Biomaterials with low endotoxin content could lead to a more accurate representation of the safety and potency of novel therapeutics.
by Dr Bjorn Vergauwen
In vitro 3D cancer models are becoming increasingly important for studying the tumour microenvironment (TME) and testing the potential efficacy of new cancer drugs, but research2 led jointly by the University of Twente, The Netherlands, and Rousselot Biomedical shows that the reliability of the models is significantly impacted by the presence of endotoxins.
Recent changes in legislation and increased ethical considerations1 are driving a shift from pre-clinical animal trials to 3D models created from biomaterials and human cells and tissues. The models use 3D bioprinting to reproduce a pathophysiological environment that aims to mimic cell interactions that would happen in the body. Since they are replacing other types of models used in development, it’s essential that the efficacy of novel drugs on both cancer cells and macrophages can be measured accurately.
Endotoxins are a known problem in the world of biomaterials and medical devices. Their levels differ across materials, but so far, no study has investigated the effects of endotoxins on the predictive value of 3D cancer models.
The researchers studied the impact of endotoxins on 3D breast cancer/immune models, looking at both the effect on the crosstalk between macrophages and cancer cells and the effect on the measured efficacy of immunotherapies.2 These are both important factors in measuring immune-response and drug validity because tumour-associated macrophages (TAMs) are abundant in the TME and are known to promote an immunosuppressive state, making them an interesting target for new immunotherapies.
Endotoxins, or lipopolysaccharides, are large, highly immunogenic molecules and the major component of the outer membrane of Gram-negative bacteria. It’s known that even at low levels, endotoxins can pose a serious health risk. When exposed to endotoxins, the immune system initiates an inflammatory response that can lead to tissue inflammation, increased sensitivity to other allergens, and the risk of fatal shock.3
For some applications, endotoxin limits have been defined to ensure patient safety. Complying with the limit values for endotoxins in these applications is a challenge: the endotoxin levels in traditionally manufactured gelatin and collagen are typically >100 times higher than those recommended by the United States’ Food and Drug Administration (USFDA), for example. Although the presence of endotoxins is already regulated by the FDA for in-body applications, this issue has been overlooked in 3D models.
The study found that endotoxin levels have a marked effect on the reliability of the 3D-printed model in several ways. The standard gels create a more artificial inflammatory environment than the purified gels. In the biomaterial with high endotoxin levels, macrophages were 2.16 times larger and produced three times the amount of nitrous oxide, indicating a strong inflammatory response to the bioink. However, the endotoxins also reduced the crosstalk between macrophages and cancer cells, making the macrophages less responsive to the cancer cells. In contrast, the low endotoxin biomaterial allowed significant upregulation of genes involved in the crosstalk between macrophages and cancer cells, demonstrating successful crosstalk between macrophages and cancer cells.
Studying the immunotherapies in these models further, the researchers saw even more disparity. The high endotoxin environment artificially increased the measured efficacy of the therapy that was designed to inhibit the expression of anti-inflammatory markers and seemed to show non-efficacy in the therapy that induced pro-inflammatory markers.
The research brings to light the overlooked issue of endotoxicity in cancer models, and it also demonstrates how certain types of gelatin are more suitable for 3D models than others. For example, standard, commercially available gelatin has naturally high endotoxin levels compared with a purified biomaterial such as Rousselot’s X-Pure® gelatin methacrylate (GelMA).
Dr Kathleen Jacobs, Global Regulatory Affairs Director, Rousselot, explains the dangers that this can bring: “This one study revealed that novel breast cancer immunotherapies display clear therapeutic efficacy differences in low and high endotoxin gels. Biomaterials with high endotoxin content could lead to misinterpretation of the safety and potency of novel therapeutics, and this can certainly put the validity of the model into question.”
Dr Bjorn Vergauwen, Scientific Director, Product and Process Development, Rousselot, says, “The use of purified biomaterials such as Rousselot’s X-Pure or X-Pure GelMA in in vitro 3D models is critical in producing a biologically relevant environment while also helping to reduce the costs, time, and ethical concerns associated with drug development.”
Endotoxins are part of bacteria and therefore difficult to avoid. Once present, they are extremely hard to remove due to the fact that endotoxins can form large supramolecular structures (up to 100 nm) that are UV- and heat-stable.4 Successful removal requires temperatures over 180 °C, which raises the risk of altering or damaging the biomaterial itself. This is why most commercially available biomaterials are non-endotoxin purified.
Vergauwen concludes, “As we move towards greater dependency on 3D models, this study is a critical step in ensuring that they more accurately reflect the human condition.”
This study further highlights how low endotoxin levels are important for both in-body applications and research. For example, research has already shown improved cell viability and differentiation in an endotoxin-purified cell growth environment.5 Pharma and medical developers are continually developing new uses for gelatin- and collagen-based applications, both human and veterinary.
With X-Pure, Rousselot has developed a range of highly purified pharmaceutical gelatins with low endotoxin levels that they believe can contribute to the exciting and growing applications of 3D cancer models. [APBN]
- European Commission. (2022). JRC supporting alternatives to animal testing. https://joint-research-centre.ec.europa.eu/jrc-news/jrc-supporting-alternatives-animal-testing-2022-04-07_en
- Heinrich, M. A., Heinrich, L., Ankone, M. J. K., Vergauwen, B., Prakash, J. (2023). Endotoxin contamination alters macrophage-cancer cell interaction and therapeutic efficacy in pre-clinical 3D in vitro models, Biomaterials Advances. (144). https://doi.org/10.1016/j.bioadv.2022.213220
- Weil, M. H., & Spink, W. W. (1957). A comparison of shock due to endotoxin with anaphylactic The Journal of laboratory and clinical medicine, 50(4), 501–515.
- Heinrich, M.A., M. Mangia, and J. Prakash. (2021). Impact of endotoxins on bioengineered tissues and models. Trends Biotechnol.
- Olijve, J. & Gevaert, E. (2021). Stem cell proliferation on 3D printed discs improved with low endotoxin and low molecular weight gelatin coating. https://d1ip4j1950xau.cloudfront.net/Rousselot/Rousselot%20Biomedical/Stem%20cell%20proliferation%20on%203D%20printed%20discs.pdf
About the Author
Dr. Bjorn Vergauwen is the Scientific Director for Product and Process Development at Rousselot. He leads R&D projects focused on unlocking the full clinical potential of gelatin. His expertise lies in the biophysical and biochemical principles underlying gelatin’s behavior, which he applies to various applications in the regenerative and pharmaceutical space.