Ratiometric Nanoviscometers: Applications for Measuring Cellular Physical Properties in 3D Cultures.

New insights into the biomechanical properties of cells are revealing the importance of these properties and how they relate to underlying molecular, architectural, and behavioral changes associated with cell state and disease processes. However, the current understanding of how these in vitro biomechanical properties are associated with in vivo processes has been developed based on the traditional monolayer (two-dimensional [2D]) cell culture, which traditionally has not translated well to the three-dimensional (3D) cell culture and in vivo function. Many gold standard methods and tools used to observe the biomechanical properties of 2D cell cultures cannot be used with 3D cell cultures. Fluorescent molecules can respond to external factors almost instantaneously and require relatively low-cost instrumentation. In this review, we provide the background on fluorescent molecular rotors, which are attractive tools due to the relationship of their emission quantum yield with environmental microviscosity. We make the case for their use in both 2D and 3D cell cultures and speculate on their fundamental and practical applications in cell biology.

Secretome-Based Prediction of Three-Dimensional Hepatic Microtissue Physiological Relevance.

Early biomarkers for indication of the complex physiological relevance (CPR) of a three-dimensional (3D) tissue model are needed. CPR is detected late in culture and requires different analytical techniques. Albumin production, CYP3A4 expression, and formation of bile canaliculi structures are commonly used to compare in vitro hepatic cells to their in vivo counterpart. A universal biomarker independent of the cell type would bring this to a common detection platform. We make the case that these hepatic characteristics are not sufficient to differentiate traditional (2D) cell culture from the more complex 3D culture. We explored the cytokine secretion profile (secretome) for its potential as a 3D early culture biomarker. PDGF-AB/BB and vascular endothelial growth factor (VEGF) were found to be upregulated in 3D compared to 2D cultures at early time points (days 3 and 4). These observations provide a foundation upon which in vivo validation of cytokines can lead to physiologically relevant 3D in vitro cell culture.

Evaluation of cellular adhesion and organization in different microporous polymeric scaffolds.

The lack of prediction accuracy during drug development and screening risks complications during human trials, such as drug-induced liver injury (DILI), and has led to a demand for robust, human cell-based, in vitro assays for drug discovery. Microporous polymer-based scaffolds offer an alternative to the gold standard flat tissue culture plastic (2D TCPS) and other 3D cell culture platforms as the porous material entraps cells, making it advantageous for automated liquid handlers and high-throughput screening (HTS). In this study, we optimized the surface treatment, pore size, and choice of scaffold material with respect to cellular adhesion, tissue organization, and expression of complex physiologically relevant (CPR) outcomes such as the presence of bile canaliculi-like structures. Poly-l-lysine and fibronectin (FN) coatings have been shown to encourage cell attachment to the underlying substrate. Treatment of the scaffold surface with NaOH followed with a coating of FN improved cell attachment and penetration into pores. Of the two pore sizes we investigated (A: 104 ± 4 µm; B: 175 ± 6 µm), the larger pore size better promoted cell penetration while limiting tissue growth from reaching the hypoxia threshold. Finally, polystyrene (PS) proved to be conducive to cell growth, penetration into the scaffold, and yielded CPR outcomes while being a cost-effective choice for HTS applications. These observations provide a foundation for optimizing microporous polymer-based scaffolds suitable for drug discovery. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:505-514, 2018.