2D vs 3D cell culture: What’s best for scalable biopharmaceutical production?

Exploring the differences between 2D and 3D culture systems and their impact on efficiency, scalability, and relevance in modern cell-based manufacturing.

Properties of 2D and 3D cell culture systems impact the scalability, robustness, and product quality of processes, making them a crucial decision impacting products success.

Cell culture systems are the foundation of modern biopharmaceutical production, playing a crucial role in advanced therapy medicinal products (ATMPs), viral vector manufacturing, and cell-based therapies. The decision between 2D and 3D cell culture impacts scalability, process robustness, and final product quality, making it essential for manufacturers to select the right approach.

While 3D culture systems have gained attention for their ability to mimic in vivo conditions, 2D cell culture remains the dominant platform in industrial manufacturing. This is primarily due to its proven scalability, process control, and regulatory acceptance. However, as biopharma companies explore 3D technologies, several technical challenges have emerged, particularly regarding cell harvesting, post-translational modifications, and unintended differentiation.

This article explores the fundamental differences between 2D and 3D cell culture, the challenges of scaling up 3D systems in biopharmaceutical production, and how optimized 2D systems remain a practical choice for industrial-scale adherent cell culture.

2D vs. 3D Cell Culture: Understanding the Differences

The primary distinction between 2D and 3D cell culture lies in how cells grow within the system. In 2D culture, cells attach to flat surfaces such as T-flasks, roller bottles, and multi-layer stacks, where they form direct cell-cell interactions that are essential for many biological processes. These systems are widely used due to their well-characterized performance. However, traditional 2D methods require multiple vessels to achieve large-scale production, increasing labor intensity and contamination risks.

By contrast, 3D culture systems allow cells to grow within a three-dimensional structure, such as microcarriers in suspension, scaffolds, or fixed-bed reactors. These systems are designed to increase volumetric cell densities. However, cell-cell interactions in 3D cultures or suspension are often weaker than in 2D. Additionally, the complexity of 3D systems introduces challenges in cell harvesting, which can create significant hurdles in industrial biomanufacturing (1).

Challenges of Scaling Up 3D Cell Culture in Biopharmaceutical Production

Cell Harvesting Limitations in Fixed-Bed Bioreactors

One of the biggest challenges in 3D bioprocessing is the difficulty of harvesting cells from fixed-bed bioreactors. These systems offer a high surface area for adherent cell growth but make cell retrieval extremely difficult. Unlike in 2D systems, where cells can be easily detached using enzymatic or mechanical methods, cells in fixed-bed bioreactors are embedded deep within the scaffold structure.

Harvesting cells from fixed beds or microcarriers often requires enzymatic digestion (e.g., trypsinization), which not only prolongs processing time but also introduces risks of residual enzyme contamination. Furthermore, monitoring cells during culture is challenging, as traditional sampling techniques do not work effectively in fixed-bed environments. These factors make fixed-bed bioreactors unsuitable for applications where the cells themselves are the final product, such as cell-based therapies.

Post-Translational Modifications (PTMs) and Viral Vector Potency

Shifting from 2D to 3D culture can also impact post-translational modifications (PTMs), which are critical for the potency and stability of biopharmaceuticals. The cell environment in 3D systems differs significantly from 2D, with variations in oxygen gradients, nutrient availability, and mechanical forces. These factors influence protein folding and glycosylation, which in turn affect the biological activity of viral vectors and recombinant proteins (2-3).

For example, AAV vectors produced in 3D bioreactors can exhibit different glycosylation patterns compared to those produced in 2D systems. These modifications can alter infectivity and transduction efficiency, potentially leading to variability in therapeutic outcomes. Regulatory agencies closely monitor these changes, requiring extensive characterization to ensure consistent product quality across different culture platforms.

Unintended Cell Differentiation in 3D Systems

Another critical challenge of scaling up 3D culture is the risk of unintended cell differentiation, particularly when using microcarriers or scaffold-based systems. The structural environment of 3D culture can introduce mechanical cues that alter cell behavior, leading to phenotypic drift.

For instance, stem cells expanded on microcarriers have been found to lose key pluripotency markers, reducing their regenerative potential. Similarly, shear forces in stirred bioreactors can cause epigenetic changes, making cells less suitable for therapeutic applications. These issues highlight the need for careful optimization of 3D expansion conditions, particularly in cell therapy and regenerative medicine (4-5).

Why 2D Systems Remain the Gold Standard in Industrial Bioprocessing

Despite advancements in 3D culture technologies, 2D systems continue to be the preferred choice for large-scale adherent cell culture - especially when the cells themselves are the product and must be harvested efficiently.

One key advantage of optimized 2D systems is that they allow seamless scalability from R&D to manufacturing without requiring extensive process re-development. In contrast, switching to 3D systems demands significant resources for process optimization, including adjustments to cell attachment, media flow, and bioreactor parameters. By using a scalable 2D system, manufacturers can avoid these process development hurdles, ensuring consistency, regulatory compliance, and cost-effectiveness.

While 3D cell culture offers promising advantages, its challenges in preserving cell identity and during cell harvesting make it less practical for large-scale industrial bioprocessing. In contrast, 2D systems remain the most reliable solution for commercial manufacturing, particularly in cell-based therapies.

For companies looking to scale adherent cell cultures without major process modifications, optimized 2D systems provide a straightforward path to industrial-scale production. By maintaining a consistent culture environment from R&D to manufacturing, biopharma companies can save resources while ensuring high-quality, reproducible outcomes.

References

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2. Hall MK, Burch AP, Schwalbe RA. Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures. PLoS One. 2021 Nov 8;16(11):e0259743. doi: 10.1371/journal.pone.0259743. PMID: 34748597; PMCID: PMC8575246.
   

3. Xie Y, Butler M. N-glycomic profiling of capsid proteins from Adeno-Associated Virus serotypes. Glycobiology. 2024 Mar 19;34(1):cwad074. doi: 10.1093/glycob/cwad074. PMID: 37774344; PMCID: PMC10950483.

   
   

4. Chen AK, Chen X, Choo AB, Reuveny S, Oh SK. Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Res. 2011 Sep;7(2):97-111. doi: 10.1016/j.scr.2011.04.007. Epub 2011 May 11. PMID: 21763618.

   
   

5. Gareau, T., Lara, G.G., Shepherd, R.D., Krawetz, R., Rancourt, D.E., Rinker, K.D. and Kallos, M.S. (2014), Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors. J Tissue Eng Regen Med, 8: 268-278. https://doi.org/10.1002/term.1518