Table 1.

Characteristics of animal models compared to 2D and 3D cell culture systems.

	Animal Models	2D Cell Culture	3D Cell Culture
Cost	Relatively high expenses are relied on for the purchase of animals but also for housing and breeding, in addition to being time consuming.	Has potential for lower costs than in vivo experiments and involves relatively simple manipulations.	Often more expensive than 2D cell cultures. Some techniques can be technically demanding and time consuming.
Ethical concerns regarding animal welfare	Many ethical concerns are involved in animal testing because of the pain and distress experienced by models in certain protocols.	Cell culture has the potential to reduce animal testing and spare animal lives. However, the possible use of animal serum raises concerns for animal welfare and human biosafety.	Similar to 2D platforms.
Gene expression	Reflective of animals in vivo. Can differ from humans	Lower expression levels and numerous variations from in vivo and 3D gene expression.	More reflective of in vivo gene expression, thus contributing to better tissue-specific functions than 2D systems.
Morphology	Reflective of animals in vivo. Can differ from humans	Restrictions encountered in 2D environments cause changes in cell morphology and induce an artificial apical–basal polarity.	A three-plane environment allows for the development of complex morphologies.
In vivo imitation	N/A	2D systems do not accurately mimic the natural 3D microenvironment of cells. This leads to misleading and unpredictable data for in vivo responses.	3D cell cultures allow for a better representation of the in vivo organization than 2D systems, resulting in more physiologically relevant data.
Transferability to humans	Controversial. Some very important discoveries for humans had been made using animals. However, animal models are sometimes inefficient to predict human in vivo responses, especially for toxicity studies.	Using human cells minimizes the questionability of transferability of in vitro data to humans and opens doors for personalized medicine.	3D platforms can produce results using human cells in physiological contexts which can lead to high translational potential of the discoveries.
Complexity of environment	Whole organisms are highly complex, thus implying potential unknown interactions.	Low complexity. Often more easily interpretable results.	Intermediate, leading to more relevant data than 2D systems, while controlling most interactions.
Tumour modeling	Helpful models to study tumours.	Cannot accurately replicate characteristics of the tumoural microenvironment.	Suitable for the development of tumour models.
Reproducibility	Not satisfactory, especially in preclinical research.	High reproducibility potential, but decreased by the use of animal serum.	Various 3D techniques offer lower reproducibility than 2D platforms, although high reproducibility is achievable (e.g., hanging drop technique). Reproducibility seems diminished by the use of animal-derived scaffolds or serums.
High throughput agreement	Small animals can be suitable for high throughput screening.	Easily suitable for high throughput screening.	For a long time, difficult to adapt for high throughput screening. New technologies (e.g., tissue chips/microphysiological systems) render them more accessible. Automation is possible but at higher costs.
Vascularization	Reflective of in vivo. Advantageous for tumour and angiogenesis studies.	Lack of vascularization.	Endothelialization of 3D tissues is possible for certain techniques and could improve graft take in addition to being useful for tumour and angiogenesis studies.
Immune system interactions	Presence of interactions. However, immunodeficient models cannot adequately reflect interactions normally encountered with an entirely intact immune component.	Usually no interactions. Low complexity interactions can be encountered in 2D co-cultures with immune cells.	Potential for higher interactions than 2D systems. Incorporation of immunogenic components like immune cells and lymphatic capillaries are being explored to establish 3D systems with more complex in vivo-like interactions with the immune system.