Researchers in South Korea have developed an organoid disease model and non-destructive stiffness analysis technology for evaluating the efficacy of new drugs for fatty liver disease.

The research team led by Dr. Hyunwoo Kim and Dr. Myungae Bae at the Korea Research Institute of Chemical Technology (KRICT) has developed the nano-probe-based quantitative stiffness measurement technique for a non-alcoholic fatty liver disease (NAFLD) simulate artificial organoid model while minimizing tissue damage.

Non-alcoholic fatty liver disease occurs when excessive fat accumulates in liver cells due to overeating or lack of exercise, making the liver softer. Over time, the condition can progress to fibrosis, cirrhosis, and ultimately life-threatening diseases such as liver cancer. Therefore, in drug development for liver diseases, identifying effective treatments at the early fatty liver stage is crucial.

Drug development for liver diseases involves repeated testing of candidate drugs on disease-mimicking organoids and analyzing their responses. Especially, physical stiffness of liver tissues are useful maker indicating progress of NAFLD; however, continuous and in-situ measurements of their stiffness are challenging, because current measurement methods are mainly relying on press down the entire area of organoid until destruction.

The research team has developed a technique that enables the non-destructive and in-situ measurement of liver organoids modeled with fatty liver disease while keeping them alive. By applying a few nano-Newton forces onto probe-organoid interface using nano-scale probes, the team was able to quantitatively measure localized stiffness without damaging the organoid.

To prove feasibility of nano-probe based local stiffness measurement technique, they stained the artificial organoid with a dye to identify fat accumulation rich/poor regions, where strong/weak fluorescence intensities are observed, respectively.

For the stiffness measurement, “nano-probe” with a apex size of tens of nanometers attached on a minuscule cantilever was slowly indented into organoid tissues.

The degree of bending of the nano-probe while pressing the organoid was accurately measured through laser reflection on the probe surface. By correlating the degree of bending to stiffness, quantified stiffness value was acquired as parameterized in Young’s modulus.

Unlike conventional methods that required chemical fixation, which killed the organoid, the new nano-probe technique allows “Live” measurements while maintaining the organoid’s viability in culture media. Additionally, by applying a shallow indentation of approximately 5 micrometers, the method does not damage the liver tissue at all.

Applying the newly developed “nano-probe stiffness measurement technology” to non-alcoholic fatty liver organoid models revealed that the stiffness of fat-accumulated regions emitting strong fluorescence was approximately 35% softer in terms of Young’s modulus compared to areas with weak fluorescence. This confirms the ability to precisely target specific regions.

By utilizing fluorescence imaging of lipid accumulation to determine measurement locations, the total measurement time was reduced by more than half compared to random sampling methods. Furthermore, post-measurement liver cell viability was maintained at over 97%, demonstrating minimal tissue damage.

Moving forward, the research team aims to develop a continuous drug evaluation system that allows non-destructive monitoring of disease progression in a single organoid over multiple stages.

The research team stated, “This technology enables convenient analysis of disease model changes during fatty liver drug development.” KRICT President Lee Young-Kook added, “We expect this technology to be widely applicable not only for liver disease drug development but also for other disease treatment advancements.“