2D vs 3D Cell Culture

Cell-based assays have a central role in drug discovery and development, being widely used to evaluate the efficacy and toxicity of potential therapeutic candidates. In recent years, traditional 2-dimensional (2D) cell-based assays have been complemented by more advanced 3D models that can offer many advantages for scientific research.

3D cell models evolved to bridge the gap between in vitro and in vivo environments by more closely recapitulating natural conditions than traditional 2D cultures. They achieve this by allowing cells to grow and interact with one another in a three-dimensional space, often through the use of a matrix. Not only do 3D cell models provide better predictivity of in vivo responses, potentially lowering attrition rates for drug discovery, but they also serve to minimize animal use for scientific research. Since the passing of the FDA Modernization Act 2.0 in December 2022, there has been a heightened drive toward implementing 3R (reduction, refinement, replacement) practices, such as through replacing animal testing with alternative technologies^1,2.

Because 3D cell models can vary in terms of accessibility and ease of use, it is important to understand the different options that are available. These include the following:

• Spheroids are formed by allowing one or more cell types to self-aggregate in low-attachment cultureware or propagate within a matrix. They are commonly derived from established tumor cell lines or primary tumor cells, enabling researchers to replicate the tumor microenvironment. However, spheroids may also be generated from other cell sources. For example, human gingiva-derived progenitor cells have been co-cultured with human umbilical vein endothelial cells (HUVEC) to create spheroids for studying angiogenesis³.
• Organoids are derived from embryonic stem cells, induced pluripotent stem cells, or adult stem cells that have been harvested from primary tissues. Adding different cocktails of cytokines and growth factors to the culture medium induces differentiation into miniaturized versions of organs including the lung, kidney, and pancreas.  Depending on the type of model that is generated, organoids serve different purposes. For example, liver organoids can be used for assessing the risk of hepatotoxicity, which is estimated to be responsible for around 22% of clinical trial failures⁴.
• Microfluidic-based 3D cell culture systems are designed to control cell microenvironments, including fluid flow and gradients of nutrients and oxygen, so that tissue-specific functions are maintained in vitro. They encompass organ-on-a-chip platforms, which allow for modeling cellular interactions within specific organs, and more sophisticated body-on-a-chip systems consisting of multiple organoid types⁵. Recent advances within the field include the development of pumpless fluid recirculation and the introduction of biocompatible thermoplastic materials, both of which are intended to minimize drug loss due to surface adsorption⁶.
• 3D bioprinting, which can be broadly categorized as extrusion-, droplet-, or laser-based, uses computer-aided processes for precise layer-by-layer assembly of biomaterials. Compared with methods that allow the cells to grow randomly in culture, 3D bioprinting gives researchers tight control over cellular orientation. This can, for example, be important when modeling the intestinal mucosa, which is known to be a critical site for absorption, distribution, metabolism, and excretion (ADME) studies in drug development⁷.

Working with 3D model systems presents several inherent challenges. First, the higher density of 3D structures compared with cell monolayers makes imaging more difficult, although this can be addressed through paraffin-embedding and immunohistochemical staining or by performing optical clearing⁸. Second, assay steps involving the transfer of liquids may require more optimization to ensure that the growing cells are not perturbed. Other issues center around the cost of 3D cell culture compared with traditional 2D cell-based assays, although demand for 3D technology looks set to drive prices down.