Three-dimensional (3D) cell culture matrices, also known as scaffolds, have emerged as a solution to the limitations of traditional two-dimensional (2D) cell culture methods. These matrices provide a more accurate representation of the extracellular matrix (ECM) in terms of geometry, chemistry, and signaling environment, enabling better cell growth, organization, and differentiation. This review discusses the current state of 3D cell culture techniques, focusing on their applications in tissue engineering and in vitro modeling of human organs. It also outlines key challenges and future directions for the development of 3D cell culture over the next 5–10 years. Traditional 2D culture systems, while useful, have limitations in replicating the complex 3D microenvironments found in living tissues. The ECM in the body is a complex network of fibers and gaps that provide biochemical and physical signals essential for cell function. 3D scaffolds, on the other hand, can mimic these environments more accurately, allowing cells to respond to their surroundings in a more natural way. The design of 3D scaffolds involves considering multiple scales, from macro-scale structures that determine the overall shape and size of the scaffold to micro-scale features that influence cell behavior, and nano-scale surface topography that affects cell adhesion and differentiation. The design of 3D scaffolds requires careful consideration of materials properties, including biocompatibility, mechanical strength, and degradation rates. Biomaterials used in 3D scaffolds can be natural or synthetic, and their selection depends on the specific application. For example, natural materials like Matrigel, fibrin gel, and alginate gel are commonly used due to their biocompatibility, while synthetic materials like poly(ethylene glycol) (PEG) hydrogels offer greater control over physical and chemical properties. Various fabrication techniques, such as electrospinning, particulate-leaching, and solid free-form (SFF) fabrication, are used to create 3D scaffolds with specific architectures and properties. These techniques allow for the precise control of scaffold structure, porosity, and surface topology, which are crucial for mimicking the ECM. Additionally, the incorporation of functional nano-materials, such as carbon nanotubes, can enhance the mechanical and biological properties of 3D scaffolds. Surface properties of 3D scaffolds are also important, as they influence cell adhesion, proliferation, and differentiation. Surface chemistry and topography can be modified to enhance cell interactions with the scaffold, improving the overall effectiveness of 3D cell culture systems. The development of 3D scaffolds with tailored properties is essential for advancing tissue engineering and in vitro modeling of human organs.Three-dimensional (3D) cell culture matrices, also known as scaffolds, have emerged as a solution to the limitations of traditional two-dimensional (2D) cell culture methods. These matrices provide a more accurate representation of the extracellular matrix (ECM) in terms of geometry, chemistry, and signaling environment, enabling better cell growth, organization, and differentiation. This review discusses the current state of 3D cell culture techniques, focusing on their applications in tissue engineering and in vitro modeling of human organs. It also outlines key challenges and future directions for the development of 3D cell culture over the next 5–10 years. Traditional 2D culture systems, while useful, have limitations in replicating the complex 3D microenvironments found in living tissues. The ECM in the body is a complex network of fibers and gaps that provide biochemical and physical signals essential for cell function. 3D scaffolds, on the other hand, can mimic these environments more accurately, allowing cells to respond to their surroundings in a more natural way. The design of 3D scaffolds involves considering multiple scales, from macro-scale structures that determine the overall shape and size of the scaffold to micro-scale features that influence cell behavior, and nano-scale surface topography that affects cell adhesion and differentiation. The design of 3D scaffolds requires careful consideration of materials properties, including biocompatibility, mechanical strength, and degradation rates. Biomaterials used in 3D scaffolds can be natural or synthetic, and their selection depends on the specific application. For example, natural materials like Matrigel, fibrin gel, and alginate gel are commonly used due to their biocompatibility, while synthetic materials like poly(ethylene glycol) (PEG) hydrogels offer greater control over physical and chemical properties. Various fabrication techniques, such as electrospinning, particulate-leaching, and solid free-form (SFF) fabrication, are used to create 3D scaffolds with specific architectures and properties. These techniques allow for the precise control of scaffold structure, porosity, and surface topology, which are crucial for mimicking the ECM. Additionally, the incorporation of functional nano-materials, such as carbon nanotubes, can enhance the mechanical and biological properties of 3D scaffolds. Surface properties of 3D scaffolds are also important, as they influence cell adhesion, proliferation, and differentiation. Surface chemistry and topography can be modified to enhance cell interactions with the scaffold, improving the overall effectiveness of 3D cell culture systems. The development of 3D scaffolds with tailored properties is essential for advancing tissue engineering and in vitro modeling of human organs.