3D culture systems allow for cell-cell and cell-extracellular matrix (ECM) interactions, therefore more accurately mimicking the tissue environment in vivo. This approach allows for monoculture as well as co-culture of multiple cell types.

Using 3D culture systems, more representative knowledge can be obtained regarding cell behaviors and pharmacological responses, as well as tissue function. Thus, these systems are becoming increasingly important in drug discovery, cancer research, and personalized medicine as they continue to advance to meet the more stringent preclinical testing standards, and may thus aid in the development of individualized therapeutic techniques.

Basics of 3D cell culture systems

A 3D cell culture system is designed to replicate the natural structure of tissues. Unlike in a monolayer, 3D cell culture systems use scaffolds, hydrogels, and microfluidic devices to create complex cellular environments that mimic in vivo conditions.

Types of 3D cell culture systems: 3D cell culture techniques have evolved to provide more physiologically relevant models for studying cellular behavior and interactions. These methods can be broadly categorized into different types:

• Scaffold-based systems: Cells are supported by a physical structure that provides a three-dimensional environment for growth. These scaffolds can be made from various materials, including hydrogels and synthetic polymers.

• Scaffold-free systems: Scaffold-free methods allow cells to grow without any supporting structure, relying on self-aggregation to form three-dimensional structures.

• Microfluidic devices: Manipulate small volumes of fluids through channels on the micrometer scale. They enable precise control over the cellular microenvironment and can be used to mimic a blood supply.

  • Cell culture platforms: Microfluidic devices can be designed to create controlled environments for 3D cell cultures, allowing for the study of cell interactions under dynamic conditions.
  • Perfusion systems: These devices can facilitate nutrient and waste exchange by continuously perfusing media through the culture system, enhancing cell viability and function.

Micro-fluidic surfaces and rotating bioreactors provide a regulated environment for cell development. Magnetic levitation and 3D bioprinting are advanced approaches that use magnetic fields to arrange cells into 3D patterns, providing a scalable and customizable approach to tissue engineering and cell interaction investigations.

Several types of scaffolds have been developed for use in 3D cell culture systems:

• Scaffolds: Provide support for cell attachment and growth. They can be made from natural or synthetic materials and are designed to facilitate tissue development and regeneration.

  • Natural scaffolds: Derived from biological materials such as collagen, gelatin, or chitosan. These materials often provide bioactive cues that promote cell adhesion and proliferation.
  • Synthetic scaffolds: Made from polymers like polylactic acid (PLA) or polycaprolactone (PCL). These materials can be engineered to have specific mechanical properties and degradation rates, offering greater control over the cellular microenvironment.

• Hydrogels: Water-swollen networks of polymers that can mimic the ECM found in tissues. They provide a soft, hydrated environment conducive to cell growth. Hydrogels are generally biocompatible, allowing cells to survive and function within them. Their mechanical and biochemical properties can be tailored to match those of specific tissues, including stiffness and degradation rates.

  • Natural hydrogels: Such as alginate, hyaluronic acid, and collagen-based gels that provide natural biochemical cues.
  • Synthetic hydrogels: Such as polyethylene glycol (PEG) or methacrylated hydrogels, allow for precise control over their properties.

Abcam offers an alginate hydrogel kit for 3D cell culture (ab241011), which serves as a 3D cell culture matrix.

Applications of 3D cell culture systems in research

3D systems replicate complex cellular interactions, mechanical forces, and microenvironments found in living tissues. This enables more accurate modeling of human physiology, disease progression, and treatment responses, making them invaluable across multiple research disciplines.

Drug discovery and toxicity testing

3D cell culture systems are becoming increasingly used for drug discovery, providing a more accurate method to study how drugs interact with human tissues. By mimicking the 3D architecture of tissues, these models offer a more reliable method for testing drug efficacy, toxicity, distribution, and take-up, enabling earlier and more effective identification of potential issues compared to traditional 2D cultures.

For example, 3D spheroid cultures of SW1353 cells were reported to serve as a reliable in vitro model for analyzing drug effectiveness in chondrosarcoma. In another report, the chronic toxicity of fialuridine (a compound that in previous in vitro studies did not exhibit direct hepatotoxicity) was highlighted using a 3D primary human hepatocyte culture. The study outlined the potential of 3D cultures in studying drug-induced liver injury and liver diseases like viral hepatitis.

Cancer biology and personalized medicine

In cancer research, 3D cell cultures are used to better understand tumor behavior, growth, resistance to treatments, invasion and metastasis, and interactions of cancer cells with the surrounding non-cancerous cells and extracellular matrix. For example, several genes responsible for tumor dormancy in breast cancer were identified using a 3D in vitro model of the endosteal bone niche (endothelial, bone marrow stromal cells, and fetal osteoblasts) in a 3D collagen matrix (GELFOAM) (research phase).

In another study, biomimetic scaffolds (Mg-doped hydroxyapatite and collagen) and cancer stem cells from osteosarcoma cell lines were used to construct 3D osteosarcoma models that closely mimicked the stem cell niche and tumor microenvironment (research phase). They can be used to recreate the tumor microenvironment, allowing for more precise drug screening.

Multicellular tumor spheroids from epithelial ovarian cancer cells were used to screen clinically repurposed drugs, and licofelone (an anti-inflammatory drug) was identified. This lead compound reversed the stemness of cancer cells and extended the survival of mice models when combined with paclitaxel (research phase).

Tissue engineering and regenerative medicine

For tissue engineering, 3D cultures are essential in creating functional tissues that can be used for regenerative medicine. By providing scaffolds for cells to grow and organize, these systems help develop tissues like skin, cartilage, and even organs, which can eventually be used for transplants or healing damaged tissues. An interesting example is the construction of scaffold-free 3D cell constructs of dental pulp stem cells (DPSCs) capable of self-organization.

When these constructs were filled in human tooth root canals and implanted in mice, human pulp-like tissues were formed, showing promise in pulp generation (research phase). In another study, islet cells cultured in 3D hydrogel had functions and β-cell mass close to freshly isolated islets. They restored the glucose balance when transplanted in diabetic mice (research phase). With the potential for scale-up, further optimization can pave the way for islet cell transplantation.

Disease modeling and other research applications

3D cell cultures play an important role in disease modeling by accurately simulating the complex cellular interactions and tissue architecture found in vivo. These models provide better insights into the developmental progression of diseases by replicating the in vivo environment.

Amid the chaos caused by the COVID-19 pandemic, 3D cultures played a crucial role in understanding disease pathogenesis. For example, an advanced 3D microfluidic model of the human blood-brain barrier was used to identify the loss of barrier effect induced by the S1 subunit of the SARS-CoV-2 spike protein, explaining the neurological complications of the disease (research phase).

In another report, researchers developed a novel 3D culture model of Alzheimer's disease, constructed with induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) in poly(lactic-co-glycolic acid) (PLGA) microtopographic scaffolds for disease modeling (research phase).

Key models in 3D cell culture

Various models in 3D cell culture are designed to represent in vivo biology in various ways, ranging from simple cellular interactions to very intricate tissue architectures. Different types of models provide unique advantages to researchers depending on their specific needs.

Types of 3D cell culture models

• Spheroids: Spherical aggregates of cells produced in non-adherent environments and mimic tissues and microtumors. They are commonly used to examine cell-cell interactions, drug screening, and, in general, cancer research. They simulate early-stage tissue architecture and offer a simple 3D model.
• Organoids: 3D structures formed by stem cells that self-organize into organ-like tissues, replicating the functional features and topologies of real organs. As a result, they could be used in both disease modeling and tailored medicine.
• Specialized and emerging models: Created to meet the specific needs of a study, such as multicellular models or cell lines representing specific tissue types.Emerging approaches, like microfluidic devices, offer precise control over the cell environment. These systems manipulate fluid flow, chemical gradients, and physical stimuli within tiny channels, enabling more detailed analysis of cellular behavior than traditional cell culture methods.
• Bioprinted models: Customized cell structure designs by layering biomaterials and cells to create complex tissues and organs. The process allows the synthesis of bioidentical tissue that could integrate smoothly into natural tissue, assisting in the restoration of organ function. These models can recreate complex tissues and open new possibilities in tissue engineering, regenerative medicine, and drug testing.
• Tumor-on-a-chip:Incorporates 3D tumor tissues into microfluidic chips to imitate the tumor microenvironment. These small devices possess channels that allow precise control over fluid flow, enabling the delivery of nutrients and drugs to the tumor tissue on the chip.
  By incorporating various cell types (like endothelial cells and immune cells) and ECM components, these models can mimic the complex environment around a tumor. These models facilitate the investigation of cancer progression, medication efficacy, and cancer therapy in a highly regulated and repeatable manner.
• Microtissues: Small clusters of seed cells that assemble themselves into 3D structures due to cell-cell and cell-ECM interactions in upside-down droplets, low-adhesion cell culture plates, microcarriers, microgels, or other culture media.

This system is frequently used for drug testing and disease modeling; it has a compact architecture that allows for high-throughput screening while retaining tissue-like characteristics. Microtissues can be injected directly, combined with 3D scaffolds, or mixed into bio-ink, which is a biological material, cells, or a combination of both that mimics the extracellular environment.

These include gelatin and its derivatives, such as methacrylated gelatin, collagen, and multicomponent inks like alginate-based bioink for HepG2 cells in 3D printing, demonstrating promising potential for repairing tissues and possibly even organs.

Choosing the right 3D cell model

Selecting the appropriate 3D cell model depends mainly on the objectives of the research, the level of complexity needed, and the resources available. Spheroids are a good starting point, being cost-effective and suitable for basic studies, though they can be less standardized in size and less accurately represent an in vivo environment.

Organoids offer more complexity and better mimic organ-like structures, but they require more resources and specialized protocols and may be more suited to studying diseases or patient-specific conditions.

For studies involving multiple cell types or specific physical properties, bio-printed tissues or microfluidic models offer greater precision in controlling cell arrangement and the cellular environment. In general, simpler models still provide valuable insights at a lower cost, while more complex systems are better suited to in-depth studies where accuracy and reproducibility are vital.

Spheroids vs. organoids: Differences and applications

Advantages of 3D cell culture systems

• Higher physiological relevance

3D cell culture conditions are closer to the in vivo environment than 2D cultures. In a 3D system, cells interact with other cells and with the ECM, similarly to in vivo conditions. Data from 3D cell cultures are much more relevant for drug development and disease modeling. For example, the traditional monolayer cannot be representative of the in vivo environment, making the interpretation of apoptosis or differentiation challenging; for example, the characteristics of tumor cells are not fully represented in 2D cultures.

• Improved cell-cell and cell-matrix interactions

Cells in 3D cell culture show more physiologically accurate behaviors regarding cell migration, differentiation, and apoptosis. Such processes are vital in accurately testing a drug’s efficacy and the disease mechanism. Cells confined to a 2D plane cannot mimic tissue structure; it is through the 3D systems that cells can establish tissue-like networks, which enable more accurate evaluation of physiologically relevant cellular processes and responses.

This physiological relevance of 3D cultures over 2D cultures can be seen in a study where the effect of the extracellular matrix was compared between these culture types for colorectal cancer (CRC) cells. The researchers discovered differences in the expression of key pathways, such as the mitogen-activated protein kinases (MAPK) pathway, and that epidermal growth factor receptor (EGFR) inhibition in wild-type Kirsten rat sarcoma viral oncogene homolog (KRAS) cells was less effective in 3D cultures than 2D cultures, suggesting the use of 3D cultures in research under more physiologically relevant conditions (research phase).

In another study, functional enrichment analyses of 2D and 3D cultures of five CRC cell lines revealed that altered gene expression for genes associated with cell maintenance in the 3D microenvironment in 3D cultures, such as extracellular matrix organization, cell-cell contact, and cell adhesion, highlighting the better mimicking of in vivo conditions by 3D cultures (research phase).

• Cell culture stability and viability for longer periods

In general, 3D cell cultures are likely to support stable long-term growth in comparison to 2D cultures. The cells in 3D systems experience less stress and have more extended culture periods with better maintenance of their original functions. For example, analysis of five head and neck squamous cell carcinoma (HNSCC) cell lines revealed higher viability for 3D spheroids compared to 2D cultures after cisplatin and cetuximab treatment (research phase).

In another study, researchers compared cell viability among other factors for B16 F10 murine melanoma cells and 4T1 murine breast cancer cells in 2D and 3D cultures. Cells grown in 3D culture systems demonstrated increased resistance to drug treatment with dacarbazine, with around a 30% higher viability rate than the 2D monolayer group (research phase).

Challenges and limitations of 3D cell culture systems

• Technical and biological challenges: Optimization and maintenance of the 3D model remain a challenge, given the differences and specificities of different cell lines. Differences in cell seeding, matrix composition, and environmental conditions have an impact on reproducibility, and the optimal conditions will vary by cell line. Additionally, the third dimension in a cell culture model introduces additional parameters to consider, potentially affecting reproducibility.
  For example, spheroid size can affect the oxygen and nutrient supply to cells in different layers. given that oxygen can diffuse through 100-150 µm of tissue; thus, the presence of a hypoxic core in spheroids larger than 300 µm is an issue. However, this also more accurately represents the early stages of the growth of a tumor before neovascularization occurs. Another technical challenge is that images of 2D cultures are taken only in the X and Y-axes, while 3D cultures necessitate a Z-axis, which increases the time and complexity of data collection and analysis.
  Further, the development of 3D multicellular tumor spheroids using liquid overlay and agitation-based protocols requires significant time and labor.
• Standardization and reproducibility: The 3D cell culture system needs standardization to ensure more consistent results. Further, guidance and standardization are needed for cell types, biocompatible scaffold materials, and assemblies that mimic the extracellular matrix for more accurate replication of the conditions in vivo.
  A report stated that the formation of 3D multicellular tumor spheroids depends on cell-cell interactions, cell type, medium composition, technique used, and cell density, which need to be optimized for successful culture.

• Cost and complexity: 3D cell culture systems tend to be more expensive and technically demanding than traditional 2D cultures. Specialized equipment, reagents, and expertise can render them impractical for widespread use. For example, while co-culture systems can help study multiple cell types, the complexity level increases given that the cell ratios, media requirements, and growth rates of the cell types used need to be optimized, challenging automation and high-throughput analyses.
  Another example of the complexity can be seen with a study that showed the method used for generating spheroids impacted the chemosensitivity, ie, chemoresistance to cisplatin was higher in spheroids generated on nutator or hanging drop plates than spheroids on ultra-low attachment plates (20-60% viability vs. 10-20% viability, respectively).

Future directions and innovations in 3D cell culture

Advances in scaffold materials and bioprinting will drive future 3D cell culture improvements, allowing for more precise and complicated tissue models to be created. New scaffold materials enhance cell integration, while bioprinting enables personalized cell placement and tissue building. Researchers are paying increasing attention to decellularized extracellular matrix (dECM) scaffolds, which are tissue/organ biomaterials without immunogenic components.

The biological/physicochemical signals and functions are maintained, making them candidates for tissue regeneration. For example, human skin-derived decellularized dermal matrix (DDM) with human umbilical cord perivascular cells could increase wound healing and faster re-epithelization in wounds in diabetic rats, showing the potential of this approach for skin healing (research phase).

Nanofibrillar cellulose is a novel 3D culture scaffold material used for isolating extracellular vesicles from cancer spheroids. In addition to obtaining batch isolation, a continuous culture was also reported with successive harvesting based on time (with potential applications in tailormade studies such as drug delivery) (research phase).

Researchers are testing various approaches in bioprinting. A few examples of developments in bioinks being studied are covalent cross-linking systems to produce new functionalized materials with filament fabrication (FFF) with a thermally reversible dynamic covalent Diels-Alder reaction, composites like alginate-silver nanoparticles and gold nanoparticles with methacrylated gelatin and “sacrificial templates” where “fugitive inks” are used to print vasculature, such as glass filaments that dissolve to form vessels (research phase).

The combination of CRISPR and 3D cultures may enhance the possibilities of gene editing studies. This combination allows researchers to introduce or correct specific genetic mutations in organoids or spheroids, enabling the study of gene-disease relationships in a more physiologically relevant context. For example, genome-wide CRISPR screens of 2D and 3D lung cancer spheroids revealed that 3D spheroids recapitulated the in vivo tumors more accurately.

Further analyses identified a new potential biomarker, carboxypeptidase D, that was correlated with patient survival (research phase). In another study, the CRISPR–human organoids–single-cell RNA sequencing (CHOOSE) system was used to analyze high-risk autism spectrum disorder genes and identified that the most susceptible cells to these genetic alterations were intermediate progenitor cells and L2/3 excitatory neurons.‘

The screening revealed ventral telencephalon progenitor cell enrichment due to alterations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex and subunit ARID1B mutation influenced the progenitor fate, which was also verified in patient cell organoids, facilitating gene regulatory network analysis (research phase).

FAQs

What are the principal advantages of 3D cell culture models compared to 2D models?

3D cell culture models provide a more physiological environment compared to 2D cultures. In 3D cultures, cells interact in all dimensions due to their similarity with tissue architecture. Interactions between cells and matrix enhance interactions between cells and matrices and provide more precise models of diseases, better drug responses, and higher improvement in tissue engineering. Compared to 2D cultures, 3D models exhibit complex cellular behaviors, drug resistance, and tumor progression, which are highly relevant for studying human diseases and testing therapeutics.

How do spheroids and organoids differ in application in 3D cell culture?

Spheroids are small, spherical cell clusters made of a single or few cell types and are often used to study cell behavior, drug responses, and cancer research. Organoids are more complex 3D structures derived from stem cells, capable of self-organizing into tissue-like structures that resemble organs. Organoids are used in the study of tissue development, organ function, and disease modeling, which gives more detailed insights into human biology, including organ-specific diseases and personalized medicine.

In what ways does 3D cell culture enhance drug efficacy and toxicity testing?

3D cell culture mimics the natural architecture and complexity of human tissue. Therefore, its more realistic cellular environment is applied during drug testing. This allows for the more accurate evaluation of drugs in terms of efficacy and toxicity as cells responding to these treatments are present in a more “in vivo-like” environment. Unlike 2D models, 3D systems capture signaling pathways and cellular responses more effectively and can replicate how a drug diffuses through tissues, interacts with cell layers, and penetrates tumors. This leads to improved prediction of drug absorption, distribution, metabolism, and potential side effects, ultimately enhancing clinical trial outcomes.

What is the role of microfluidic technology in 3D cell cultures?

Microfluidics facilitates the use of organ-on-a-chip models through controlled fluid flow in tiny channels under physiological conditions. Microfluidics provides in vitro tissue models within a 3D cell culture system with highly controlled nutrient delivery, waste removal, and cell signaling. Beyond that, real-time dynamic processes such as drug diffusion, cell migration, and the interaction of the cells with an extracellular matrix can be studied. Thus, microfluidics increases the complexity of 3D cell cultures and, thus, their relevance in drug testing and modeling.

What is the difference between an organoid and a 3D cell culture?

Organoids are a defined 3D cell culture type derived from stem cells and capable of self-organization into functional, tissue-like structures that closely resemble organs. Although there is a broader range of 3D cell culture systems, ranging from spheroids to scaffold-based systems, organoids have more complexity within them and better recapitulate the architecture and function of native organs. Organoids are being increasingly used in the study of disease models, organ development, drug testing, and personalized medicine, whereas 3D cultures correspond to less complex models, which in turn have simpler self-organization dynamics and are used for relatively less complex studies like cell-cell interactions or assessing drug toxicity.

What are spheroids in cell culture?

Spheroids are dense, three-dimensional aggregates of cells that can spontaneously form in vitro. Cell aggregates, with the typical composition usually being single or only a few different types of cells, are widely used in cancer research, drug screening applications, and tissue engineering. Spheroids mimic tissue growth, cell-cell interactions, and oxygen gradients observed in tissues. They are less complex than organoids but valuable in modeling cellular behaviors like proliferation, migration, and response to therapies in a 3D context.

What are the challenges of bioprinted 3D models?

3D bioprinted models have several challenges, including correct cell positioning, sustaining high cell viability during the printing process, and using materials that accurately imitate the features of tissue-like structures. Scaling up to larger tissues is especially challenging since adequate vascularization and replicating the complex architecture of genuine organs is difficult to achieve. To overcome these difficulties, it is critical to improve the mechanical qualities and biocompatibility of printing materials to promote long-term culture and functional integration. Furthermore, standardizing techniques to improve repeatability and consistency is a considerable difficulty.

How is the tumor-on-a-chip model applied for drug testing?

Tumor-on-a-chip models integrate 3D cultures of cancer cells within the microfluidic device that mimics the actual in vivo environment. These systems enable researchers to model the growth, metastasis, and drug response of tumors in controlled dynamic environments. Microfluidics enables not only the introduction of fluid flow as blood circulation but also mimics the transport of drugs in tissues. The tumor-on-a-chip platforms are broadly applied to personalized drug screening while developing the efficacy and toxicity of cancer therapies under more representative conditions than can be tested in clinical trials. This approach facilitates simultaneous testing of multiple drug candidates on a single chip, enabling rapid evaluation of different drug combinations and concentrations.