Organ-on-a-chip (OOC) technology aims to more accurately reproduce human physiology in vitro, addressing the limitations of current approaches. This field is advancing rapidly.
Image Credit: CN Bio Innovations Limited
Major Pharma and Biotech companies have expressed interest in expediting the introduction of new medicines to the market, and regulators are keen on new alternative methodologies (NAMs), like OOC technology.1
Academic researchers are also actively working in this area, supported by an increasing number of grant-funded opportunities.
The number of publications citing the use or development of OOC technologies is growing exponentially, and several well-established companies are designing and producing commercially viable systems and services.
While these advanced OOC models significantly enhance physiological relevance and culture longevity, compared to standard 2D cell cultures, the question arises - do they compare favorably against in vivo models? It is often asked, will OOC replace animal models, and do we have data available that proves OOC models are better than in vivo ones?
Despite being ethically desirable and an overarching aim for many in the OOC field, realistically, the complete replacement of animal usage is unlikely to happen for quite some time. Before this can become a reality, OOC technologies will need to undergo further verification. However, by strategically integrating OOC technologies into drug discovery and development stages, these disruptive tools can be utilized to cross-validate and supplement preclinical data immediately.
Rather than asking if OOC will replace animal models completely, a more pertinent question may be to explore the disadvantages of animal models and how OOC can be utilized in combination to generate useful information that fills knowledge gaps that are unaddressed by traditional methods of preclinical testing.
Using liver research as an example, this concept can be explored further. The most common cause of chronic liver disease globally is the metabolic disorder, non-alcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction-associated steatotic liver disease (MASLD). Linked to both obesity and diabetes, this disease affects an astounding 25% of the global population.
NAFLD manifests as a spectrum of metabolic disorders, where fat accumulates in the liver tissues, as a consequence of genetics, diet and lifestyle, even in the absence of excessive alcohol consumption.
About 20% of people diagnosed with NAFLD progress to non-alcoholic steatohepatitis (NASH), also known as metabolic dysfunction-associated steatohepatitis (MASH), characterized by steatosis (fat deposits in the liver), fibrosis, inflammation, and insulin resistance.
Without treatment, NASH can advance to cirrhosis and/or hepatocellular carcinoma (HCC), with liver transplant being the only possible therapeutic intervention.
To stop NASH from becoming a worldwide economic problem, many companies have embarked on drug discovery programs, yet their high-profile compounds continually fail to meet primary endpoints in late-stage clinical trials. To this day NASH remains a disorder of unmet medical need.
What are the disadvantages of preclinical in vivo models of NASH?
Despite the availability of a wide variety of preclinical NASH models, accurately predicting the effectiveness of drug candidates in humans remains a challenge.
While in vivo models provide a clear “systems” benefit over existing in vitro models, they ultimately involve mice and not humans, raising valid concerns about cross-species differences affecting data translatability.
In preclinical studies, in vivo murine models are the most utilized tools and are generally categorized into genetic, dietary, chemical, or combinations of these models. Unfortunately, there is no simple “one size fits all” option.
1. Genetic
Genetic mouse models play a valuable role in studying specific aspects of NASH, particularly concerning obesity and HCC. Starting with obesity, mice deficient in leptin (Ob/ob mice) or with non-functioning leptin receptors (db/db mice) are commonly used to model obesity, resulting in severe steatosis, a hallmark of NASH.
These models, while effective at generating fat mice, fall short of replicating other important markers of NASH, such as hepatocellular ballooning and inflammation. Achieving a more representative NASH phenotype often requires combining leptin models with dietary or chemical elements.
Similarly, to investigate the predisposition of NASH patients to HCC, a combination of genetic models (such as tumor suppressor gene PTEN mutations) and dietary models is necessary.2
2. Dietary
Much like genetic models, dietary models capture some elements of NASH but fail to replicate the entire disease. To compensate for these constraints, dietary models are also commonly utilized in combination with genetic or chemical elements.
One extensively researched model of NAFLD is the methionine and choline-deficient (MCD) diet. This diet, rich in sugars and fats but deficient in choline and methionine, causes the accumulation of fat in hepatocytes, oxidative stress, inflammation, ballooning, and fibrosis.
It also leads to increased levels of alanine transaminase (ALT) and aspartate transaminase (AST), which are important clinical markers of NASH.
Unfortunately, the metabolic profile induced by the MCD diet differs significantly from human NASH, as it results in weight loss rather than obesity, and lacks insulin resistance, and dyslipidaemia.3
Typically, high-fat diets better replicate the overall NASH metabolic phenotype than MCD diets. Treated mice develop obesity, insulin resistance, and hyperlipidaemia, although high-fat diets fall short of MCD diets in terms of inducing steatosis, inflammation, and fibrosis.4
3. Chemical
Chemical models are often most effective when used in combination with dietary and genetic ones to address phenotypic shortcomings. For instance, Streptozotocin is used to induce diabetes and, when combined with a high-fat diet, can replicate NAFLD. Diethylnitrosamine, when added to a high-fat diet, can induce HCC.4Carbon tetrachloride (CCl4) induces an oxidative stress response resulting in hepatic fibrosis. However, it has several drawbacks such as severe toxicity and, when combined with a high-fat diet, a reduction in body weight.5 These challenges make the interpretation of experimental results quite challenging.
One question remains - how relevant are chemically induced models to the mechanisms of disease onset in humans, and could this impact the lack of data translatability?
In summary, each mouse model can replicate certain important aspects of NASH but not all. When models are combined, they can imitate important disease hallmarks, but in doing so may induce “side effects” that are not related to NASH.
The necessity to combine or modify the models to suit specific research needs is complex, expensive, time-consuming, and very challenging to validate, and ultimately, the data derived still does not reliably translate into clinical results.
How does Organ-on-a-chip complement existing disease models?
One thing is certain, there is little chance of pre-clinical drugs accomplishing clinical success if pre-clinical models lack human predictivity.
In the context of NASH, where traditional models have shown limitations, the question arises: should research and development efforts be abandoned, or is there a missing key weapon in the arsenal? Can OOC technology fill the gaps and provide a more reliable solution?
Cultured using the PhysioMimix® OOC, CN Bio’s advanced in vitro model of NASH disease employs proprietary liver-on-a-chip technology.
It provides long-term (>1 month) in vitro cultures of primary human hepatocytes, stellate, and Kupffer cells in 3D microtissue structures that capture important NAFLD/NASH stages, including intracellular fat accumulation, inflammation, and fibrosis.
This OOC model allows for precise elucidation of the mechanistic effects of compounds and easy manipulation of models to suit research needs.9
It provides long-term (>1 month) in vitro co-cultures of primary human hepatocytes, stellate, and Kupffer cells in 3D microtissue structures that capture important phenotypes of NAFLD/NASH stages, including intracellular fat accumulation, inflammation, and fibrosis.
This OOC model allows for precise elucidation of the mechanistic effects of compounds and easy manipulations to suit research needs.9
A co-authored research publication, led by the University of Cambridge in 2020,10 shows the direct conversion between this NASH model, human clinical studies, and in vivo mouse models.
Their findings demonstrate that the PhysioMimix OOC NASH disease model provides more human-relevant data compared to standard in vitro liver tissue studies. It also offers a quicker and more cost-effective option for studying NASH pathophysiology than lengthy and expensive animal testing, while yielding similar results.
The data generated using the NASH model not only provides valuable insights into the complex cellular mechanisms that cause liver fibrosis but also identified a new pathway that regulates the underlying mechanism of NASH, potentially revealing new therapeutic targets.
The results speak for themselves, OOC is certainly a useful tool to help reduce, refine, and complement existing tests. While mice capture the complexity of a full organism, OOC models excel at demonstrating how disease mechanisms or the effects of drugs differ in a human setting.
Combining these two approaches offers a more robust and comprehensive insight, enhancing translational research, and potentially paving the way for a successful (NASH) therapeutic to make it to market. In December 2023, CN Bio announced that their PhysioMimix NASH assay was used to provide human-relevant data on compound efficacy to support the initiation of Inipharm’s Phase 1 clinical trial for INI-822. The submission represents the first example of an OOC provider’s data supporting the clinical progression of a drug for a complex metabolic liver disease and demonstrates the transformative potential of the approach within preclinical programs
As advanced in vitro technologies continue to increase in complexity - from individual OOC models, operating in isolation, to multi-organ models that begin systemic functions, and ultimately, human body-on-a-chip configurations, the future vision is more decisively focused on reducing and replacing the dependence on animal models.
Adopting CN Bio’s NASH model into the workflow
CN Bio has developed a distinctive product called NASH in-a-box, which is tailored for use with its PhysioMimix® OOC microphysiological systems (MPS). The kit includes all the necessary components, including 3D validated primary human cells, media, and consumables, to recreate CN Bio’s NASH disease model in the customer’s laboratory.
Combined with software-guided protocols (which walk users through the experimental process step-by-step), the kit simplifies the entire workflow, permitting rapid adoption. Alternatively, users can access the model through CN Bio’s portfolio of Contract Research Services, where their team of experienced scientists works with customers to design and optimize OOC experiments, run endpoint assays, and analyze the data to generate comprehensive reports.
References and further reading
- Rubiano, A., Indapurkar, A., Yokosawa, R., Miedzik, A., Rosenzweig, B., Arefin, A., Moulin, C.M., Dame, K., Hartman, N., Volpe, D.A., Matta, M.K., Hughes, D.J., Strauss, D.G., Kostrzewski, T. and Ribeiro, A.J.S. (2021), Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation. Clin Transl Sci, 14: 1049-1061. https://doi.org/10.1111/cts.12969
- Horie Y, Suzuki A, Kataoka E, Sasaki T, Hamada K, Sasaki J, Mizuno K, Hasegawa G, Kishimoto H, Iizuka M, Naito M, Enomoto K, Watanabe S, Mak TW, Nakano T. Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas. J Clin Invest. 2004 Jun;113(12):1774-83. doi: 10.1172/JCI20513. PMID: 15199412; PMCID: PMC420505.
- Ibrahim SH, Hirsova P, Malhi H, Gores GJ. Animal Models of Nonalcoholic Steatohepatitis: Eat, Delete, and Inflame. Dig Dis Sci. 2016 May;61(5):1325-36. doi: 10.1007/s10620-015-3977-1. Epub 2015 Dec 1. PMID: 26626909; PMCID: PMC4838538.
- Van Herck MA, Vonghia L, Francque SM. Animal Models of Nonalcoholic Fatty Liver Disease-A Starter’s Guide. Nutrients. 2017 Sep 27;9(10):1072. doi: 10.3390/nu9101072. PMID: 28953222; PMCID: PMC5691689.
- Scholten D, Trebicka J, Liedtke C, Weiskirchen R. The carbon tetrachloride model in mice. Lab Anim. 2015 Apr;49(1 Suppl):4-11. doi: 10.1177/0023677215571192. PMID: 25835733.
- Takahashi Y, Soejima Y, Fukusato T. Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol. 2012;18(19):2300-2308. doi:10.3748/wjg.v18.i19.2300
- Trak-Smayra V, Paradis V, Massart J, Nasser S, Jebara V, Fromenty B. Pathology of the liver in obese and diabetic ob/ob and db/db mice fed a standard or high-calorie diet. Int J Exp Pathol. 2011 Dec;92(6):413-21. doi: 10.1111/j.1365-2613.2011.00793.x. Epub 2011 Nov 25. PMID: 22118645; PMCID: PMC3248077.
- Criver.com – Nonalcoholic Steatohepatitis NASH Models
- Kostrzewski T, Maraver P, Ouro-Gnao L, Levi A, Snow S, Miedzik A, Rombouts K, Hughes D. A Microphysiological System for Studying Nonalcoholic Steatohepatitis. Hepatol Commun. 2019 Nov 13;4(1):77-91. doi: 10.1002/hep4.1450. PMID: 31909357; PMCID: PMC6939502.
- Vacca M, Leslie J, Virtue S, Lam BYH, Govaere O, Tiniakos D, Snow S, Davies S, Petkevicius K, Tong Z, Peirce V, Nielsen MJ, Ament Z, Li W, Kostrzewski T, Leeming DJ, Ratziu V, Allison MED, Anstee QM, Griffin JL, Oakley F, Vidal-Puig A. Bone morphogenetic protein 8B promotes the progression of non-alcoholic steatohepatitis. Nat Metab. 2020 Jun;2(6):514-531. doi: 10.1038/s42255-020-0214-9. Epub 2020 Jun 8. PMID: 32694734.
About CN Bio
CN Bio is a leading organ-on-a-chip (OOC) company that offers a portfolio of products and contract research services to optimise the accuracy and efficiency of bringing new medicines to market. With more than a decade of research and development experience, we aim to transform the way human-relevant pre-clinical data is generated through the development of advanced in vitro human organ models.
CN Bio's PhysioMimix® OOC range of microphysiological systems (MPS) enable researchers to recreate human biology in the lab. The technology bridges the gap between traditional cell culture and human studies, to support the development of safer and more efficacious therapeutics, whilst reducing the dependence on animal model usage.
CN Bio’s portfolio of products (MPS, 3D validated cells, consumable plates) and services support researchers that require reliable, data-rich, in vitro studies, to uncover novel mechanistic insights into drug or disease mechanism of action.
Sponsored Content Policy: News-Medical.net publishes articles and related content that may be derived from sources where we have existing commercial relationships, provided such content adds value to the core editorial ethos of News-Medical.Net which is to educate and inform site visitors interested in medical research, science, medical devices and treatments.