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Cell Culture


ties and provides some further plasticity, such as tissue remodelling. The next level is to include liver sinusoidal endothelial cells (LSECs), which are important modulators of liver health and critical for both toxicity and inflammatory applications. As key regulators of tissue inflammation, home- ostasis and repair, hepatic stellate cells should be incorporated in models used for any chronic inflammatory studies.


Validation, standardisation and translatability 3D models and their components, the cells, must be well validated to ensure cell-specific marker expression and functionality, both of which are prerequisites for robust model performance and assay readouts. Using adult cell lots requires good QCs, well-


Figure 1


Inducible fatty liver disease model. In humans, the progression of fatty liver


diseases, such as non-alcoholic steatohepatitis (NASH) and fibrosis, starts with lipid accumulation within the


hepatocytes (steatosis). This 3D human liver hepatocyte model shows tight tissue formation and change in


hepatocyte phenotype under healthy control conditions


(left) and diabetic conditions (right) induced with a


specialised media that contains high glucose levels and free fatty acids. Nile Red staining


captures a normal amount of lipids in the healthy


microtissue (green), whereas steatotic hepatocytes are


abnormally enlarged and filled with lipid vacuoles. Hoechst


staining for nuclei (blue) and Cell Mask Deep Red for


plasma membrane structures (red) further illustrate how


disease induction in the liver model mimics the fatty liver


disease state in humans. (Photo credit: ETH Zurich, imaged on a high-content Visitron system)


46


defined pass/fail criteria parameters and often requires testing multiple lots. QC criteria may vary depending on the cell type and must ensure the qualified lot is fit for purpose for downstream applications when integrated in a tissue. This is especially challenging as cell functionality assess- ment in 2D does not always correlate with the same features in multicellular 3D models. Critical parameters for hepatocytes include albu-


matrices, conventional cell culture substrates, in comparison, are quite primitive, consisting only of flat, transparent plastic. The relocated cells must either secrete their own extracellular matrix and rebuild the tissue structure – which we propone – or one can provide artificial assistance through exoge- nous extracellular matrices, such as Matrigel or other, more advanced, formulations. Equally important, blood needs to be replaced by a surro- gate. Sophisticated medium compositions with defined supplements play a key role to support in vitro adaptation and further medium transfers may be needed to comply with in vitro endpoints.


Human biology is complex. Effective models must be, too Cell type composition determines the viability and limitations of in vitro models and usually stands in opposition to costs and quality controls (QCs). More complex models of multiple cell types cost more to create and validate, but they are usually worth the extra time and investment. For example, traditional hepatocyte toxicity assays rely on a sim- ple, single-cell-type model of hepatocytes. A model composed only of hepatocytes, however, cannot detect inflammation-dependent toxicities. Adding Kupffer cells to this model enables the detection of a wider range of inflammation-dependent toxici-


min secretion, inducibility and expression of a large panel of CYP genes, while for Kupffer cells the acti- vation of inflammatory pathways upon exogenous inflammatory stimuli should be assessed. Hepatic stellate cells must fulfill stringent criteria for mor- phology, activation state and activation potential. As with cell lots, the 3D model must perform


according to well-defined QC metrics. Defined metrics are critical when changing models, whether to use a new cell lot, change the model cell compo- sition or introduce new culture conditions. It is crucial to establish a harmonised model def-


inition. Such a definition should clearly state the minimum criteria required to answer defined scien- tific questions by specific experimental approach- es. For example, high throughput screening approaches require a model with high scalability, while physiological cell composition and interac- tion are more relevant for mechanistic studies. In most situations, a model is intended for several applications requiring a compromise between model features and experimental feasibility. Knowing the limitations of your model is as impor- tant as the performance metrics.


3D assay guidelines and considerations ‘Thinking in 3D’ is key to making the transition from monolayers to 3D tissues. Most in vitro bio-


Drug Discovery World Winter 2018/19


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