, circuit formation, and neurological disease. However, each oxygenation strategy presents intrinsic biological and practical limitations that must be considered when interpreting experimental outcomes. Apical-exposure systems, including air–liquid improve oxygenation and experimental accessibility at the tissue surface but remain constrained in their ability to fully recapitulate the interface (ALI) configurations, complex internal spatial organization of the developing brain. Enclosed ventricular-like zones, deep radial architecture, and long-range cytoarchitectural patterning are not uniformly accessible in intact ALI-cultured organoids, and altered boundary conditions at the air-exposed surface may impose non-physiological stresses that influence neuronal maturation or regional identity. Critically, physiological oxygen levels in the human brain are substantially lower than atmospheric conditions (~21% O2), and exposure to higher oxygen levels in standard culture systems can introduce non-physiological effects, including altered cellular responses and oxidative stress (Barakat et al., 2025). Although air–liquid interface (ALI) culture enhances oxygen diffusion and reduces hypoxic core formation, it does not reflect the native brain environment, which is not exposed to air, therefore, ALI should be considered primarily as a technical strategy rather than a physiologically accurate configuration, and its non-physiological boundary conditions should be into account when interpreting experimental outcomes. taken Sliced cerebral organoid cultures effectively address diffusionlimited hypoxia by exposing internal tissue compartments, thereby enhancing metabolic stability and experimental access. However, these advantages are accompanied by disruption of intact threedimensional geometry and long-range tissue continuity. Mechanical perturbation introduced during slicing can elicit cellular stress responses, and thin organoid sections typically exhibit reduced longevity compared with intact constructs. Although vibratomebased slicing improves oxygen diffusion, it is typically performed at low frequency (e.g., approximately once per month) to minimise cumulative mechanical stress, as it may cause localized disruption to neuronal processes and induce transient injury-related responses interpreting experimental that should be considered when outcomes.

  Air–liquid interface–centered oxygen engineering in human brain organoids: intact, sliced, and microfluidic extensions · 2026 · DOI

• Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Shao et al. Microsystems & Nanoengineering (2026) 12:183 Page 2 of 17 limiting their applicability for multilayer fabrication, functional screening6,24. Hydro- large-scale single-cell dynamic trap arrays enable deterministic single-cell cap- ture and relatively high throughput; however, trapped cells are commonly exposed to shear stress due to con- tinuous perfusion during trapping and culture, which also removes autocrine and paracrine factors, potentially altering cellular states and limiting studies of shear- sensitive or autocrine/paracrine-dependent behaviors8,25. Microwell and microchamber arrays offer high-density layouts for secretion profiling or short-term cell–cell interaction studies; however, stochastic Poisson-limited loading results in low single-cell occupancy, wasting a large fraction of assay units17. In addition, closed-format wells suffer from restricted nutrient and waste exchange, making them unsuitable for long-term proliferation or drug-response assays, whereas open-format wells sacrifice the cellular microenvironment15,16. precise control of Droplet-based microfluidic systems achieve ultrahigh throughput but encapsulate cells in isolated aqueous droplets surrounded by oil, which severely limits sus- tained culture, cell adhesion, migration and controlled cell–cell interactions, in addition to Poisson-limited low single-cell occupancy21–23. Electrical, optical, acoustic, and magnetic methods have also been integrated with microfluidics for single-cell manipulation and analysis but are more complicated and typically require additional instrumentation or specialized setups26,27. Importantly, most existing platforms are function-specific, designed to interrogate only one or two cellular behaviors, such as rather proliferation, cytotoxicity, or than functional profiling. enabling integrated, multimodal the lack of reliable and selective live-cell Moreover, retrieval in closed microfluidic formats prevents direct linkage between dynamic functional phenotypes and characterization10. Together, downstream molecular these limitations underscore the unmet need for a single- cell functional analysis platform that combines determi- nistic loading, high throughput, shear-free long-term culture, modular assay geometries, and selective live-cell retrieval within a single integrated system. secretion, To overcome these limitations, we developed the High- throughput Single-Cell Omni-functional Profiling Engine (HiSCOPE), a modular microfluidic platform designed for large-scale, multimodal single-cell functional analysis. HiSCOPE integrates deterministic single-cell and cell-pair loading with centrifugation-directed homing into dead- end, no-flow assay chambers, enabling shear-free long- term culture and spatially programmable cell–cell con- figurations within a unified trapping array. Importantly, the decoupled modular design allows diverse functional assays to be implemented without redesigning the core trapping architecture, while supporting 12,800 parallel assay units per chip and selective retrieval of single cells of loading, shear-free interest after on-chip interrogation for downstream clonal expansion or molecular profiling. This combination of deterministic long-term culture, modular assay flexibility, and live-cell retrieval is, to our knowledge, not simultaneously available on existing single-cell microfluidic platforms. Using this platform, we demonstrate high-throughput single-cell proliferation and drug susceptibility profiling, integrated analysis of immune-cell cytotoxicity and cytokine secretion, and spatially controlled dissection of contact-dependent and paracrine cell–cell interactions. Together, these results establish HiSCOPE as a versatile toolbox for system- atically interrogating functional heterogeneity at the single-cell level.

  A high-throughput modular microfluidic platform for versatile functional assays at single-cell level · 2026 · DOI

• Microsphere-based technologies have evolved into multi- faceted platforms with transformative potential across breast cancer modeling, diagnosis, and therapy. For instance, Spe- cific miRNA expression profiles serve as potential biomark- ers for TNBC diagnosis, but conventional methods require large samples and are complex. To overcome this, Liu et al. (2023) [120] developed a high-throughput, sensitive method using rolling circle amplification on fluorescence-encoded microspheres to simultaneously quantify multiple TNBC- associated miRNAs (miR-16, miR-21, miR-92, miR-199, miR-342). The method exhibits broad linearity, high sensi- tivity, specificity, and reproducibility in serum, facilitating early TNBC detection. Figure 5 depicts a fluorescence-encoded microsphere- based isothermal amplification system for sensitive, high- throughput detection of TNBC-associated miRNAs. Their ability to integrate structural support, biological signaling, and controlled delivery within a single system offers clear advantages over conventional approaches. From enabling physiologically relevant three-dimensional tumor models to delivering chemotherapeutics, biologics, and radionuclides with spatial precision, microspheres address critical unmet needs in breast cancer research and clinical care. The grow- ing sophistication of microsphere design, including stimuli- responsive, multifunctional, and immune-active systems, signals a shift toward highly personalized and adaptive therapeutic strategies. In particular, the success of micro- spheres in locoregional interventions for metastatic breast cancer underscores their clinical relevance, especially in settings where systemic therapies have failed. Parallel advances in diagnostic and biosensing applications further highlight the versatility of microspheres as tools for disease monitoring and treatment guidance. Looking forward, the next generation of microsphere platforms should prioritize biological integration, incorporating immune components, vascular mimetics, and patient-derived cells to better reflect in vivo tumor ecosystems. Rational combination strategies that align microsphere-based delivery with systemic thera- pies, immunotherapy, and radiotherapy will be essential to maximize therapeutic benefit. Equally important is the development of scalable, standardized manufacturing pipe- lines to facilitate regulatory approval and clinical adoption. DARU Journal of Pharmaceutical Sciences (2026) 34:26 1 3 Additionally, emerging technologies such as artificial intel- ligence for predictive modeling, advanced real-time imag- ing modalities, and organoid-based personalized platforms are expected to synergize with microsphere-based systems, enabling highly adaptive, precise, and patient-specific therapeutic strategies in the near future. Future advances in microsphere technologies will require improved targeting ligands, more robust and precise stimuli-responsive materi- als, and integration with emerging therapies such as immu- notherapy and CAR-T cells. Priority areas include clinical validation of personalized microsphere systems tailored to individual tumor characteristics, along with addressing regulatory and manufacturing challenges to enable safe and scalable clinical translation. In summary, microsphere tech- nologies represent a convergence point between materials engineering and precision oncology. Continued interdis- ciplinary collaboration and rigorous translational research will be crucial for unlocking their full potential and estab- lishing microspheres as integral components of future breast cancer diagnostics and therapeutics. Acknowledgements None.

  Engineering microspheres for breast cancer: integrating tumor modeling, diagnostics, and targeted treatment · 2026 · DOI

• The design parameters of spheroid-based in vitro tumor models determine their biomimetic properties (as noted by Lugovoi et al. 2025), but quantitative benchmarking frameworks comparing biomimetic fidelity across different scaffold materials (alginate hydrogels, collagen vitrigel, methylcellulose) and culture geometries remain underdeveloped for standardizing model selection in preclinical oncology.

  An overview of the developments in 3D cancer cell models, assay techniques, and imaging modalities · 2026 · DOI

• The integration of flexible electrodes into 3D spheroid and organoid culture systems has been initiated but the paper excerpt is incomplete regarding the specific electrophysiological monitoring or bioelectrical assessment capabilities. The technical implementation of multi-electrode arrays with 3D cancer cell models for real-time functional readouts during spheroid development and drug response remains partially undefined.

  An overview of the developments in 3D cancer cell models, assay techniques, and imaging modalities · 2026 · DOI

• Patient-derived organoids have been established for cervical, biliary tract, colorectal, and ovarian cancers for drug screening and chemotherapy response prediction, but systematic comparative validation across cancer types and treatment modalities is lacking. The generalizability of organoid-based drug response predictions to other tumor entities and across different therapeutic classes (beyond chemotherapy and PARP inhibitors) remains unexplored.

  An overview of the developments in 3D cancer cell models, assay techniques, and imaging modalities · 2026 · DOI

• The relationship between inter-spheroid proximity and cancer-associated fibroblast-mediated invasion requires further characterization in heterotypic tumor spheroid systems. Mehta et al. (2026) identified that inter-spheroid proximity and matrix remodeling determine invasive behavior, but the specific spatial thresholds and biomolecular mechanisms governing this communication across different cancer types remain incompletely defined.

  An overview of the developments in 3D cancer cell models, assay techniques, and imaging modalities · 2026 · DOI

• The influence of experimental variables on spheroid attributes requires systematic investigation across different 3D cancer cell model systems. While Zhu et al. (2025) examined how variables affect spheroid properties, there is a need to establish standardized protocols that account for how culture conditions, scaffold materials, and cell composition variability impact the biological relevance and reproducibility of 3D spheroid models for drug screening.

  An overview of the developments in 3D cancer cell models, assay techniques, and imaging modalities · 2026 · DOI

• Longitudinal single-cell sequencing and metabolic profiling are required to reveal how immune pressure drives CAFs toward pro-fibrotic or pro-inflammatory phenotypic transitions and triggers upregulation of alternative immune checkpoint pathways (TIGIT and TIM-3).

  Engineering the tumor microenvironment via 3D multicellular bioprinting for personalized immunotherapy assessment and resistance deciphering · 2026 · DOI

• Rigorous experimental evidence is needed to test the hypothesis that a 'spatial barrier' underlies therapeutic resistance and support combination strategies targeting CAF-derived barriers such as FAK or CXCR4 inhibitors.

  Engineering the tumor microenvironment via 3D multicellular bioprinting for personalized immunotherapy assessment and resistance deciphering · 2026 · DOI

• Need to quantify T-cell infiltration efficiency and analyze how CAF paracrine signaling (TGF-β and CXCL12) and matrix remodeling are enhanced under defined spatial configurations.

  Engineering the tumor microenvironment via 3D multicellular bioprinting for personalized immunotherapy assessment and resistance deciphering · 2026 · DOI

• There are no published reports specifically evaluating the in vitro interactions of 13–93 BG compositions with HT-29 colon carcinoma or HIF (human intestinal fibroblast) cell lines.

  Fabrication of silicate-based bioactive glass-containing sodium alginate hydrogel microfibers for tissue engineering applications · 2026 · DOI

Most-cited papers in 3D Printing in Biomedical Research

• Harnessing the potential of hydrogels for advanced therapeutic applications: current achievements and future directions · Signal Transduction and Targeted Therapy · 2024 · 529 citations
• A microfluidic platform integrating functional vascularized organoids-on-chip · Nature Communications · 2024 · 296 citations
• Scaffold-based 3D cell culture models in cancer research · Journal of Biomedical Science · 2024 · 285 citations
• Advancements in tissue and organ 3D bioprinting: Current techniques, applications, and future perspectives · Materials & Design · 2024 · 159 citations
• Integrating organoids and organ-on-a-chip devices · Nature Reviews Bioengineering · 2024 · 143 citations
• Nanoformulations in Pharmaceutical and Biomedical Applications: Green Perspectives · International Journal of Molecular Sciences · 2024 · 142 citations
• AI energized hydrogel design, optimization and application in biomedicine · Materials Today Bio · 2024 · 136 citations
• Engineering Large‐Scale Self‐Mineralizing Bone Organoids with Bone Matrix‐Inspired Hydroxyapatite Hybrid Bioinks · Advanced Materials · 2024 · 128 citations
• 3D bioprinting of human neural tissues with functional connectivity · Cell stem cell · 2024 · 122 citations
• Human organoids-on-chips for biomedical research and applications · Theranostics · 2024 · 121 citations

Most recent work

• Microphysiological Systems: Transforming Biomedical and Clinical Research · Biotechnology and Bioengineering · 2026
• High Dense Cell Patches from Acoustically Assisted 3D Bioprinting for Wound Healing · Advanced Healthcare Materials · 2026
• Exploring the role of 4D printing materials, techniques, and characteristics for personalized oncology · Translational Oncology · 2026
• High-density extracellular recordings from the interior of intact brain organoids enable automated high-throughput functional assay · bioRxiv · 2026
• On-Chip modeling of drug-gut interactions in Oral drug delivery · Advanced Drug Delivery Reviews · 2026
• Ambient temperature storage of encapsulated cells in alginate microspheres and core–shell capsules · One Health Journal · 2026
• Fabrication of silicate-based bioactive glass-containing sodium alginate hydrogel microfibers for tissue engineering applications · Applied Physics A · 2026
• Engineering the tumor microenvironment via 3D multicellular bioprinting for personalized immunotherapy assessment and resistance deciphering · HepatoBiliary Surgery and Nutrition · 2026
• 3D-printed alginate/gelatin/PCL/hyaluronic acid scaffolds with metal–organic frameworks for quercetin delivery: Improving mechanical properties and cytocompatibility for cartilage tissue engineering applications · Bioprinting · 2026
• A compatible gravity-driven organoid perfusion (GDOP) platform for drug screening with sensitivity and toxicity process evaluation · Communications Biology · 2026

Related topics in Engineering