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. 2025 Jul 28;10(32):36267–36275. doi: 10.1021/acsomega.5c04198

Hyaluronic Acid Microparticles Promote Uniform Differentiation and Survival of Stem Cells in Spheroid Cultures

Hye-Eun Shim †,‡, Yong-Jin Kim §, Kyoung Hwan Park ‡, Jean Choi ∥, Yu Bin Lee ⊥,#, Kang Moo Huh ‡,*, Sun-Woong Kang †,#,*
PMCID: PMC12368670  PMID: 40852269

Abstract

Three-dimensional (3D) spheroid cultures offer a physiologically relevant environment for stem cell differentiation but face challenges with oxygen and nutrient delivery, leading to uneven differentiation and apoptosis in larger spheroids. This study addresses these limitations by incorporating hyaluronic acid (HA) microparticles into AdMSC spheroids. HA microparticles, synthesized via an inverse emulsion method, were confirmed to be cross-linked and porous, enhancing diffusion and microenvironmental support. Spheroids containing HA microparticles showed significantly improved cell viability and reduced apoptosis, evidenced by TUNEL staining and the upregulation of BCL2, alongside the downregulation of Caspase3 and Caspase7. Enhanced chondrogenic and adipogenic differentiation was confirmed through histological staining, immunohistochemistry, and gene expression analysis, with the 30% HA group demonstrating the most uniform and robust differentiation. These findings highlight HA microparticles as an effective tool for overcoming diffusion limitations in spheroid cultures, enabling uniform differentiation and improved cell survival. This approach holds promise for regenerative medicine applications such as cartilage repair, adipose tissue engineering, and advanced tissue modeling.

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1. Introduction

Regenerative medicine has gathered significant interest due to its potential to repair or replace damaged tissues and organs. Among the various strategies employed, stem cell therapy stands out for its ability to differentiate into multiple cell types, offering a promising cell source for tissue engineering and regenerative applications. However, achieving efficient differentiation and functional integration of stem cells remains a critical challenge. Traditional two-dimensional (2D) cell cultures fail to accurately mimic the in vivo cellular environment, often leading to suboptimal cell survival and differentiation outcomes.

Three-dimensional (3D) cell culture systems have emerged as a superior alternative, providing a more physiologically relevant environment that promotes cell–cell interactions and better mimics the extracellular matrix (ECM) of native tissues. , Spheroid cultures, a type of 3D cell culture, offer numerous advantages, including enhanced cell viability, improved differentiation potential, and the ability to be used in minimally invasive transplantation techniques. Despite these benefits, one of the major limitations of spheroid cultures is the efficient delivery of differentiation factors to the innermost cells, which can hinder uniform differentiation and overall functionality.

The development of effective methods for the differentiation of stem cells into specific cell types such as chondrocytes and adipocytes are crucial for advancing regenerative therapies. Chondrocytes are essential for cartilage repair and regeneration, while adipocytes are important for reconstructive surgery and metabolic studies. , Enhancing the differentiation efficiency of stem cells into these cell types can significantly impact the treatment of degenerative diseases and injuries. In particular, differentiation of stem cells into chondrocytes and adipocytes within 3D culture systems has significant therapeutic relevance. Chondrogenic differentiation is essential for cartilage repair in degenerative diseases such as osteoarthritis, traumatic joint injuries, and congenital cartilage defects. Current cartilage repair strategies are often limited by poor regenerative capacity of native chondrocytes, making stem cell-derived chondrocytes a promising alternative. Similarly, adipogenic differentiation has wide applications in reconstructive surgery, soft tissue augmentation, and metabolic disease modeling. Adipose tissue engineering also holds great potential for treating soft tissue defects resulting from trauma, tumor resection, or congenital anomalies. Therefore, establishing efficient and uniform differentiation into these lineages within 3D systems is highly desirable for regenerative medicine and translational applications.

Hyaluronic acid (HA), a naturally occurring glycosaminoglycan, has been extensively studied for its biocompatibility, biodegradability, and ability to interact with a variety of cell surface receptors. , HA hydrogels and microparticles have shown promise in enhancing cell survival, proliferation, and differentiation by providing a supportive microenvironment that facilitates nutrient and signal molecule exchange. The incorporation of HA microparticles into 3D spheroid cultures represents a novel approach to overcoming the diffusion limitations of conventional spheroid systems. ,

In this study, we investigate the use of HA microparticles to enhance the differentiation potential of stem cells in 3D spheroid cultures. Our in vitro experiments demonstrate that HA microparticles significantly improve the differentiation of stem cells into chondrocytes and adipocytes, ensuring uniform differentiation throughout the spheroid structure. Additionally, the presence of HA microparticles was associated with a reduction in the expression of apoptosis-related genes, indicating improved cell survival. These findings suggest that HA microparticles can effectively address some of the critical challenges in stem cell-based tissue engineering, paving the way for improved regenerative therapies.

2. Materials and Methods

2.1. Preparation and Characterization of HA Microparticles

HA microparticles were synthesized using an inverse emulsion method. Briefly, hyaluronic acid (HA) with a molecular weight of 1,500 kDa (Hyundai Bioland, Cheongju, South Korea and Durae, Anyang, South Korea) was dissolved in 0.1 N NaOH and cross-linked with poly­(ethylene glycol) diglycidyl ether (PEGDG; Sigma-Aldrich, USA). The solution was gently stirred at room temperature for 2 h before being emulsified into a heptane-based solution containing ARL P135 (PEG-30 dipolyhydroxystearate; Croda, UK) as the emulsifier. The emulsion was homogenized at 60 °C for 30 min, followed by further stirring at the same temperature for 2 h to complete the cross-linking reaction. Neutralization was achieved with acetic acid, and the reaction was allowed to continue overnight at room temperature. The resulting microparticles were purified through three cycles of washing with a water-acetone mixture (1:3 v/v) and pure acetone, then collected by vacuum filtration and dried at 60 °C for 24 h.

The chemical modifications and cross-linking of HA microparticles were confirmed by Fourier-transform infrared (FT-IR) spectroscopy using a Nicolet iS5 spectrometer (Thermo Fisher Scientific, MA, USA) over a range of 4000–650 cm–1 with a resolution of 4 cm–1. Morphological analysis was conducted with scanning electron microscopy (SEM; JSM6330F, JEOL, Tokyo, Japan). Samples were coated with platinum under an argon atmosphere before imaging at an accelerating voltage of 10.0 kV. Microparticle size and porosity were analyzed before and after swelling in culture medium.

2.2. Cytotoxicity Assessment of HA Microparticles

Adipose-derived mesenchymal stem cells (AdMSCs; ATCC, SCRC-4000) were cultured in MSC Basal Media (ATCC, PCS-500-030) supplemented with an MSC Growth Kit (ATCC, PCS-500-040) under standard conditions (5% CO2, 37 °C). Cells were passaged at 80–90% confluence using Trypsin-EDTA for Primary Cells (ATCC, PCS-999-003) and Trypsin Neutralizing Solution (ATCC, PCS-999-004).

To validate the identity of the AdMSCs, flow cytometry (FCM) analysis was performed to assess the expression of surface markers. Cells were harvested and washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). The cell suspension was then incubated with fluorochrome-conjugated antibodies against specific mesenchymal stem cell (MSC) markers. After incubation with antibodies for 30 min at 4 °C in the dark, cells were washed twice with PBS containing 1% BSA and resuspended in PBS for analysis. FCM was conducted using a BD FACSCalibur flow cytometer (BD Biosciences, USA), and data were analyzed with FlowJo software (BD Biosciences, USA). Positive marker expression was defined as the percentage of cells expressing CD44, CD73, CD90, and CD105, while the absence of CD45 expression confirmed the lack of hematopoietic contamination.

Cytotoxicity of HA microparticles was evaluated using the MTT assay, following ISO 10993-5 guidelines for in vitro cytotoxicity testing. AdMSCs were seeded at a density of 5 × 103 cells per well in 96-well plates, and the medium containing varying concentrations of HA microparticles was replaced every 2 days. Cell viability was determined after exposure to the microparticles, and cytotoxicity was assessed based on the percentage of viable cells.

2.3. Spheroid Formation and Differentiation into Adipocytes and Chondrocytes

Three-dimensional (3D) spheroids were generated by mixing 1 × 105 AdMSCs with 10% (v/v) or 30% (v/v) HA microparticles in 1.5 mL Eppendorf tubes. The mixtures were centrifuged at 1200 rpm for 3 min to facilitate spheroid formation and then cultured for 2 days to stabilize the structures. The schematic in Figure illustrates the overall process of spheroid generation, including the addition of HA microparticles and centrifugation steps, leading to the formation of compact spheroids. For differentiation studies, spheroids were transferred into specific differentiation media. Adipogenic differentiation was induced using lipid differentiation medium (StemPro, Gibco, A1007001) for 2 weeks, while chondrogenic differentiation was carried out using chondrogenic medium (StemPro, Gibco, A1007101) for 3 weeks. Media were refreshed every 2 days during the differentiation periods. After differentiation, the spheroids were harvested and analyzed for viability, histological staining, and gene expression to evaluate their differentiation potential.

3.

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Schematic illustration of AdMSC spheroid formation incorporating HA microparticles. (A) Preswelling of HA microparticles prior to spheroid assembly. (B) Mixing of AdMSCs and HA microparticles for spheroid formation. (C) Centrifugation and culture process to generate stable spheroids.

2.4. Histological Analysis

Spheroids, both with and without HA microparticles, were fixed in 4% paraformaldehyde (PFA) for tissue analysis, and paraffin-embedded or frozen sections were prepared for subsequent staining protocols. Apoptotic cells were identified using the TUNEL assay (In Situ Apoptosis Detection Kit; Cell Signaling Technology, Danvers, MA, USA) following the manufacturer’s instructions. Hematoxylin and eosin (H&E) staining was performed by deparaffinizing sections in xylene, rehydrating them through a graded alcohol series, and staining with hematoxylin for 5 min. The sections were differentiated in 1% acid alcohol, blued in 0.2% ammonia–water, counterstained with eosin for 1–2 min, dehydrated, and mounted. For Alcian Blue staining, deparaffinized sections were immersed in acetic acid for 3 min, stained with Alcian Blue solution (pH 2.5) for 15–30 min, counterstained with Nuclear Fast Red, dehydrated, and mounted. Oil Red O staining was conducted on frozen sections using freshly prepared Oil Red O working solution, where sections were stained for 10 min, rinsed with distilled water, counterstained with hematoxylin, and mounted in aqueous medium.

2.5. Gene Expression Analysis

Total RNA was extracted from spheroids using TRIzol reagent (Invitrogen) and reverse-transcribed into cDNA using the SuperScript II Reverse Transcription Kit (Invitrogen). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green reagents (Roche, Mannheim, Germany) with GAPDH as the internal control. Primer sequences for key chondrogenic and apoptotic markers included: Aggrecan (AGG), Collagen Type II Alpha 1 Chain (COL2A1), SOX9, BCL2, Caspase3, and Caspase7. Gene expression was calculated using the ΔΔCt method, with triplicate samples for each condition.

2.6. Statistical Analysis

Quantitative data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. Results were considered statistically significant for p-values <0.05.

3. Results

3.1. Preparation and Characterization of HA Microparticles

HA microparticles were successfully synthesized using an inverse emulsion method, with poly­(ethylene glycol) diglycidyl ether (PEGDG) as a cross-linking agent for hyaluronic acid (HA) (Figure A,B). Fourier-transform infrared (FT-IR) spectroscopy confirmed the cross-linking process and structural modifications in the microparticles (Figure C). Peaks at 2920, 1088, and 1740 cm–1 corresponded to methylene (−CH2) stretching vibrations, ether (C–O–C) linkages, and carbonyl (CO) groups, respectively. The peak at 2920 cm–1 indicated the incorporation of alkyl chains introduced by PEGDG, while the peak at 1088 cm–1 confirmed the formation of ether bonds. The strong peak at 1740 cm–1 suggested ester bond formation, likely resulting from esterification between HA’s carboxylic acid groups and PEGDG. Comparative analysis of cross-linked and non-cross-linked HA samples verified that these structural changes were specific to the cross-linking process, ensuring the structural stability of the microparticles.

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Synthesis and characterization of HA microparticles. (A) Schematic illustration of HA microparticle synthesis via inverse emulsion cross-linked with PEGDG. (B) Chemical structure of cross-linked HA microparticles showing ether and ester bond formations. (C) FT-IR spectra comparing cross-linked and non-cross-linked HA microparticles. (D) SEM images of HA microparticles before swelling (dry state) and after swelling in culture medium.

Scanning electron microscopy (SEM) revealed spherical microparticles with diameters ranging from 1 to 30 μm before swelling. After immersion in culture medium, the microparticles swelled to a range of 40 to 150 μm, forming porous structures on their surfaces (Figure D). The observed porosity indicated potential pathways for material exchange, suggesting their capacity to support nutrient and oxygen diffusion within biological systems.

3.2. Cytotoxicity of HA Microparticles on AdMSCs

The mesenchymal stem cell (MSC) identity of adipose-derived mesenchymal stem cells (AdMSCs) was confirmed via flow cytometry (FCM), which showed high expression of MSC markers CD44, CD73, CD90, and CD105, and minimal expression of the hematopoietic marker CD45 (Figure A). These results validated the suitability of the cells for spheroid formation and differentiation studies.

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AdMSC characterization and cytotoxicity of HA microparticles. (A) Flow cytometry analysis confirming MSC marker expression in AdMSCs (CD44, CD73, CD90, CD105 positive; CD45 negative). (B) MTT assay demonstrating cell viability of AdMSCs exposed to various concentrations (0%, 10%, and 30% v/v) of HA microparticles.

The biocompatibility of HA microparticles was assessed using an MTT assay, where AdMSCs were exposed to various concentrations of microparticles (Figure B). Cell viability remained consistently above 90% across all concentrations tested, confirming that HA microparticles are noncytotoxic and safe for use in spheroid cultures.

3.3. Spheroid Formation and Enhanced Viability of AdMSC Spheroids Incorporating HA Microparticles

AdMSC spheroids were generated using the pellet culture method in e-tubes (Figure ). Within 24 h, cells aggregated to form compact spheroids irrespective of the presence of HA microparticles. However, spheroid formation with HA microparticles was limited to one-third of the total cell volume; attempts to exceed this capacity were unsuccessful. The resulting spheroids maintained their structural integrity for the duration of the culture period.

The inclusion of HA microparticles significantly enhanced cell viability and reduced apoptosis, as evidenced by TUNEL staining and gene expression analysis. TUNEL staining revealed abundant apoptotic cells in spheroids without HA microparticles, whereas HA-containing spheroids exhibited markedly fewer TUNEL-positive cells, particularly at higher HA concentrations (Figure A). The reduction in apoptosis was most pronounced in the outer regions of these spheroids.

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Cell viability and apoptosis in AdMSC spheroids containing HA microparticles. (A) TUNEL staining of AdMSC spheroids after 2 weeks of culture. Apoptotic cells (green) are indicated by TUNEL-positive staining, and nuclei are counterstained with DAPI (blue). Representative images are shown for Control (without HA microparticles), 10% HA microparticles, and 30% HA microparticles groups. Reduced apoptosis is observed with increasing HA microparticle concentration. (B) Gene expression analysis of apoptosis-related markers. Expression levels of antiapoptotic gene BCL2 and pro-apoptotic genes Caspase3 and Caspase7 are presented for Control, 10% HA, and 30% HA groups. Data are presented as mean ± SD; *p < 0.05, **p < 0.01.

Gene expression analysis further confirmed these findings (Figure B). Spheroids with higher HA concentrations showed increased expression of the antiapoptotic gene BCL2 and decreased expression of pro-apoptotic genes Caspase3 and Caspase7. These results suggest that HA microparticles enhance cell survival by creating a more supportive microenvironment, potentially facilitating oxygen and nutrient diffusion to mitigate hypoxia-induced apoptosis.

3.4. Chondrogenic Differentiation of AdMSC Spheroids

Chondrogenic differentiation was evaluated through histological staining, immunohistochemistry, and gene expression analysis. H&E staining revealed well-organized, densely packed cells in HA-containing spheroids, with the 30% HA group displaying the most pronounced chondrocyte-like morphology (Figure A). Alcian Blue staining confirmed increased glycosaminoglycan (GAG) deposition in the 30% HA group, reflecting enhanced cartilage matrix production compared to the 10% HA group and control spheroids.

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Chondrogenic differentiation of AdMSC spheroids with HA microparticles. (A) Histological staining after 3 weeks of chondrogenic induction. H&E staining shows spheroid morphology, and Alcian Blue staining indicates glycosaminoglycan (GAG) deposition. Representative cross-sectional images are shown for Control, 10% HA, and 30% HA groups. (B) Immunohistochemical staining for chondrogenic markers collagen type II (Col2, red) and aggrecan (green). Nuclei are counterstained with DAPI (blue). Stronger and more uniform staining is observed in the 30% HA group. (C) Gene expression levels of Aggrecan (AGG) and SOX9 analyzed by qRT-PCR for Control, 10% HA, and 30% HA groups. Data are presented as mean ± SD; *p < 0.05, **p < 0.01.

Immunohistochemistry demonstrated stronger and more uniform expression of chondrogenic markers collagen type II (Col2) and aggrecan in HA-containing spheroids (Figure B), with the 30% HA group showing the most intense staining. Gene expression analysis further supported these findings (Figure C), with the 30% HA group exhibiting the highest levels of Aggrecan (AGG) and SOX9, a master regulator of chondrogenesis. While Col2 expression was comparable between HA-containing and control spheroids, SOX9 expression was significantly higher in the HA groups, indicating the enhanced chondrogenic potential of HA microparticles.

3.5. Adipogenic Differentiation of Spheroids

Adipogenic differentiation was assessed using Oil Red O staining to identify lipid droplet accumulation. Surface staining showed robust lipid accumulation in all groups, including the control, 10% HA, and 30% HA microparticle groups (Figure A). However, cross-sectional staining revealed differences in differentiation patterns. In the control group, lipid accumulation was largely confined to the surface, with minimal staining in the core, suggesting limited differentiation due to poor nutrient and factor diffusion (Figure B). In contrast, the 30% HA group exhibited uniform staining throughout the spheroid, indicating complete and consistent adipogenic differentiation.

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Adipogenic differentiation of AdMSC spheroids incorporating HA microparticles. Oil Red O staining was performed after 2 weeks of adipogenic induction. (A) Surface views of spheroids show lipid accumulation in Control, 10% HA, and 30% HA groups. (B) Cross-sectional views of spheroids reveal differences in internal lipid droplet distribution. The 30% HA group demonstrates uniform lipid deposition throughout the entire spheroid, while lipid accumulation in the Control group is mostly restricted to the outer layers.

These findings demonstrate that HA microparticles, particularly at higher concentrations, enhance the microenvironment within spheroids, facilitating uniform differentiation and overcoming the diffusion limitations observed in the control group. The ability of HA microparticles to support both adipogenic and chondrogenic differentiation highlights their utility in tissue engineering and regenerative medicine applications.

4. Discussion

3D spheroid culture has emerged as an essential tool for enhancing stem cell differentiation and survival, providing a more physiologically relevant environment compared to traditional 2D cultures. , While 3D cultures offer significant advantages, achieving uniform differentiation throughout the spheroid structure remains a considerable challenge, especially in larger spheroids where limited diffusion of oxygen, nutrients, and differentiation factors to the core can lead to heterogeneous differentiation and increased apoptosis. These limitations reduce the overall efficiency of stem cell-based regenerative therapies, highlighting the need for innovative strategies to overcome these diffusion barriers.

In this study, we addressed these challenges by incorporating hyaluronic acid (HA) microparticles into the spheroid culture system. HA was chosen for its natural presence in the human extracellular matrix (ECM), its ability to absorb water, and its excellent biocompatibility. Our findings demonstrated that HA microparticles significantly enhanced cell viability and promoted uniform differentiation of AdMSCs into chondrocytes and adipocytes. Spheroids containing HA microparticles exhibited reduced apoptosis, as evidenced by fewer TUNEL-positive cells and the downregulation of pro-apoptotic genes such as Caspase3 and Caspase7, alongside the upregulation of the antiapoptotic gene BCL2. , These results suggest that HA microparticles create a more supportive microenvironment by facilitating the diffusion of essential molecules, mitigating hypoxia-induced apoptosis, and enhancing overall cell survival.

The enhanced differentiation observed in HA-containing spheroids aligns with previous studies highlighting the role of HA in promoting cell attachment, proliferation, and differentiation. Unlike conventional 2D cultures, which fail to replicate the native tissue microenvironment, HA microparticles enable the creation of a 3D structure that mimics the ECM more effectively, allowing for better cell–cell and cell-ECM interactions. , Our results further support the hypothesis that HA microparticles can overcome the diffusion limitations of traditional 3D cultures, enabling uniform distribution of growth factors and nutrients. This property is particularly advantageous for applications requiring larger spheroids or a higher number of differentiated cells, such as cartilage repair, adipose tissue engineering, and organoid development. Additionally, the ability to maintain viability and uniform differentiation within larger spheroids expands their potential use in drug screening and disease modeling, where accurately mimicking native tissue architecture is critical.

Moreover, the HA microparticles used in this study demonstrated swelling and porosity, which may facilitate the diffusion of not only nutrients and oxygen but also therapeutic agents or bioactive molecules. HA-based materials can act as reservoirs for growth factors, enabling their controlled release. Integrating such bioactive molecules into HA microparticles could further enhance the differentiation efficiency and uniformity of stem cells within spheroids. In our previous studies, incorporating TGF-β or BMP-2 into HA microparticles could boost chondrogenic differentiation, while factors like insulin or IGF-1 could enhance adipogenic differentiation. This approach would allow precise control over the differentiation process, opening new avenues for tissue engineering and organoid development.

In terms of scalability and translational potential, the inverse emulsion method employed for HA microparticle synthesis is readily adaptable to large-scale manufacturing. Process parameters such as reaction volume, stirring speed, and emulsification conditions can be systematically optimized for batch or continuous production. Furthermore, the raw materials used in the synthesis, including hyaluronic acid and polyethylene glycol derivatives, are commercially available in pharmaceutical-grade purity, facilitating compliance with good manufacturing practice (GMP) standards. Importantly, hyaluronic acid is a clinically approved, biocompatible, and biodegradable material with established safety profiles in various FDA-approved medical applications such as dermal fillers, ophthalmic surgery, and viscosupplementation. These properties support the feasibility of translating HA microparticle-based spheroid systems into in vivo therapeutic applications, including injectable cell delivery platforms and scaffold-free tissue engineering constructs.

Although HA was the primary focus of this study, other natural polymers such as gelatin and collagen could offer complementary or alternative benefits. Like HA, these polymers are biocompatible and possess ECM-mimicking properties. They could be used to create hybrid scaffolds, combining the strengths of multiple materials to optimize the 3D microenvironment for specific cell types or applications. For instance, gelatin and collagen-based microparticles could support differentiation pathways that require distinct ECM components or mechanical properties. Exploring these materials in combination with HA could lead to the development of advanced 3D culture systems with even greater versatility and functionality.

Despite these promising findings, this study has several limitations. The results are limited to in vitro observations for chondrogenic and adipogenic differentiation, and further research is needed to evaluate the performance of HA microparticles in vivo for these specific cell types. Future studies should focus on testing the engraftment efficiency, functionality, and long-term stability of HA-containing spheroids in animal models. Additionally, optimizing the concentration, size, and degradation rate of HA microparticles could further refine their performance, enabling better control over spheroid growth and differentiation.

5. Conclusions

Our study demonstrates that the incorporation of HA microparticles into 3D spheroid cultures effectively overcomes the challenges of achieving uniform differentiation and maintaining cell viability within the spheroid structure. By enhancing nutrient and growth factor delivery, HA microparticles support consistent differentiation into chondrocytes and adipocytes, even in the spheroid core. This approach offers significant advantages for regenerative medicine applications, including cartilage repair, adipose tissue engineering, and the development of complex tissue models for drug screening. Future research should explore the incorporation of bioactive molecules and alternative biomaterials to further expand the utility and efficacy of this system.

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00405287 and RS-2022NR069564), the Ministry of Food and Drug Safety (RS-2024-00331852), and Korean Fund for Regenerative Medicine (KFRM) Grant (RS-2025-02222978 and 22A0105L1-11) by the Korean government. This work was also supported by Chungnam National University.

H.-E.S. and Y.-J.K.: data curation, writing– original draft, investigation, methodology, formal analysis. K.H.P.: data curation, methodology, formal analysis. C.J. and Y.B.L.: methodology, formal analysis. K.M.H.: Writing – review and editing, Supervision, Project administration, Funding acquisition, Conceptualization. S.-W.K.: Writing – review and editing, Supervision, Project administration, Funding acquisition, Conceptualization. H.-E.S. and Y.-J.K. contributed equally to this work as cofirst authors. K.M.H. and S.-W.K. contributed equally to this work as corresponding authors.

The authors declare no competing financial interest.

References

  1. Mahla R. S.. Stem Cells Applications in Regenerative Medicine and Disease Therapeutics. Int. J. Cell Biol. 2016;2016(1):6940283. doi: 10.1155/2016/6940283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Mao A. S., Mooney D. J.. Regenerative medicine: Current therapies and future directions. Proc. Natl. Acad. Sci. U. S. A. 2015;112(47):14452–14459. doi: 10.1073/pnas.1508520112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Jensen C., Teng Y.. Is It Time to Start Transitioning From 2D to 3D Cell Culture? Front. Mol. Biosci. 2020;7:33. doi: 10.3389/fmolb.2020.00033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Pampaloni F., Reynaud E. G., Stelzer E. H. K.. The third dimension bridges the gap between cell culture and live tissue. Nature Reviews. Molecular Cell Biology. 2007;8(10):839–845. doi: 10.1038/nrm2236. [DOI] [PubMed] [Google Scholar]
  5. Lee S.-Y., Koo I.-S., Hwang H. J., Lee D. W.. In Vitro three-dimensional (3D) cell culture tools for spheroid and organoid models. SLAS Discovery. 2023;28(4):119–137. doi: 10.1016/j.slasd.2023.03.006. [DOI] [PubMed] [Google Scholar]
  6. Białkowska K., Komorowski P., Bryszewska M., Miłowska K.. Spheroids as a Type of Three-Dimensional Cell CulturesExamples of Methods of Preparation and the Most Important Application. International Journal of Molecular Sciences. 2020;21(17):6225. doi: 10.3390/ijms21176225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Johnson K., Zhu S., Tremblay M. S., Payette J. N., Wang J., Bouchez L. C., Meeusen S., Althage A., Cho C. Y., Wu X.. et al. A stem cell–based approach to cartilage repair. Science. 2012;336(6082):717–721. doi: 10.1126/science.1215157. [DOI] [PubMed] [Google Scholar]
  8. Minteer D., Marra K. G., Rubin J. P.. Adipose-derived mesenchymal stem cells: biology and potential applications. Adv. Biochem. Eng. Biotechnol. 2013;129:59–71. doi: 10.1007/10_2012_146. [DOI] [PubMed] [Google Scholar]
  9. Luo Y., Yu P., Liu J.. The efficiency of stem cell differentiation into functional beta cells for treating insulin-requiring diabetes: Recent advances and current challenges. Endocrine. 2024;86(1):1–14. doi: 10.1007/s12020-024-03855-8. [DOI] [PubMed] [Google Scholar]
  10. Yue L., Lim R., Owens B. D.. Latest Advances in Chondrocyte-Based Cartilage Repair. Biomedicines. 2024;12(6):1367. doi: 10.3390/biomedicines12061367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Patrick C. W. Jr.. Engineering Adipose Tissue for Regenerative and Reparative Therapies. Semin Plast Surg. 2005;19(3):207–215. doi: 10.1055/s-2005-919716. [DOI] [Google Scholar]
  12. Abatangelo G., Vindigni V., Avruscio G., Pandis L., Brun P.. Hyaluronic Acid: Redefining Its Role. Cells. 2020;9(7):1743. doi: 10.3390/cells9071743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ye H., Zhang R., Zhang C., Xia Y., Jin L.. Advances in hyaluronic acid: Bioactivity, complexed biomaterials and biological application: A review. Asian Journal of Surgery. 2025;48(1):49–61. doi: 10.1016/j.asjsur.2024.08.100. [DOI] [PubMed] [Google Scholar]
  14. Fang Y., Shi L., Duan Z., Rohani S.. Hyaluronic acid hydrogels, as a biological macromolecule-based platform for stem cells delivery and their fate control: A review. Int. J. Biol. Macromol. 2021;189:554–566. doi: 10.1016/j.ijbiomac.2021.08.140. [DOI] [PubMed] [Google Scholar]
  15. Park K. H., Ryu B., Song J. J., Shim H.-E., Song S.-S., Han J.-S., Kim C.-Y., Huh K. M., Kang S.-W.. Hyaluronic acid microparticles for effective spheroid culture and transplantation in liver tissue. Chemical Engineering Journal. 2023;464:142666. doi: 10.1016/j.cej.2023.142666. [DOI] [Google Scholar]
  16. Shim H.-E., Kim Y.-J., Park K. H., Park H., Huh K. M., Kang S.-W.. Enhancing cartilage regeneration through spheroid culture and hyaluronic acid microparticles: A promising approach for tissue engineering. Carbohydr. Polym. 2024;328:121734. doi: 10.1016/j.carbpol.2023.121734. [DOI] [PubMed] [Google Scholar]
  17. Yen B. L., Hsieh C. C., Hsu P. J., Chang C. C., Wang L. T., Yen M. L.. Three-Dimensional Spheroid Culture of Human Mesenchymal Stem Cells: Offering Therapeutic Advantages and In Vitro Glimpses of the In Vivo State. Stem cells translational medicine. 2023;12(5):235–244. doi: 10.1093/stcltm/szad011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Edmondson R., Broglie J. J., Adcock A. F., Yang L.. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay and drug development technologies. 2014;12(4):207–218. doi: 10.1089/adt.2014.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Vasvani S., Kulkarni P., Rawtani D.. Hyaluronic acid: A review on its biology, aspects of drug delivery, route of administrations and a special emphasis on its approved marketed products and recent clinical studies. Int. J. Biol. Macromol. 2020;151:1012–1029. doi: 10.1016/j.ijbiomac.2019.11.066. [DOI] [PubMed] [Google Scholar]
  20. Lakhani S. A., Masud A., Kuida K., Porter G. A. Jr., Booth C. J., Mehal W. Z., Inayat I., Flavell R. A.. Caspases 3 and 7: key mediators of mitochondrial events of apoptosis. Science. 2006;311(5762):847–851. doi: 10.1126/science.1115035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Qian S., Wei Z., Yang W., Huang J., Yang Y., Wang J.. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Frontiers in oncology. 2022;12:985363. doi: 10.3389/fonc.2022.985363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Gai T., Zhang Y., Li G., Zhou F., He C., Wang X., Su J.. Engineered hydrogel microspheres for spheroids and organoids construction. Chemical Engineering Journal. 2024;498:155131. doi: 10.1016/j.cej.2024.155131. [DOI] [Google Scholar]
  23. Kang S.-W., Kim J.-S., Park K.-S., Cha B.-H., Shim J.-H., Kim J. Y., Cho D.-W., Rhie J.-W., Lee S.-H.. Surface modification with fibrin/hyaluronic acid hydrogel on solid-free form-based scaffolds followed by BMP-2 loading to enhance bone regeneration. Bone. 2011;48(2):298–306. doi: 10.1016/j.bone.2010.09.029. [DOI] [PubMed] [Google Scholar]
  24. Tiwari S., Bahadur P.. Modified hyaluronic acid based materials for biomedical applications. Int. J. Biol. Macromol. 2019;121:556–571. doi: 10.1016/j.ijbiomac.2018.10.049. [DOI] [PubMed] [Google Scholar]
  25. Kang S.-W., Choi J.-J., Kim H.-N., Seo J., Park S.-J., Kim E.-Y., Park S.-P., Huh K. M., Chung H.-M., Chung H. Y.. et al. EGF-Loaded Hyaluronic Acid Based Microparticles as Effective Carriers in a Wound Model. Particle & Particle Systems Characterization. 2017;34(5):1600320. doi: 10.1002/ppsc.201600320. [DOI] [Google Scholar]
  26. Bi Y., Xie S., Li Z., Dong S., Teng L.. Precise nanoscale fabrication technologies, the “last mile” of medicinal development. Acta Pharmaceutica Sinica B. 2025;15(5):2372–2401. doi: 10.1016/j.apsb.2025.03.040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Huerta-Ángeles G., Nešporová K., Ambrožová G., Kubala L., Velebný V.. An Effective Translation: The Development of Hyaluronan-Based Medical Products From the Physicochemical, and Preclinical Aspects. Front Bioeng Biotechnol. 2018;6:62. doi: 10.3389/fbioe.2018.00062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kang M., Liang H., Hu Y., Wei Y., Huang D.. Gelatin-based hydrogels with tunable network structure and mechanical property for promoting osteogenic differentiation. Int. J. Biol. Macromol. 2024;281:136312. doi: 10.1016/j.ijbiomac.2024.136312. [DOI] [PubMed] [Google Scholar]

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