3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection

3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection Tianyi Zhang, Yuqing Xu, Yiwei Sun, Kun Ye, Jiarong Liu, Rui Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7659713/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted 10 You are reading this latest preprint version Abstract Viral organisms characterized by elevated transmissibility and pathogenicity constitute a substantial public health risk. Three-dimensional (3D) cell culture systems better mimic the in vivo microenvironment than traditional two-dimensional (2D) models, offering significant potential for virological research. Therefore, a method for natural viral growth based on 3D cell culture need be developed. In this study, we developed an integrated microfluidic platform for the efficient generation of highly uniform alginate hydrogel microspheres (AHMs) encapsulating HeLa cells, enabling robust 3D cell culture and subsequent infection with Coxsackievirus B3 expressing enhanced green fluorescent protein (CVB3-eGFP). Our results demonstrate that AHMs support high cell viability and facilitated cell proliferation within a biomimetic 3D matrix. By systematically reducing the alginate concentration from 1.0% to 0.6%, we enhanced viral accessibility while maintaining microstructural integrity, thereby significantly improving CVB3-eGFP infection rates, as confirmed by fluorescence imaging and western blot analysis. This study establishes a tunable, reproducible, and physiologically relevant 3D model for studying virus–host interactions, with broad applications in antiviral drug screening and infectious disease modeling. Microfluidic chip Alginate hydrogel microspheres 3D cell culture CVB3 Host–pathogen interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Viruses with high transmissibility and pathogenicity pose a substantial threat to public health. In recent decades, multiple viral pandemics have occurred worldwide. Pathogens such as the influenza virus, Ebola virus, and coronaviruses exert profound impacts on socioeconomic stability and human health [ 1 – 4 ]. Enterovirus , which belongs to the family Picornaviridae , primarily include 10 Enterovirus species (EV A–J) and three Rhinovirus species (RV A–C) [ 5 , 6 ]. Among them, Coxsackievirus B3 (CVB3), a non-enveloped, single-stranded, positive-sense RNA virus, is the primary pathogen responsible for myocarditis [ 7 , 8 ]. Moreover, the persistence of CVB3-RNA residuing in tissues can lead to sustained immune responses and the production of autoantibodies, thereby inducing the progression of chronic disease [ 9 – 11 ]. Consequently, CVB3 infections have received increasing attention. Understanding the viral infection process in host cells is a crucial step in virology research, which is of great significance for the development of antiviral drugs and formulation of therapeutic strategies. Establishing appropriate viral infection models is fundamental for elucidating virus-host interactions. Conventional infection models have been established in vitro using cell culture [ 12 , 13 ]. However, two-dimensional (2D) cell culture methods can only provide data under simplified monolayer conditions, which fail to recapitulate the actual in vivo infection processes. Moreover, these approaches are relatively tedious and yield results of limited biological significance [ 1 , 14 ]. Animal models (for in vivo studies) can also be used. However, this may raise ethical concerns. Additionally, other obstacles exist, such as high costs and prolonged experimental duration [ 15 – 17 ]. Encapsulating cells within hydrogel microspheres for culture presents a straightforward and readily implementable novel cultivation method [ 18 – 20 ]. As a method for three-dimensional (3D) cell culture, this system provides an environment that closely mimics in vivo conditions, allowing for more direct observation of cellular behavior and responses to experimental treatments [ 20 ]. Consequently, it has gained increasing application in regenerative medicine, drug delivery, and tissue engineering [ 21 – 24 ]. Among these materials, alginate hydrogel microspheres (AHMs) have attracted increasing attention. Alginate is characterized by excellent biocompatibility, low cost, safety, and biodegradability, making it an ideal coating material for simulating an in vivo microenvironment [ 25 , 26 ]. The preparation of relatively uniform AHMs primarily involves three methods: extrusion, electrostatic dripping, and droplet-based microfluidics [ 27 – 29 ]. Extrusion is the most classical approach, whereby an alginate solution is dripped into a gelling bath containing Ca²⁺ ions, with manual control over the formation of AHMs droplets. Although simple and controllable, this method often results in AHMs with broad size distributions and relatively rough surfaces. Electrostatic dripping can effectively regulate the size of AHMs, yielding highly uniform microspheres. However, this technique is predominantly applied in drug encapsulation and has limited applications in cell culture [ 24 ]. The use of microfluidic chips to prepare monodisperse droplet-based microspheres has garnered considerable attention. By leveraging chip architecture and fluid flow control, this technique enables the high-throughput and rapid production of uniformly sized microspheres, offering operational simplicity, high efficiency, and excellent controllability [ 30 , 31 ]. The process mainly consists of two key steps: shearing of the aqueous phase by an oil phase to form uniformly sized gel microspheres, and subsequent demulsification to separate the microspheres from the oil medium. Studies have demonstrated that microfluidic technology exhibits significant advantages in generating monodisperse AHMs, representing an advanced method characterized by high throughput, stability, and automation [ 29 , 32 , 33 ]. Previously, we developed a flexible and tunable microfluidic platform capable of generating and isolating AHMs to provide a stable microenvironment for bacterial growth and enable real-time monitoring of bacterial proliferation within the microspheres [ 34 ]. Building on this foundation, we optimized the chip architecture to better accommodate cell encapsulation within AHMs, establishing a robust 3D cell culture model for further applications. In summary, we constructed a microfluidic platform for generating highly uniform AHM-based cell culture microspheres, investigated cellular growth under 3D culture conditions, and conducted preliminary applications using AHM-encapsulated cells in viral infection studies under 3D settings. Materials and methods 2.1 Materials and reagents Sodium alginate (90.0%), ethylene diamine-N, N'-diacetic acid (EDDA, 99%), ethylene diamine tetraacetic acid (EDTA, 99%), 3-(N-morpholino)-propane sulfonic acid (MOPS), CaCl 2 , and zinc acetate were purchased from Al. Ltd. (China). Latex beads (sulfate-modified polystyrene, fluorescent red) were purchased from Sigma-Aldrich (USA). 1H,1H,2H,2H-perfluoro-1-octanol (PFO) was purchased from Alfa Aesar (USA). All the reagents were used as received. Deionized water was used in all experiments. 2.2 One-step generation of microspheres AHMs were produced via internal gelation using a complex ligand-exchange crosslinking method [ 35 ]. The crosslinking ion solution contained 1% sodium alginate, 30 mM Ca²⁺ chelated with 30 mM EDTA, and 14 mM MOPS buffer (pH 7). The exchange ion solution contained 1% sodium alginate, 30 mM zinc acetate, 30 mM EDDA, and 14 mM MOPS buffer (pH 7). Red fluorescent polystyrene microspheres (0.1 µm, 0.1% v/v) were added to visualize the microspheres. The continuous phase was fluorocarbon oil (HFE 7500) with 3% (v/v) fluorosurfactants. The flow rates of the continuous phase were varied (10, 300, 500, 700, and 900 µL h⁻¹), while that of the dispersed phase was fixed at 20 µL h⁻¹. The solutions were infused using a syringe-pump. The droplets were broken using 10% (v/v) perfluorooctanol (PFO). The size and coefficient of variation (CV) of the microspheres were measured using the ImageJ software. 2.3 Calcein/PI staining assay in HeLa cells The Calcein/PI Kit (Beyotime Biotechnology, China) was used to assess cell viability and cytotoxicity. First, HeLa cells captured by AHMs were cultivated for 12h. Upon completion, the cells were stained with the Calcein/PI Kit. The culture medium was aspirated, and the cells were gently washed once with phosphate-buffered saline (PBS). According to the manufacturer’s instructions, the calcein/PI detection working solution was prepared and added. The cells were incubated at 37°C in the dark for 30 min. After incubation, cell viability and cytotoxicity were assessed by observing fluorescence under a fluorescence microscope (Axio Vert A1, ZEISS, Germany). 2.4 Virus infection For CVB3-eGFP construction, eGFP, a reporter protein, was encoded in the viral genome after the start codon of the viral VP4 protein. Due to the presence of a protease cleavage site, eGFP and VP4 are expressed as single proteins and can be detected. In the infection studies of HeLa cells, the cells were plated in a six-well culture plate, and 1.5×106 plaque-forming units (PFU) of CVB3-eGFP were applied per well. After an incubation period of 1 h, the virus inoculum was replaced with fresh DMEM. 2.5 Western blot analysis The cells were lysed in RIPA buffer (Beyotime Biotechnology, China) supplemented with protease inhibitor cocktails (Beyotime Biotechnology, China), and 20 µg of total protein was subjected to 10% SDS polyacrylamide gel electrophoresis. The samples were transferred to PVDF Western Blotting membranes and blotted with anti-VP1 (GeneTex, USA) and GAPDH (GeneTex, USA) antibodies. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H + L) (Cell Signaling, USA) was used as a secondary antibody. The membranes were analyzed using a Western Lumax LightTM Superior (ZETA) with an enhanced chemiluminescence (ECL) imager. Results 3.1 Performance of the integrated microfluidic system In our platform, we employed droplet-based microfluidics to generate AHMs. HeLa cells were encapsulated in a gel-microsphere system. A dispersed phase consisting of 1% sodium alginate complexed with Ca–EDTA and 1% sodium alginate complexed with Zn–EDDA was mixed with the pre-treated HeLa cell suspension, forming aqueous phases that were injected into channels A and B of the chip. Simultaneously, fluorocarbon oil containing 3% surfactant was introduced as the continuous phase through channel C. The aqueous phases in channels A and B were infused at the same low flow rate. Under stable conditions, the two streams merged at the junction to form a cross-linked structure, which was sheared by the high-speed oil flow from Channel C. The resulting microspheres were propelled by the oil phase into the collection channel. After confirming stable microsphere generation using microscopy, the collected oil-encapsulated gel microspheres were demulsified. A medium containing 10% PFO was used as the demulsifier for the water-in-oil-in. In the presence of PFO, the microspheres rapidly separated from the oil phase. To minimize the potential adverse effects on cell viability caused by prolonged exposure to the demulsifier, we ensured thorough yet rapid contact between the demulsifier and microspheres after removing most of the oil phase. Finally, the demulsifier was removed, and the microspheres were repeatedly washed and resuspended in cell culture medium. The fundamental principles and setup of the experiment are illustrated (Fig. 1 ). 3.2 Generation of AHMs Owing to the chip architecture and shear force provided by the oil phase, alginate microspheres with a uniform size distribution were successfully formed at the junction where the oil and aqueous phases converged. To further validate the reliability of our chip design and the stability of the flow rate control, we investigated the effect of different flow rate ratios between the continuous (oil) and dispersed (aqueous) phases on the size of the generated microspheres. The flow rate of the aqueous phase was maintained at a relatively low value of 20 µL/h, whereas the flow rate of the oil phase was adjusted to regulate droplet size. When the flow rate ratio of the aqueous phase to the oil phase was set to 1:5, spherical droplets were stably generated with a relatively larger average diameter of 109.0 ± 3.3 µm and a coefficient of variation (CV) of 3.0% (Fig. 2 A). When the oil flow rate was increased to 300 µL/h (resulting in a ratio of 1:15), the microsphere size decreased significantly, yielding an average diameter of 88.5 ± 3.3 µm and a CV of 3.8% (Fig. 2 B). Subsequently, the oil flow rate was incrementally increased at equal intervals of time. The results showed only minor changes in size, consistent with previous reports. At a ratio of 1:25, the average diameter was 80.3 ± 4.2 µm (CV = 5.3%) (Fig. 2 C); at 1:35, it was 75.3 ± 2.9 µm (CV = 3.9%) (Fig. 2 D); and at 1:45, the average diameter was 74.6 ± 3.0 µm (CV = 4.0%) (Fig. 2 E). These results demonstrate that droplets can be stably generated across various flow rate ratios, with all CV values remaining relatively low (Fig. 2 F), confirming the stability and reliability of our microfluidic chip design and platform operation. Furthermore, as the oil flow rate increased, the enhanced shear force led to a consistent reduction in the size of the AHMs, which aligned with previously published studies and met our expectations [ 36 , 37 ]. 3.3 Characterization of AHMs To evaluate the size and structural strength of the AHMs for practical cell culture applications, we demulsified the droplets generated by the microfluidic chip and analyzed the size and coefficient of variation (CV) of the resulting microspheres. The results showed that at a flow rate ratio of Falg/Foil = 1:5, the microspheres exhibited the largest average diameter of 145.9 ± 4.3 µm with a CV of 2.9% (Fig. 3 A). When the ratio was increased to 1:15, the average diameter decreased to 119.0 ± 4.3 µm (CV = 3.6%) (Fig. 3 B). At Falg/Foil = 1:25, the average diameter was 111.3 ± 3.5 µm (CV = 3.2%) (Fig. 3 C); at 1:35, it was 109.4 ± 3.0 µm (CV = 3.0%) (Fig. 3 D); and at 1:45, the average diameter was 108.2 ± 3.9 µm (CV = 3.6%) (Fig. 3 E). The influence of different Falg/Foil ratios on the microsphere size is summarized in the corresponding figure. Compared to emulsified droplets, demulsified AHMs exhibited an overall increase in size (Fig. 3 F). This swelling behavior was consistent with expectations and can be attributed to the hydrophilic hydroxyl groups in alginate, which enhance the affinity for water molecules in a dispersed environment. Excessively large microspheres not only exhibit lower production efficiency but also, according to the Poisson distribution, are less conducive to effective cell encapsulation [ 38 ]. Moreover, oversized microspheres may impede efficient nutrient and metabolite exchange during culture [ 39 ]. Based on these considerations, we selected the microspheres generated at Falg/Foil = 1:15 as the model for subsequent experiments. 3.4 Encapsulation and culture of HeLa cells in AHMs We further explored the feasibility of 3D culture of HeLa cells using AHMs. To demonstrate the viability of the 3D cell culture, we performed bright-field microscopic observations at 12-hour intervals. As shown in the corresponding figure, cell growth under 3D culture conditions was monitored at 12, 24, 36, 48, and 60 h to confirm that the cells within the AHMs maintained active division and proliferation. Optical images revealed that small groups and clusters of cells continuously emerged within the AHMs with prolonged culture periods (Supplement Fig S1 ). The 3D structure of AHMs, which encapsulates cells in isolated compartments, allows efficient exchange of nutrients and metabolites. By 48 h, the HeLa cells showed robust growth in the AHMs. After 48 h, a small number of cells began to grow outside the microspheres. Furthermore, we compared the differences between the 3D and conventional 2D cultures. Cells in 2D culture exhibited typical spread morphology, whereas those encapsulated in AHMs presented various forms, including single cells, small groups, and clusters (Supplementary Fig. S2). To confirm that the encapsulation process and AHMs culture did not significantly affect cell viability, we used a Calcein-AM/PI staining kit to assess cell survival (Fig. 4 ). Fluorescence images indicated that HeLa cells grew well within the AHMs over a three-day culture period. The viability of the encapsulated cells was evaluated by measuring the area of green fluorescence (live cells) and red fluorescence (dead cells) at the focal planes using ImageJ software. Viability was determined by dividing the area of green fluorescence by the total area of green and red fluorescence. Quantitative analysis of cells cultured for 1–3 days showed viability ranging from 95% to 97%, demonstrating that AHMs provide a feasible environment for cell culture. In summary, the AHM-based culture model established in this study offers a versatile 3D culture tool for microorganisms and cells. The high cytocompatibility of alginate hydrogels provides suitable sites for cell-matrix integration, thereby better simulating in vivo cell division and proliferation than gelatin hydrogels. 3.5 Establishment of a CVB3-eGFP infection model in HeLa cell cultured in AHMs We further explored the application of AHM-encapsulated HeLa cells in viral infection research. Initially, we characterized the infection of monolayer-cultured HeLa cells with CVB3-eGFP. As expected, infection of 2D-cultured HeLa cells in 12-well plates with CVB3-eGFP (1000 TCID₅₀) resulted in a time-dependent increase in eGFP-positive cells, plateauing at 48 h post-infection. These preliminary experiments confirmed the effectiveness of CVB3-eGFP in HeLa cells (Supplementary Fig. S3). Based on previous results demonstrating that AHMs provide a favorable in vivo microenvironment for cell culture, we hypothesized that 3D-cultured cells could better mimic tissue infection conditions than 2D cultures, thereby enabling the establishment of a 3D infection model. We specifically investigated CVB3-eGFP infection in HeLa cells that were cultured in AHMs. Prior to infection, HeLa cells in good condition were encapsulated in 1.0% alginate AHMs using a microfluidic platform. After cell stabilization and complete gelation, the culture medium was replaced. After 12 h of static culture, CVB3-eGFP was added to initiate the infection. Infection progression was monitored using fluorescence microscopy at 12, 24, 36, 48, and 60 h post-infection (Fig. 5 A). The results showed that the cells encapsulated in the AHMs exhibited good morphology and proliferated over time within the microspheres. Although viral infection increased slightly, eGFP expression levels remained low, indicating partial inhibition of infection. We speculate that the dense structure of AHMs may hinder interactions between viral particles and cell surface receptors, thereby impeding viral adsorption and entry into host cells. Thus, while AHMs provide a favorable environment for cell growth, they may pose a barrier to viral infection, particularly during the viral adsorption stage of infection. To address this issue, we further optimized the AHMs system by testing a gradient of alginate concentrations. In addition to the original 1.0% alginate AHMs, we prepared 0.8%, 0.6%, and 0.4% alginate AHMs to encapsulate actively proliferating HeLa cells for comparison. The results showed that 1.0%, 0.8%, and 0.6% alginate AHMs maintained good structural integrity and supported cell proliferation, which improved with decreasing alginate concentration. However, the reduced alginate content also weakened crosslinking, resulting in softer microspheres prone to swelling and rupture, which is consistent with our expectations. Notably, 0.4% alginate AHMs were too fragile to withstand demulsification and handling, showing structural failure within 12 h post-encapsulation (Supplement Fig S4), and were therefore excluded from further studies. For viral infection experiments, HeLa cells encapsulated in 0.8% and 0.6% alginate AHMs were subjected to the same infection protocol. The results demonstrated improved viral infection efficiency, confirming that alginate concentration optimization is a viable strategy for reducing the physical barriers to infection (Fig. 5 B-C). Western blotting of cell lysates further confirmed that the infection efficiency increased with decreasing alginate concentration, indicating that a lower matrix density enhanced viral accessibility while still supporting cell growth (Fig. 5 D-F). In conclusion, by modulating the alginate concentration of AHMs, we achieved a better balance between simulating in vivo -like cell growth and facilitating viral infection. This approach enables the development of a more robust and physiologically relevant 3D HeLa cell model for studying CVB3-eGFP infections. Conclusion In summary, we developed an integrated microfluidic platform for the efficient generation, demulsification, and 3D culture of AHMs encapsulating HeLa cells. This system enables the production of highly uniform microspheres under controlled flow conditions and preserves the interactions between cells with the extracellular matrix, which provides a biocompatible microenvironment conducive to cell proliferation and viral infection studies. By optimizing the alginate concentration, we achieved a balance between mimicking the in vivo cellular environment and facilitating viral accessibility, thereby establishing a robust 3D model for CVB3-eGFP infection. Our results demonstrate that lower alginate concentrations enhance viral infection efficiency while maintaining structural integrity and supporting cell growth. This platform not only offers a reliable tool for studying virus–host interactions in a physiologically relevant context, but also holds great potential for applications in antiviral drug screening, tissue engineering, and infectious disease modeling. Future work will focus on further refining material properties and integrating multi-omics approaches to enhance the functionality and applicability of the system. Declarations CRediT authorship contribution statement Tianyi Zhang: Writing – original draft, Writing – review & editing, Visualization, Validation, Supervision, Software, Methodology. Yuqing Xu: Writing – original draft, Writing – review & editing, Methodology, Software. Yiwei Sun: Writing – review & editing, Software, Methodology. Kun Ye: Writing – review & editing, Software, Methodology. Jiarong Liu: Writing – review & editing, Software. Rui Zhang: Writing – review & editing, Software. Sirong Yu: Writing – review & editing, Software. Ziqiao Wang: Writing – review & editing, Software. Min Li: Writing – review & editing, Project administration, Funding acquisition. Hua Wang: Writing – review & editing. Hongxing Shen: Writing – review & editing. Xiaoxiang Zhou: Writing – review & editing, Visualization, Validation, Supervision, Software, Methodology, Project administration, Funding acquisition, Conceptualization. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Natural Science Foundation of Jiangsu Province (BK20230527), China Postdoctoral Science Foundation (2023M731366), Jiangsu Commission of Prevention medical research projects (Ym2023094). References R.M. Meganck, R.S. Baric, Developing therapeutic approaches for twenty-first-century emerging infectious viral diseases, Nat Med, 27 (2021) 401-410. D. Wang, B. Hu, C. Hu, F. Zhu, X. Liu, J. Zhang, B. Wang, H. Xiang, Z. Cheng, Y. Xiong, Y. Zhao, Y. Li, X. Wang, Z. 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Supplementary Files supplementaryfile.docx GraphicalAbstracts.png Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Microchimica Acta → Version 1 posted Editorial decision: Revision requested 19 Dec, 2025 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviews received at journal 15 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 30 Sep, 2025 Reviewers invited by journal 29 Sep, 2025 Editor assigned by journal 23 Sep, 2025 Submission checks completed at journal 23 Sep, 2025 First submitted to journal 19 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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14:27:57","extension":"html","order_by":32,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106422,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/b635f287efbc5c3c51b3a84f.html"},{"id":93339805,"identity":"ee25d114-7b6c-4edd-bb09-5cefb10826ee","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":167837,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of our study, which consists of droplet generation, droplet demulsification, alginate microsphere capture and 3D cell culture for virus infection. Images of our device and the channels of the microfluidic chip are vividly shown. Cell growth and infection were observed by microscopy. The bright-field and fluorescence photos are clearly demonstrated.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/94a9a989f452f16628eb0161.png"},{"id":93339806,"identity":"b3472d0e-8636-41f9-82d3-14936fd672ba","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470921,"visible":true,"origin":"","legend":"\u003cp\u003eGeneration of alginate droplets in our integrated microfluidic device. (A–E) Images of alginate droplets; the scale bar is 100 μm. Corresponding size distribution of the monodisperse alginate droplets in the presence of oil at (A) Foil/Falg =5, (B) Foil/Falg =15, (C) Foil/Falg =25, (D) Foil/Falg =35, (E) Foil/Falg =45. Falg =20 μL h⁻¹ (100 alginate droplets were counted, which were used to calculate their average diameters). (F) Varying alginate droplet size as a function of the flow rate ratio Foil/Falg. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"FIG02.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/21e331aa0f93775bad330c18.png"},{"id":93342671,"identity":"46e0cf2a-a720-4a3e-96ef-f08ba0df124c","added_by":"auto","created_at":"2025-10-12 14:43:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":436625,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of alginate microspheres after demulsification. (A–E) Images of alginate microspheres; the scale bar is 100 μm. Corresponding size distribution of the monodisperse alginate microspheres at (A) Foil/Falg =5, (B) Foil/Falg =15, (C) Foil/Falg =25, (D) Foil/Falg =35, (E) Foil/Falg =45. Falg =20 μL h⁻¹ (100 alginate droplets were counted, which were used to calculate their average diameters). (F) Varying alginate microsphere size as a function of the flow rate ratio Foil/Falg. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"FIG03.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/159753e02073469b9abacd08.png"},{"id":93339815,"identity":"6a5235e4-7b0d-464c-9edf-158d4b87241a","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":290148,"visible":true,"origin":"","legend":"\u003cp\u003eCell growth in captured AHMs. (A–E) Fluorescence images of HeLa cells in a 3D culture. Green fluorescence indicates live cells, and red fluorescence indicates dead cells. Scale bar = 100 μm.\u003c/p\u003e","description":"","filename":"FIG04.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/10a86402a95ec8ea30622231.png"},{"id":93339820,"identity":"c74798e1-3b48-41aa-b2a7-36589a090ea4","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":559293,"visible":true,"origin":"","legend":"\u003cp\u003eCVB3-eGFP infection of HeLa cells captured in AHMs. (A–C) Images of CVB3-eGFP infection to HeLa cells captured in AHMs; scale bar = 100 μm. (A) Concentration of alginate = 1.0%, (B) Concentration of alginate = 0.8%, (C) Concentration of alginate = 0.6%. (D-F) Representative immunoblot of CVB3-eGFP infected cell lysates infected at the indicated time points. Twenty micrograms of total lysate were separated on a 12% SDS-PAGE gel. (D) Alginate concentration = 1.0 %, (E) Alginate concentration = 0.8 %, (F) Alginate concentration = 0.6%.\u003c/p\u003e","description":"","filename":"FIG05.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/35fc93af25fa930f41761938.png"},{"id":102785482,"identity":"13a84bc9-d578-4304-ae18-7afa89ea8cdc","added_by":"auto","created_at":"2026-02-16 16:07:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2565437,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/95cebd5f-5708-4462-b4db-a8644c01b565.pdf"},{"id":93339807,"identity":"8891de35-7d7b-42f3-8fd9-1c1d6c9817e9","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1161667,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/a6d66fcd855ff0169d6906bb.docx"},{"id":93339809,"identity":"6fdd323c-50c4-4cb7-83a4-657be02ce6b6","added_by":"auto","created_at":"2025-10-12 14:27:56","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":504304,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstracts.png","url":"https://assets-eu.researchsquare.com/files/rs-7659713/v1/207cb179c9fe1e54e65f8fab.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection","fulltext":[{"header":"Introduction","content":"\u003cp\u003eViruses with high transmissibility and pathogenicity pose a substantial threat to public health. In recent decades, multiple viral pandemics have occurred worldwide. Pathogens such as the influenza virus, Ebola virus, and coronaviruses exert profound impacts on socioeconomic stability and human health [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. \u003cem\u003eEnterovirus\u003c/em\u003e, which belongs to the family \u003cem\u003ePicornaviridae\u003c/em\u003e, primarily include 10 Enterovirus species (EV A\u0026ndash;J) and three Rhinovirus species (RV A\u0026ndash;C) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Among them, Coxsackievirus B3 (CVB3), a non-enveloped, single-stranded, positive-sense RNA virus, is the primary pathogen responsible for myocarditis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, the persistence of CVB3-RNA residuing in tissues can lead to sustained immune responses and the production of autoantibodies, thereby inducing the progression of chronic disease [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Consequently, CVB3 infections have received increasing attention.\u003c/p\u003e\u003cp\u003eUnderstanding the viral infection process in host cells is a crucial step in virology research, which is of great significance for the development of antiviral drugs and formulation of therapeutic strategies. Establishing appropriate viral infection models is fundamental for elucidating virus-host interactions. Conventional infection models have been established in vitro using cell culture [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, two-dimensional (2D) cell culture methods can only provide data under simplified monolayer conditions, which fail to recapitulate the actual \u003cem\u003ein vivo\u003c/em\u003e infection processes. Moreover, these approaches are relatively tedious and yield results of limited biological significance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Animal models (for \u003cem\u003ein vivo\u003c/em\u003e studies) can also be used. However, this may raise ethical concerns. Additionally, other obstacles exist, such as high costs and prolonged experimental duration [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEncapsulating cells within hydrogel microspheres for culture presents a straightforward and readily implementable novel cultivation method [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a method for three-dimensional (3D) cell culture, this system provides an environment that closely mimics \u003cem\u003ein vivo\u003c/em\u003e conditions, allowing for more direct observation of cellular behavior and responses to experimental treatments [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Consequently, it has gained increasing application in regenerative medicine, drug delivery, and tissue engineering [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Among these materials, alginate hydrogel microspheres (AHMs) have attracted increasing attention. Alginate is characterized by excellent biocompatibility, low cost, safety, and biodegradability, making it an ideal coating material for simulating an \u003cem\u003ein vivo\u003c/em\u003e microenvironment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe preparation of relatively uniform AHMs primarily involves three methods: extrusion, electrostatic dripping, and droplet-based microfluidics [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Extrusion is the most classical approach, whereby an alginate solution is dripped into a gelling bath containing Ca\u0026sup2;⁺ ions, with manual control over the formation of AHMs droplets. Although simple and controllable, this method often results in AHMs with broad size distributions and relatively rough surfaces. Electrostatic dripping can effectively regulate the size of AHMs, yielding highly uniform microspheres. However, this technique is predominantly applied in drug encapsulation and has limited applications in cell culture [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The use of microfluidic chips to prepare monodisperse droplet-based microspheres has garnered considerable attention. By leveraging chip architecture and fluid flow control, this technique enables the high-throughput and rapid production of uniformly sized microspheres, offering operational simplicity, high efficiency, and excellent controllability [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The process mainly consists of two key steps: shearing of the aqueous phase by an oil phase to form uniformly sized gel microspheres, and subsequent demulsification to separate the microspheres from the oil medium. Studies have demonstrated that microfluidic technology exhibits significant advantages in generating monodisperse AHMs, representing an advanced method characterized by high throughput, stability, and automation [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePreviously, we developed a flexible and tunable microfluidic platform capable of generating and isolating AHMs to provide a stable microenvironment for bacterial growth and enable real-time monitoring of bacterial proliferation within the microspheres [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Building on this foundation, we optimized the chip architecture to better accommodate cell encapsulation within AHMs, establishing a robust 3D cell culture model for further applications. In summary, we constructed a microfluidic platform for generating highly uniform AHM-based cell culture microspheres, investigated cellular growth under 3D culture conditions, and conducted preliminary applications using AHM-encapsulated cells in viral infection studies under 3D settings.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e\u003cp\u003eSodium alginate (90.0%), ethylene diamine-N, N'-diacetic acid (EDDA, 99%), ethylene diamine tetraacetic acid (EDTA, 99%), 3-(N-morpholino)-propane sulfonic acid (MOPS), CaCl\u003csub\u003e2\u003c/sub\u003e, and zinc acetate were purchased from Al. Ltd. (China). Latex beads (sulfate-modified polystyrene, fluorescent red) were purchased from Sigma-Aldrich (USA). 1H,1H,2H,2H-perfluoro-1-octanol (PFO) was purchased from Alfa Aesar (USA). All the reagents were used as received. Deionized water was used in all experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 One-step generation of microspheres\u003c/h2\u003e\u003cp\u003eAHMs were produced via internal gelation using a complex ligand-exchange crosslinking method [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The crosslinking ion solution contained 1% sodium alginate, 30 mM Ca\u0026sup2;⁺ chelated with 30 mM EDTA, and 14 mM MOPS buffer (pH 7). The exchange ion solution contained 1% sodium alginate, 30 mM zinc acetate, 30 mM EDDA, and 14 mM MOPS buffer (pH 7). Red fluorescent polystyrene microspheres (0.1 \u0026micro;m, 0.1% v/v) were added to visualize the microspheres. The continuous phase was fluorocarbon oil (HFE 7500) with 3% (v/v) fluorosurfactants.\u003c/p\u003e\u003cp\u003eThe flow rates of the continuous phase were varied (10, 300, 500, 700, and 900 \u0026micro;L h⁻\u0026sup1;), while that of the dispersed phase was fixed at 20 \u0026micro;L h⁻\u0026sup1;. The solutions were infused using a syringe-pump. The droplets were broken using 10% (v/v) perfluorooctanol (PFO). The size and coefficient of variation (CV) of the microspheres were measured using the ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Calcein/PI staining assay in HeLa cells\u003c/h2\u003e\u003cp\u003eThe Calcein/PI Kit (Beyotime Biotechnology, China) was used to assess cell viability and cytotoxicity. First, HeLa cells captured by AHMs were cultivated for 12h. Upon completion, the cells were stained with the Calcein/PI Kit. The culture medium was aspirated, and the cells were gently washed once with phosphate-buffered saline (PBS). According to the manufacturer\u0026rsquo;s instructions, the calcein/PI detection working solution was prepared and added. The cells were incubated at 37\u0026deg;C in the dark for 30 min. After incubation, cell viability and cytotoxicity were assessed by observing fluorescence under a fluorescence microscope (Axio Vert A1, ZEISS, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Virus infection\u003c/h2\u003e\u003cp\u003eFor CVB3-eGFP construction, eGFP, a reporter protein, was encoded in the viral genome after the start codon of the viral VP4 protein. Due to the presence of a protease cleavage site, eGFP and VP4 are expressed as single proteins and can be detected. In the infection studies of HeLa cells, the cells were plated in a six-well culture plate, and 1.5\u0026times;106 plaque-forming units (PFU) of CVB3-eGFP were applied per well. After an incubation period of 1 h, the virus inoculum was replaced with fresh DMEM.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Western blot analysis\u003c/h2\u003e\u003cp\u003eThe cells were lysed in RIPA buffer (Beyotime Biotechnology, China) supplemented with protease inhibitor cocktails (Beyotime Biotechnology, China), and 20 \u0026micro;g of total protein was subjected to 10% SDS polyacrylamide gel electrophoresis. The samples were transferred to PVDF Western Blotting membranes and blotted with anti-VP1 (GeneTex, USA) and GAPDH (GeneTex, USA) antibodies. Horseradish peroxidase (HRP)-conjugated AffiniPure goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Cell Signaling, USA) was used as a secondary antibody. The membranes were analyzed using a Western Lumax LightTM Superior (ZETA) with an enhanced chemiluminescence (ECL) imager.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Performance of the integrated microfluidic system\u003c/h2\u003e\u003cp\u003eIn our platform, we employed droplet-based microfluidics to generate AHMs. HeLa cells were encapsulated in a gel-microsphere system. A dispersed phase consisting of 1% sodium alginate complexed with Ca\u0026ndash;EDTA and 1% sodium alginate complexed with Zn\u0026ndash;EDDA was mixed with the pre-treated HeLa cell suspension, forming aqueous phases that were injected into channels A and B of the chip. Simultaneously, fluorocarbon oil containing 3% surfactant was introduced as the continuous phase through channel C.\u003c/p\u003e\u003cp\u003eThe aqueous phases in channels A and B were infused at the same low flow rate. Under stable conditions, the two streams merged at the junction to form a cross-linked structure, which was sheared by the high-speed oil flow from Channel C. The resulting microspheres were propelled by the oil phase into the collection channel. After confirming stable microsphere generation using microscopy, the collected oil-encapsulated gel microspheres were demulsified.\u003c/p\u003e\u003cp\u003eA medium containing 10% PFO was used as the demulsifier for the water-in-oil-in. In the presence of PFO, the microspheres rapidly separated from the oil phase. To minimize the potential adverse effects on cell viability caused by prolonged exposure to the demulsifier, we ensured thorough yet rapid contact between the demulsifier and microspheres after removing most of the oil phase. Finally, the demulsifier was removed, and the microspheres were repeatedly washed and resuspended in cell culture medium. The fundamental principles and setup of the experiment are illustrated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Generation of AHMs\u003c/h2\u003e\u003cp\u003eOwing to the chip architecture and shear force provided by the oil phase, alginate microspheres with a uniform size distribution were successfully formed at the junction where the oil and aqueous phases converged. To further validate the reliability of our chip design and the stability of the flow rate control, we investigated the effect of different flow rate ratios between the continuous (oil) and dispersed (aqueous) phases on the size of the generated microspheres.\u003c/p\u003e\u003cp\u003eThe flow rate of the aqueous phase was maintained at a relatively low value of 20 \u0026micro;L/h, whereas the flow rate of the oil phase was adjusted to regulate droplet size. When the flow rate ratio of the aqueous phase to the oil phase was set to 1:5, spherical droplets were stably generated with a relatively larger average diameter of 109.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 \u0026micro;m and a coefficient of variation (CV) of 3.0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). When the oil flow rate was increased to 300 \u0026micro;L/h (resulting in a ratio of 1:15), the microsphere size decreased significantly, yielding an average diameter of 88.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 \u0026micro;m and a CV of 3.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Subsequently, the oil flow rate was incrementally increased at equal intervals of time. The results showed only minor changes in size, consistent with previous reports. At a ratio of 1:25, the average diameter was 80.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.2 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;5.3%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC); at 1:35, it was 75.3\u0026thinsp;\u0026plusmn;\u0026thinsp;2.9 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;3.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD); and at 1:45, the average diameter was 74.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;4.0%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eThese results demonstrate that droplets can be stably generated across various flow rate ratios, with all CV values remaining relatively low (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), confirming the stability and reliability of our microfluidic chip design and platform operation. Furthermore, as the oil flow rate increased, the enhanced shear force led to a consistent reduction in the size of the AHMs, which aligned with previously published studies and met our expectations [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Characterization of AHMs\u003c/h2\u003e\u003cp\u003eTo evaluate the size and structural strength of the AHMs for practical cell culture applications, we demulsified the droplets generated by the microfluidic chip and analyzed the size and coefficient of variation (CV) of the resulting microspheres. The results showed that at a flow rate ratio of Falg/Foil\u0026thinsp;=\u0026thinsp;1:5, the microspheres exhibited the largest average diameter of 145.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 \u0026micro;m with a CV of 2.9% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). When the ratio was increased to 1:15, the average diameter decreased to 119.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4.3 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;3.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At Falg/Foil\u0026thinsp;=\u0026thinsp;1:25, the average diameter was 111.3\u0026thinsp;\u0026plusmn;\u0026thinsp;3.5 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;3.2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC); at 1:35, it was 109.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;3.0%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD); and at 1:45, the average diameter was 108.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.9 \u0026micro;m (CV\u0026thinsp;=\u0026thinsp;3.6%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The influence of different Falg/Foil ratios on the microsphere size is summarized in the corresponding figure.\u003c/p\u003e\u003cp\u003eCompared to emulsified droplets, demulsified AHMs exhibited an overall increase in size (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This swelling behavior was consistent with expectations and can be attributed to the hydrophilic hydroxyl groups in alginate, which enhance the affinity for water molecules in a dispersed environment. Excessively large microspheres not only exhibit lower production efficiency but also, according to the Poisson distribution, are less conducive to effective cell encapsulation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, oversized microspheres may impede efficient nutrient and metabolite exchange during culture [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Based on these considerations, we selected the microspheres generated at Falg/Foil\u0026thinsp;=\u0026thinsp;1:15 as the model for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Encapsulation and culture of HeLa cells in AHMs\u003c/h2\u003e\u003cp\u003eWe further explored the feasibility of 3D culture of HeLa cells using AHMs. To demonstrate the viability of the 3D cell culture, we performed bright-field microscopic observations at 12-hour intervals. As shown in the corresponding figure, cell growth under 3D culture conditions was monitored at 12, 24, 36, 48, and 60 h to confirm that the cells within the AHMs maintained active division and proliferation. Optical images revealed that small groups and clusters of cells continuously emerged within the AHMs with prolonged culture periods (Supplement Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The 3D structure of AHMs, which encapsulates cells in isolated compartments, allows efficient exchange of nutrients and metabolites. By 48 h, the HeLa cells showed robust growth in the AHMs. After 48 h, a small number of cells began to grow outside the microspheres.\u003c/p\u003e\u003cp\u003eFurthermore, we compared the differences between the 3D and conventional 2D cultures. Cells in 2D culture exhibited typical spread morphology, whereas those encapsulated in AHMs presented various forms, including single cells, small groups, and clusters (Supplementary Fig. S2). To confirm that the encapsulation process and AHMs culture did not significantly affect cell viability, we used a Calcein-AM/PI staining kit to assess cell survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Fluorescence images indicated that HeLa cells grew well within the AHMs over a three-day culture period. The viability of the encapsulated cells was evaluated by measuring the area of green fluorescence (live cells) and red fluorescence (dead cells) at the focal planes using ImageJ software. Viability was determined by dividing the area of green fluorescence by the total area of green and red fluorescence. Quantitative analysis of cells cultured for 1\u0026ndash;3 days showed viability ranging from 95% to 97%, demonstrating that AHMs provide a feasible environment for cell culture.\u003c/p\u003e\u003cp\u003eIn summary, the AHM-based culture model established in this study offers a versatile 3D culture tool for microorganisms and cells. The high cytocompatibility of alginate hydrogels provides suitable sites for cell-matrix integration, thereby better simulating \u003cem\u003ein vivo\u003c/em\u003e cell division and proliferation than gelatin hydrogels.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Establishment of a CVB3-eGFP infection model in HeLa cell cultured in AHMs\u003c/h2\u003e\u003cp\u003eWe further explored the application of AHM-encapsulated HeLa cells in viral infection research. Initially, we characterized the infection of monolayer-cultured HeLa cells with CVB3-eGFP. As expected, infection of 2D-cultured HeLa cells in 12-well plates with CVB3-eGFP (1000 TCID₅₀) resulted in a time-dependent increase in eGFP-positive cells, plateauing at 48 h post-infection. These preliminary experiments confirmed the effectiveness of CVB3-eGFP in HeLa cells (Supplementary Fig. S3).\u003c/p\u003e\u003cp\u003eBased on previous results demonstrating that AHMs provide a favorable \u003cem\u003ein vivo\u003c/em\u003e microenvironment for cell culture, we hypothesized that 3D-cultured cells could better mimic tissue infection conditions than 2D cultures, thereby enabling the establishment of a 3D infection model. We specifically investigated CVB3-eGFP infection in HeLa cells that were cultured in AHMs. Prior to infection, HeLa cells in good condition were encapsulated in 1.0% alginate AHMs using a microfluidic platform. After cell stabilization and complete gelation, the culture medium was replaced. After 12 h of static culture, CVB3-eGFP was added to initiate the infection. Infection progression was monitored using fluorescence microscopy at 12, 24, 36, 48, and 60 h post-infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe results showed that the cells encapsulated in the AHMs exhibited good morphology and proliferated over time within the microspheres. Although viral infection increased slightly, eGFP expression levels remained low, indicating partial inhibition of infection. We speculate that the dense structure of AHMs may hinder interactions between viral particles and cell surface receptors, thereby impeding viral adsorption and entry into host cells. Thus, while AHMs provide a favorable environment for cell growth, they may pose a barrier to viral infection, particularly during the viral adsorption stage of infection.\u003c/p\u003e\u003cp\u003eTo address this issue, we further optimized the AHMs system by testing a gradient of alginate concentrations. In addition to the original 1.0% alginate AHMs, we prepared 0.8%, 0.6%, and 0.4% alginate AHMs to encapsulate actively proliferating HeLa cells for comparison. The results showed that 1.0%, 0.8%, and 0.6% alginate AHMs maintained good structural integrity and supported cell proliferation, which improved with decreasing alginate concentration. However, the reduced alginate content also weakened crosslinking, resulting in softer microspheres prone to swelling and rupture, which is consistent with our expectations. Notably, 0.4% alginate AHMs were too fragile to withstand demulsification and handling, showing structural failure within 12 h post-encapsulation (Supplement Fig S4), and were therefore excluded from further studies.\u003c/p\u003e\u003cp\u003eFor viral infection experiments, HeLa cells encapsulated in 0.8% and 0.6% alginate AHMs were subjected to the same infection protocol. The results demonstrated improved viral infection efficiency, confirming that alginate concentration optimization is a viable strategy for reducing the physical barriers to infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Western blotting of cell lysates further confirmed that the infection efficiency increased with decreasing alginate concentration, indicating that a lower matrix density enhanced viral accessibility while still supporting cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F).\u003c/p\u003e\u003cp\u003eIn conclusion, by modulating the alginate concentration of AHMs, we achieved a better balance between simulating \u003cem\u003ein vivo\u003c/em\u003e-like cell growth and facilitating viral infection. This approach enables the development of a more robust and physiologically relevant 3D HeLa cell model for studying CVB3-eGFP infections.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we developed an integrated microfluidic platform for the efficient generation, demulsification, and 3D culture of AHMs encapsulating HeLa cells. This system enables the production of highly uniform microspheres under controlled flow conditions and preserves the interactions between cells with the extracellular matrix, which provides a biocompatible microenvironment conducive to cell proliferation and viral infection studies. By optimizing the alginate concentration, we achieved a balance between mimicking the \u003cem\u003ein vivo\u003c/em\u003e cellular environment and facilitating viral accessibility, thereby establishing a robust 3D model for CVB3-eGFP infection. Our results demonstrate that lower alginate concentrations enhance viral infection efficiency while maintaining structural integrity and supporting cell growth. This platform not only offers a reliable tool for studying virus\u0026ndash;host interactions in a physiologically relevant context, but also holds great potential for applications in antiviral drug screening, tissue engineering, and infectious disease modeling. Future work will focus on further refining material properties and integrating multi-omics approaches to enhance the functionality and applicability of the system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTianyi Zhang: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Software, Methodology. Yuqing Xu: Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing, Methodology, Software. Yiwei Sun: Writing \u0026ndash; review \u0026amp; editing, Software, Methodology. Kun Ye: Writing \u0026ndash; review \u0026amp; editing, Software, Methodology. Jiarong Liu: Writing \u0026ndash; review \u0026amp; editing, Software. Rui Zhang: Writing \u0026ndash; review \u0026amp; editing, Software. Sirong Yu: Writing \u0026ndash; review \u0026amp; editing, Software. Ziqiao Wang: Writing \u0026ndash; review \u0026amp; editing, Software. Min Li: Writing \u0026ndash; review \u0026amp; editing, Project administration, Funding acquisition. Hua Wang: Writing \u0026ndash; review \u0026amp; editing. Hongxing Shen: Writing \u0026ndash; review \u0026amp; editing. Xiaoxiang Zhou: Writing \u0026ndash; review \u0026amp; editing, Visualization, Validation, Supervision, Software, Methodology, Project administration, Funding acquisition, Conceptualization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Natural Science Foundation of Jiangsu Province (BK20230527), China Postdoctoral Science Foundation (2023M731366), Jiangsu Commission of Prevention medical research projects (Ym2023094).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eR.M. Meganck, R.S. 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Lin, Multifunctional Regulation of 3D Cell-Laden Microsphere Culture on an Integrated Microfluidic Device, Anal Chem, 91 (2019) 12283-12289.\u003c/li\u003e\n \u003cli\u003eB. Namgung, K. Ravi, P.P. Vikraman, S. Sengupta, H.L. Jang, Engineered cell-laden alginate microparticles for 3D culture, Biochem Soc Trans, 49 (2021) 761-773.\u003c/li\u003e\n \u003cli\u003eM. Pires-Santos, S. Nadine, J.F. Mano, Unveiling the Potential of Single-Cell Encapsulation in Biomedical Applications: Current Advances and Future Perspectives, Small Sci, 4 (2024) 2300332.\u003c/li\u003e\n \u003cli\u003eC.C. Ahrens, Z. Dong, W. Li, Engineering cell aggregates through incorporated polymeric microparticles, Acta Biomaterialia, 62 (2017) 64-81.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Microfluidic chip, Alginate hydrogel microspheres, 3D cell culture, CVB3, Host–pathogen interaction","lastPublishedDoi":"10.21203/rs.3.rs-7659713/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7659713/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eViral organisms characterized by elevated transmissibility and pathogenicity constitute a substantial public health risk. Three-dimensional (3D) cell culture systems better mimic the \u003cem\u003ein vivo\u003c/em\u003e microenvironment than traditional two-dimensional (2D) models, offering significant potential for virological research. Therefore, a method for natural viral growth based on 3D cell culture need be developed. In this study, we developed an integrated microfluidic platform for the efficient generation of highly uniform alginate hydrogel microspheres (AHMs) encapsulating HeLa cells, enabling robust 3D cell culture and subsequent infection with Coxsackievirus B3 expressing enhanced green fluorescent protein (CVB3-eGFP). Our results demonstrate that AHMs support high cell viability and facilitated cell proliferation within a biomimetic 3D matrix. By systematically reducing the alginate concentration from 1.0% to 0.6%, we enhanced viral accessibility while maintaining microstructural integrity, thereby significantly improving CVB3-eGFP infection rates, as confirmed by fluorescence imaging and western blot analysis. This study establishes a tunable, reproducible, and physiologically relevant 3D model for studying virus\u0026ndash;host interactions, with broad applications in antiviral drug screening and infectious disease modeling.\u003c/p\u003e","manuscriptTitle":"3D alginate hydrogel microspheres with uniform micro-structure for cell culture and CVB3 infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-12 14:27:51","doi":"10.21203/rs.3.rs-7659713/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-19T10:35:41+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-17T04:50:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242292203149515162909021242468049846391","date":"2025-12-11T02:57:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-15T11:40:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84054468412053570450738408201584683409","date":"2025-10-02T08:23:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"274371210643685382989119842733413597184","date":"2025-09-30T07:02:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-29T17:14:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-24T00:42:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T00:41:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microchimica Acta","date":"2025-09-19T14:46:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microchimica-acta","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"miac","sideBox":"Learn more about [Microchimica Acta](https://link.springer.com/journal/604)","snPcode":"604","submissionUrl":"https://submission.springernature.com/new-submission/604/3","title":"Microchimica Acta","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c8b19926-1708-4604-a01f-db80c2045503","owner":[],"postedDate":"October 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:04:35+00:00","versionOfRecord":{"articleIdentity":"rs-7659713","link":"https://doi.org/10.1007/s00604-026-07865-3","journal":{"identity":"microchimica-acta","isVorOnly":false,"title":"Microchimica Acta"},"publishedOn":"2026-02-10 15:58:57","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-10-12 14:27:51","video":"","vorDoi":"10.1007/s00604-026-07865-3","vorDoiUrl":"https://doi.org/10.1007/s00604-026-07865-3","workflowStages":[]},"version":"v1","identity":"rs-7659713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7659713","identity":"rs-7659713","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}