Key considerations for getting started with 3D cell culture

3D cell culture is transforming how researchers explore biology, study disease, and screen drugs. Unlike 2D cultures, which fail to capture the complexity of native tissues, 3D cell models offer greater biological relevance by incorporating key biophysical and biomolecular properties that shape cell behavior. By recreating phenotypically relevant microenvironments, 3D models enable more predictive insights into disease progression and therapeutic response. Complex 3D co-cultures reflect intricate tissue niches, supporting studies on cell-cell interactions, matrix dynamics, and multicellular organization. Together these elements provide a powerful approach for uncovering deeper insights into cellular behavior, disease mechanisms, and therapeutic response. 

The RASTRUM™ platform enables researchers to streamline generation of complex, reproducible 3D cell models. Whether you're exploring cancer biology, drug discovery, or regenerative medicine, understanding key factors—matrix selection, model architecture, and cell preparation—is essential for success.

1. Selecting the right matrix: Recapitulating the extracellular microenvironment

The extracellular matrix (ECM) serves as a structural and biochemical foundation for cells in vivo. In 3D culture the choice of matrix composition, biofunctionality, and mechanical properties plays a key role in shaping and defining fundamental cell behaviors including growth and migration, protein expression, differentiation, and drug sensitivity​.

Key matrix considerations:

• Stiffness selection: ECM stiffness plays a crucial role in regulating cell behaviour. The RASTRUM Matrix Library provides tunable stiffness options from 0.7 to 4.8 kPa, accommodating diverse tissue models and most patient-derived, primary and continuously-cultured cell types.
• Tailored adhesion peptides and additives: Matrices can be functionalised with adhesion peptides (Fibronectin, Laminin β, Collagen I, Collagen IV) and full-length ECM components (Hyaluronic acid, Laminin-521, Laminin-211, Fibronectin) enabling cell-matrix interactions and enhancing tissue-specific relevancy.
• Xeno-Free for high reproducibility: Unlike basement membrane extracts, RASTRUM Matrices are synthetic and xeno-free, ensuring batch-to-batch consistency and eliminating variability resulting from undefined animal-derived components.

Explore RASTRUM’s tunable matrix properties.

2. Designing your 3D cell model: Architectural considerations

Beyond matrix composition, the structural organization of your 3D cell model plays a critical role in biological relevance, experimental compatibility, assay reproducibility, and downstream applications. The RASTRUM platform provides optimized cell model architectures supporting the recreation of simple and complex tissue models, ideal for investigations of fundamental biology, disease mechanisms, and drug impact. Additionally, a non-functionalized hydrogel base layer, featured as part of RASTRUM model architectures, prevents cells from interacting with the plastic culture plates and forming 2D monolayer cultures. The result of this is a defined 3D culture environment that can be maintained even as cell density increases. RASTRUM-generated 3D cell models are compatible with most standard downstream analysis strategies including high-content imaging, bulk expansion for molecular analysis (DNA, RNA, protein), and high-throughput drug screening.

3D cell model approaches optimized for research & screening

• Homogeneous or co-culture models: RASTRUM enables the generation of both single cell type (homogeneous) and multi-cell type models, in addition to multicellular spheroids/organoids with diameters <70 µm, supporting studies ranging from disease modeling to therapeutic screening.
• Defined multi-region models: By enabling the creation of multiple, spatially-distinct microenvironments within a single well, multi-region 3D cell models, such as the dual matrix and triple matrix model architectures, support investigations into cell invasion, paracrine signaling, and stromal-parenchymal cell interactions.
• Bulk expansion models for molecular analysis: Designed for DNA, RNA, and protein extraction, these models facilitate large-scale molecular studies.

Built for high-throughput & translational applications

• High-Throughput Screening (HTS): RASTRUM-generated models are formatted for 96- and 384-well plate compatibility, ensuring seamless integration into the automated workflows commonplace in compound screening and drug discovery.
• Optimized for high-content imaging: Xeno-free, transparent RASTRUM matrices allow for high-resolution imaging with no autofluorescence, making them ideal for studying cell morphology, biomarker expression, and complex multicellular interactions.
• Recapitulating the tissue microenvironment: Model architectures accurately imitate key tissue elements, promoting phenotypically-relevant cell-matrix interactions, physiological behaviours and key disease features.

Discover different 3D cell model architectures.

3. Preparing your cells for 3D culture: Ensuring viability and reproducibility

Successful 3D cell culture starts with properly prepared cells. Whether using patient-derived cultures, iPSCs, or established cell lines, high viability and uniform cell suspensions is essential for generating consistent, phenotypically-relevant models. Consistent and correct sample preparation maximizes cell health, model reproducibility, and experimental success.

Best practices for cell preparation for 3D cell models:

For adherent cells from 2D culture:

• Ensure high viability (>80%) to support robust 3D model formation and long-term culture stability.
• Maintain a uniform suspension and avoid cell clumps of no larger than 70 µm. 
• Consider the use of ROCK inhibitors when working with frozen or sensitive cells to protect cells throughout handling and printing. 
• Minimize time between harvesting and creating the 3D cell models to preserve cell integrity and optimize seeding efficiency.

For cells from tissue samples:

• Optimize enzymatic dissociation to achieve a homogenous cell suspension.
• Thoroughly wash cells to eliminate dead or damaged populations, preventing unintended experimental variability.
• Filter cell suspensions using a 70 µm sterile strainer to prepare for printing, minimise cell debris and improve post-printing culture viability.

Explore our cell preparation protocol for further recommendations. 

3. Bringing it all together: Reproducible and scalable 3D cell models

Once your matrix and cell model architecture are selected and your cells are prepared, the final step is seamlessly generating your 3D cell model with RASTRUM. Unlike manual methods, which introduce variability and workflow inefficiencies, RASTRUM’s drop-on-demand technology ensures:

• High reproducibility across experiments: Minimize variability and ensure consistent model generation with post-printing CVs of <10%.
• Recreate complex tumor and tissue microenvironments: Enable the study of dynamic interactions between distinct cell types, such as cancer and stromal cells, advancing insights into disease mechanisms and therapeutic development.
• Scalability for high-throughput screening: Compatible with 96- and 384-well formats, streamlining drug discovery and translational research.

Both RASTRUM and RASTRUM Allegro generate highly reproducible 3D cell models, ensuring consistent biological relevance and experimental reliability. RASTRUM Allegro introduces key advancements for higher throughput workflows and working with precious cell samples .

For researchers seeking flexibility and reproducibility, RASTRUM provides a trusted foundation for 3D cell culture. For those requiring higher throughput, faster processing, and enhanced handling of precious cell samples, RASTRUM Allegro is the ideal choice.

5. Next steps: Applying 3D cell culture to your research

With a strong foundation in matrix selection, model design, and cell preparation, you are ready to integrate 3D cell models into your research workflows. Whether you’re focusing on cancer, neuroscience, or regenerative medicine, RASTRUM enables biologically-relevant, scalable, and reproducible 3D models for groundbreaking discoveries.

Connect with us to schedule a consultation to get started.