Essential cultural platforms in biomedical research have been investigated particularly for drug discovery and anticancer investigations (Kim et al., 2020). The rising demand for advanced 3D cell culture models has intensified the need for sophisticated 3D cultivation techniques, from laboratory setups to industrial-scale production (Brancato et al., 2020; Wan et al., 2020). These bioreactors boast precise control systems, ensuring reproducible spheroid formation, a vital aspect for studying cellular behavior and drug responses. Beyond conventional applications, bioreactors play a crucial role in facilitating dynamic cell interactions and responses (Ferreira et al., 2018). They offer precise control over environmental conditions, including temperature, pH, and nutrient supply, ensuring optimal cell growth and function (Figure 1). Additionally, advancements in bioreactor design enable the integration of various monitoring and control systems for enhanced functionality.

Utilizing established 3D culture models, researchers have made significant strides in understanding cancer progression mechanisms (Dumont et al., 2019; Zhang et al., 2020). These models provide insights into morphological and cellular changes associated with disease progression, offering valuable platforms for drug screening and efficacy testing. Real-time monitoring of 3D cultures allows for the assessment of dynamic cellular behaviors, such as proliferation, migration, and interaction (De Leon et al., 2020). This capability is instrumental in studying complex phenomena like angiogenesis and tumor invasion, providing insights into disease mechanisms and potential therapeutic interventions.

Progression models enable the evaluation of treatment responses and the identification of novel therapeutic targets, contributing to advancements in precision medicine and personalized therapies (Xia et al., 2019). Furthermore, these models aid in deciphering intricate cellular signaling pathways involved in disease progression, paving the way for targeted interventions. Accurate measurement of structural changes in 3D cultures requires precise labeling techniques to track cell proliferation and migration over time (Zhang et al., 2019). Various labeling methods, such as fluorescent proteins and dyes, enable researchers to monitor multiple cell types simultaneously, facilitating comprehensive analyses of complex cellular interactions (Linsley et al., 2019).

Compared to traditional 2D cell cultures, 3D scaffolds provide a more realistic environment for cellular growth and interaction (Gupta et al., 2019). Crafted from diverse materials, these structures offer controlled porosity, permeability, surface chemistry, and mechanical properties, closely resembling the native extracellular matrix (ECM) that supports cellular organization in tissues (Bayir et al., 2019; Huang et al., 2020). This porous network not only supports cellular growth but also facilitates nutrient and oxygen diffusion, essential for maintaining cell viability and function (Terrell et al., 2020).

Fabricating these scaffolds entails various techniques, each tailored to achieve specific structural and mechanical properties (Ashok et al., 2020). Freeze-drying, for instance, utilizes sublimation to create a porous scaffold from a frozen solution, while electrospinning produces fibrous scaffolds through the application of electrostatic forces (Fan et al., 2019). The applications of 3D scaffolds are vast, spanning both tissue engineering and cell culture research. In tissue engineering, these scaffolds serve as a scaffold for the regeneration of damaged organs and tissues. By seeding scaffolds with appropriate cells and growth factors, researchers aim to facilitate tissue repair and functional restoration.

Beyond tissue regeneration, 3D scaffolds play a pivotal role in advancing our understanding of disease mechanisms and cell biology. By providing a more physiologically relevant environment, these scaffolds enable researchers to study cell-cell interactions, drug responses, and disease progression with greater accuracy. 3D scaffolds stand as indispensable tools in tissue engineering and cell culture for studying cellular behavior and advancing regenerative medicine.

Stem cells, particularly pluripotent stem cells (PSCs), have garnered immense interest due to their ability to generate various cell types and their potential in regenerative medicine (Costamagna et al., 2019; Turco and Moffett, 2019). They offer promising avenues for medical innovation, including drug discovery, cell therapy, and tissue regeneration (Artero Castro et al., 2019; Srivastava and Kilian, 2019). Utilizing advanced 3D cell platforms, researchers have made significant strides in understanding cell signaling and tissue development, particularly in fields.

Culturing cells in 3D models has emerged as a vital tool, providing results that closely mimic natural conditions and aiding in the translation of research findings into clinical applications (Alagarsamy et al., 2019; Balak et al., 2019; Chen and Schoen, 2019; Gibbs et al., 2019; Gopalakrishnan, 2019; Meivar-Levy and Ferber, 2019; Roberts et al., 2019; Nguyen et al., 2020; Raimondi et al., 2020). These models offer a more realistic representation of cellular behavior, ensuring better predictability when moving from the laboratory to real-world settings. Autologous, allogeneic, and xenogeneic cells are employed, with autologous cells preferred to mitigate the risk of rejection (Collins et al., 2020).

Adipose-derived stem cells (ASCs) have emerged as a significant player in regenerative medicine, offering several advantages over other sources such as bone marrow-derived mesenchymal stem cells (MSCs) (Ryu et al., 2019). ASCs, found within adipose tissue, boast higher yields and greater resistance to senescence, making them an attractive option for therapeutic applications (Seo et al., 2019). However, challenges persist in standardizing isolation methods and understanding their precise characteristics. The unique characteristics of ASCs vary depending on the tissue type and isolation method employed. Techniques like power-assisted liposuction have shown promising results in yielding high-quality ASCs, underscoring the importance of optimization in isolation protocols. Research continues to elucidate ASC behavior and identify specific markers to distinguish them from other cell types accurately.

Despite the progress made in harnessing the potential of stem cells and ASCs, several hurdles remain to be addressed. Standardization of isolation methods, precise differentiation instructions, and clarification of cell characteristics are crucial areas requiring further investigation. Nevertheless, the ongoing research holds significant promise for revolutionizing medical treatments and offering hope to patients with various degenerative diseases and injuries (Sthijns et al., 2019).

Transitioning from traditional 2D cell culture systems to more advanced 3D approaches represents a significant step towards understanding cellular behavior within a physiologically relevant context (Ndyabawe and Kisaalita, 2019; Jensen and Teng, 2020). 3D culture methods offer a more accurate representation of in vivo conditions, bridging the gap between conventional cell culture systems and the intricate physiology of living organisms (Figure 2). This advancement is particularly crucial in the medical research, where cellular interactions and the microenvironment play pivotal roles in tumorigenesis (Chaicharoenaudomrung et al., 2019).

These benefits in tissue engineering and regenerative medicine enable 3D culture to be considered for various clinical applications (Table 1). The previous studies showed the development of 3D bioprinted skin tissue, adipose microtissues, and bone scaffolds, demonstrating substantial improvements in cell viability, differentiation, and functionality (Gholipourmalekabadi et al., 2018; Yan et al., 2019; Yang et al., 2021; Li et al., 2024). Studies on esophageal, gastric, and intestinal organoids provide insights into stem cell potential and disease modeling, revealing the intricate processes of tissue development and regeneration (Spence et al., 2011; DeWard et al., 2014; McCracken et al., 2014). The generation of vascularized liver buds, lung organoids, and pancreatic organoids underscores the progress in creating functional human tissues for therapeutic applications, offering promising avenues for treating related diseases (Takebe et al., 2013; Dye et al., 2015; Boj et al., 2016). The researches on heart defect, prostate cancer, optic cup formation, inner ear sensory tissue, and lingual epithelium organoids emphasize the importance of organoid systems in understanding disease and developing personalized treatments. They suggested innovative approaches in heart patch, musculoskeletal tissue engineering, cartilage repair, and ophthalmopathy treatment, focusing on the potential of stem cell-derived tissues and hybrid scaffolds in regenerative medicine (Eiraku et al., 2011; Hisha et al., 2013; Gao et al., 2014; Koehler and Hashino, 2014; Fischer et al., 2023; Soucy et al., 2025). Some studies explore the possible usage of β -cell generated from human pluripotent stem cells to cure diabetes mellitus, the potential of salivary gland stem cell therapy for treating xerostomia, and the development of bioprinted human skin substitutes (Lee et al., 2014; Nanduri et al., 2014; Millman et al., 2016; Baltazar et al., 2023). The researches on liver organoids derived from primary human hepatocytes and the creation of hybrid scaffolds for musculoskeletal tissue demonstrate the ongoing efforts to enhance the complexity and functionality of engineered tissues (Salas-Silva et al., 2023; Enbergs et al., 2024).

One of the primary advantages of 3D culture over 2D systems is the preservation of critical extracellular matrix components and cell-cell or cell-matrix interactions (Ravi et al., 2015; Gonzalez Diaz et al., 2019; Tomas-Bort et al., 2020). These interactions are essential for various cellular processes, including differentiation, proliferation, and the expression of specific phenotypic traits. By maintaining these critical elements, 3D culture systems provide researchers with a more holistic view of cellular behavior, allowing for more accurate predictions of in vivo responses to various stimuli and treatments.

Studies have demonstrated that 3D tissue cultures can offer novel insights into tumorigenic mechanisms that may not be apparent in conventional 2D models (Liu et al., 2019; Roberts et al., 2019; Bahcecioglu et al., 2020; Zhang et al., 2020). The three-dimensional arrangement of cells in these cultures closely mimics the architecture of tumors in vivo, facilitating the study of complex processes such as invasion, metastasis, and drug resistance. Additionally, 3D culture systems enable the examination of dynamic cellular behaviors, such as migration and morphogenesis, which are challenging to replicate in 2D environments (Yamada and Sixt, 2019).

Moreover, 3D culture methods offer enhanced biomarker expression, providing researchers with valuable tools for studying cellular functions and interactions (Ravi et al., 2015). By accurately recapitulating the native cellular microenvironment, these systems enable the investigation of signaling pathways and regulatory mechanisms that are crucial for understanding disease progression and treatment response (Gonzalez Diaz et al., 2019). This improved biomarker expression also enhances the sensitivity and specificity of assays, leading to more reliable experimental results.

In addition to their utility in basic research, 3D culture systems hold promise for applications in drug development and personalized medicine (Linsley et al., 2019; Brancato et al., 2020; Fernandes et al., 2020; Fisher and Rao, 2020). These systems can serve as cost-effective screening platforms for identifying potential therapeutic agents and predicting their efficacy and safety profiles. By incorporating patient-derived cells into 3D cultures, researchers can tailor treatment strategies to individual patients, improving the likelihood of successful outcomes and minimizing adverse effects.

Overall, the transition from 2D to 3D cell culture represents a paradigm shift in biomedical research, offering researchers a more physiologically relevant model system for studying cellular behavior and disease mechanisms (Gopalakrishnan, 2019). By preserving critical cellular interactions and microenvironmental cues, 3D culture methods offer a more accurate representation of in vivo conditions. These technologies hold the potential to revolutionize medical fields, from cancer biology to regenerative medicine, paving the way for new diagnostic and therapeutic strategies.