Reducing the use of animal models using 3D cell constructs and 3D bioprinting technology

Basic and applied research commonly use animals(rodents, rabbits, monkeys, etc.) as models to understand the cellular mechanisms of diseases and to test drugs or vaccines at the preclinical stage. Still, the use of these models has continually been discussed over the years.

Besides ethical issues, animal tests are time-consuming and expensive. As a matter of fact, 40% of the newly developed drugs fail clinical trials, emphasizing animal models' poor reliability and predictability [1]. Thus, finding alternative methods is an urgent need.

Over the past few years, research in 3D cell culture and 3D bioprinting fields grew in a significant manner, leading to new technologies, such as organoids, human organ-like structures, and organ-on-a-chip, to better reflect tissue architecture and mimic cell-to-cell interaction [2]. We present here a few examples where those methods could be promising alternatives to animal models.


In the last decade, Organ-On-Chips (OOC) - 3D microfluidic systems that can be obtained using light-based 3D bioprinting - have shown great potential as platforms to represent the physiological environment and functionality of human organs. They have already been developed for many organs: brain, lung, skeletal muscle, heart, skin, kidney, liver, gut, and bone [3]. In 2017, Skardal and colleagues even described a three-tissue organ-on-a-chip system (liver, heart, lung) (Figure 1) and highlighted examples of inter-organ responses to drug administration [4].



Figure 1: Overall design and implementation strategy for the 3-tissue-representative organ-on-a-chip system using a variety of biofabrication approaches. a,b: Illustration and photographs of the modular multi-tissue organ-on-a-chip hardware system set up for maintenance of the 3 tissue model. Individual microfluidic microreactor units house each organoid or tissue model, and are connected via a central fluid routing breadboard, allowing for straightforward “plug-and-play” system preparation initialization [4].


In cancer research, animal models are used to understand the genetic mechanisms of cancer but are inefficient to study the tumor microenvironment (TME) [5]. The term TME refers to the complex cellular milieu surrounding cancer epithelium, including mesenchymal‐derived cells such as fibroblasts, blood vessels, innate and adaptive immune cellular networks, and extracellular matrix (ECM). Various tumor organoids models have already been developed to study the TME [6]. A study published by Bojin et al. modeled breast TME. They incorporated cancer cells in a central spheroid wrapped in hydrogel rings containing tumor-associated fibroblasts (TAFs) and peripheral blood mononuclear cells (PBMCs). They showed that the 3D cell culture conditions were favorable to form a tumor tissue-like structure in vitro. [7]

Another study published by Jiang T. et al. used extrusion 3D bioprinting technology to create a platform to mimic in vivo breast cancer TME. The researchers performed a co-culture of bioprinted cancer cells and fibroblasts embedded in a bioink that resembles the native tumor stroma[8]. They proved that cancer cells were able to form multicellular tumor spheroids (MCTS) with a high viability for long periods of cell culture (> 30 days) (Figure 2).


Figure2: MCTS formation within a 3D bioprinted in vitro model consisting of IMR-90-mCherry myofibroblast and MDA-MB-231-GFP breast cancer cells. (a) These photographs show the bioprinted in vitro sample (left) and the CAD model (right). (b) This representative confocal time-lapse image shows the MDA-MB-231-GFP (green) and IMR-90-mCherry (red) cells bioprinted within the model. (c,f) These zoom-ins show the MDA-MB-231-GFP cell regions (white dotted boxes). (g - j) These zoom-ins show the IMR-90-mCherry cell regions (yellow dotted boxes). The scale bars are 2 mm in panel 2a, 1 mm in panel 2b, and 500 µm in panels 2c - 2j for selected areas, and the magnification is 10X. Capital "D" in the images means "days of culture"


In virology studies, animal models are used to understand in vivo viral characteristics. The viral infection by SARS-CoV-2 has been at the center of preoccupation for the last 2 years. Not only organoids but also 3D printing scaffolds have shown promising results in this regard, improving more efficient diagnosis and vaccine development. We already discussed 3D bioprinting's relevance toward Covid-19 infection in a previous article: “3D models as a tool for studying Covid-19”.


There is no doubt that animal testing in science has significantly benefitted human beings. However, it shows some limitations, such as poor relevance, ethical problems, and high cost. 3D cell culture and 3D bioprinting technology offer new perspectives for basic research, cancer studies, and drug discovery that could help to at least reduce animal use.




References:


1. Van Norman GA et al., Limitations of animal studies for predicting toxicity in clinical trials: is it time to rethink our current approach? JACC Basic Transl Sci 4 (7), 845–854; (2019)

2. Hagenbuchner J et al., 3D bioprinting: novel approaches for engineering complex human tissue equivalents and drug testing. Essays Biochem; 65 (3): 417–427; (2021)

3. https://www.lindau-nobel.org/blog-organs-on-a-chip-the-future-of-drug-testing/

4. Skardal A et al., Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform. Nature Scientific Reports; volume 7, Article number: 8837; (2017)

5. Li Z et al. Application of Animal Models in cancer research: recent progress and future prospects. Cancer Manag Res.;13:2455-2475; (2021)

6. Xia T, Du WL, Chen XY, Zhang YN. Organoid models of the tumor microenvironment and their applications. J Cell Mol Med.;25(13):5829-5841; (2021)

7. Bojin F et al., 3D bioprinting of model tissues that mimic the tumor microenvironment. Micromachines (Basel) ;12(5):535; (2021)

8. Jiang T et al., Bioprintable alginate/gelatin hydrogel 3D in vitro model systems induce cell spheroid Formation. J Vis Exp.;(137):57826; (2018)


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