All-in-one 3D cell-based hollow liver capsules for drug screening assays: a comprehensive engineering, functional and molecular investigation – REVIL

- Design and evaluation of protocols to trigger the proliferation of primary human hepatocytes;

- Quantitative characterization of the survival, metabolism, and function of hepatocyte spheroids;

- Evaluation of the role of environmental signals

- Improvement of the encapsulation process

- Development of long-term volumetric imaging techniques

- Hepatotoxicity testing and in-depth characterization of optimal artificial lobule cultures

The production process for primary human hepatocyte (PHH) spheroids was optimized from the formation of individual spheroids to the generation of hundreds in batch format. Conditions promoting PHH proliferation (assessed by EdU incorporation, DNA content, spheroid size, and ATP levels) were initially developed outside of alginate hollow capsules. Spheroid formation from both freshly isolated and cryopreserved PHHs was highly reproducible, and the spheroids could be maintained for at least three weeks without developing necrotic cores.

The transfer to capsules was problematic due to the unexplained presence of debris, probably due to the interferance between caclcium present in the gelation bath and hepatocytes. Yet, we had optimized the formation of capsules, (article Suire et al), developed imaging techniques dedicated to long term culture (article Jana et al) and microfluidic sorting methods (article Rembotte et al.). We then pusued our efforts outside the capsules.

Under these culture conditions, hepatocytes exhibited polarization and expressed apical markers such as MRP2. PHH spheroids, whether subjected to a proliferation step or not, preserved over time key metabolic functions that, in the native liver, are spatially zonated along the lobule. Notably, the expression and activity of drug-metabolizing enzymes remained inducible by prototypical inducers, thereby recapitulating, at least in part, the metabolic heterogeneity of the hepatic lobule. Furthermore, spheroids, irrespective of prior proliferation, reliably predicted drug-induced hepatotoxicity (e.g., Bozentan, acetaminophen)

Complete the previous study with a proteomic analysis and the preparation of a manuscript in collaboration with the consortium.

Find a way to apply mechanical constraints in order to study an alternative to hepatic zonation.

Test our liver lobules in real-world hepatotoxicity assays, with the goal of identifying new active compounds for liver-related diseases.

Hepatotoxicity testing is a key challenge in drug validation. There is an urgent need for the design and development of reliable vitro human hepatic models that may be eligible for the safety evaluation of chemical compounds. While primary hepatocytes (PHHs) remain the gold standard, the long-term maintenance of viable and fully functional PHHs in culture is a bottleneck. While plethora of new products (on the market) or original technologies and protocols (in scientific journals) are blooming, significant advances in the field seem to be hampered by a lack of in-depth rationalized and comparative investigations aiming at increasing the sensitivity, sustainability and high throughput capability of drug testing assays. The first aim of REVIL is to provide an integrated and quantitative approach amenable to standardized and reliable high throughput liver drug screening. Our overarching strategy is guided by physiomimetic considerations and relies on technological advances. First, on the basis on very recent published works and preliminary findings from the consortium, we will establish robust protocols to recapitulate in vitro the proliferative in vivo capability of PHHs encountered during liver regeneration. Second, we will form hepatic spheroids using our Cellular Capsules Technology that generates hollow permeable shells enclosing cells without disrupting cell-cell and cell-matrix interactions. This versatile technique is ideally suited for i) 3D co-culture, ii) harmless culture in stirred-bioreactors, and iii) investigation of mechanotransduction processes. We will thus uniquely recapitulate in a controlled fashion the environmental cues received by hepatocytes in vivo, including the physical interactions between PHHs and non-parenchymal cells, the secreted chemical signals, the physioxic conditions and, maybe more importantly, mechanical cues such as compressive forces. This controlled micro-compartmentalization strategy is expected to ensure the formation of metabolic zonated-like architecture within 3D hepatic spheroids for the first time. Our working hypothesis is that such a biomimetic recapitulation should generate cell-based systems with unprecedented in vitro longevity, functionality and sensitivity in drug testing assays. Our interdisciplinary consortium has been assembled to gather all necessary expertise. It is composed of i) a biophysics team that pioneered and valorized the Cellular Capsule Technology, ii) a biology team expert in liver cell biology and microdissection-assisted proteomics, and iii) a second biology team specialized in the regulation pathways of zonated genes expression in hepatocytes and in the development of biotherapies for liver diseases.