A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT).

Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.

Rapid and Stable Formation Method of Human Astrocyte Spheroid in a High Viscous Methylcellulose Medium and Its Functional Advantages.

Astrocytes, a type of glial cell in the brain, are thought to be functionally and morphologically diverse cells that regulate brain homeostasis. Cell immortalization is a promising technique for the propagation of primary human astrocytes. The immortalized cells retain their astrocytic marker mRNA expression at lower levels than the primary cells. Therefore, improvement of the differentiation status is required. The use of a 3D formation technique to mimic structural tissue is a good strategy for reflecting physiological cell-cell interactions. Previously, we developed a spheroid formation method using highly viscous methyl cellulose (MC) medium. In this study, we applied this formation method to the well-established immortalized human astrocyte cell line HASTR/ci35. Stable HASTR/ci35 spheroids were successfully formed in MC medium, and laminin deposition was detected inside of the spheroids. Their functional markers were enhanced compared to conventional spheroids formed in U-bottom plates. The inflammatory response was moderately sensitive, and the ability to support neurite growth was confirmed. The HASTR/ci35 spheroid in the MC medium demonstrated the differentiation phenotype and could serve as a potent in vitro model for matured astrocytes.

Enhancement and maintenance of hepatic metabolic functions by controlling 3D aggregation of cryopreserved human iPS cell-derived hepatocyte-like cells.

Hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells are potent cells to study individual-specific hepatotoxicity for drug screening test. However, the functions of metabolic enzymes are practically low. Here, we reconstituted stable and compact 3D spheroids of commercially available cryopreserved HLCs by our original spheroid formation method with high viscous methylcellulose medium. 3D formation enhanced the hepatic functions and maintained the functions for 14 days. Especially, the expression of cytochrome P450s was 10- to 100-fold enhanced compared to conventional 2D culture, which is applicable to a typical drug-metabolizing test using liquid chromatograph-tandem mass spectrometer. In conclusion, we successfully formed human HLC spheroid from commercially available cryo-preserved cells, which realized remarkable hepatic maturation by prolonged 3D culture, especially in terms of drug-metabolizing enzymes. Our spheroid formation technology has the potential to make HLC spheroids a potent tool in aspects of pharmaceutical research, such as drug screening and pharmacokinetic studies.

Development of a tunable method to generate various three-dimensional microstructures by replenishing macromolecules such as extracellular matrix components and polysaccharides.

Multicellular spheroids (spheroids) are expected to be a promising approach to mimic in vivo organ functions and cell microenvironments. However, conventional spheroids do not fully consider the existence of extracellular matrices (ECMs). In this study, we developed a tunable method for replenishing macromolecules, including ECM components and polysaccharides, into spheroids without compromising cell viability by injecting a microvolume cell suspension into a high density of methylcellulose dissolved in the culture medium. Adjusting the ECM concentration in the cell suspension enabled the generation of different three-dimensional microstructures, such as "ECM gel capsules", which contained individually separated cells, and "ECM-loaded spheroids", which had thin ECM layers between cells. ECM-loaded spheroids with a 30-fold dilution of Matrigel (0.3 mg/ml) showed significantly higher albumin secretion than control spheroids composed of Hep G2 or HuH-7 cells. Additionally, the expression levels of major CYP genes were decreased in ECM gel capsules with undiluted Matrigel (9 mg/ml) compared to those in control spheroids. However, 0.3 mg/ml Matrigel did not disrupt gene expression. Furthermore, cell polarity associated with tight junction proteins (ZO-1 and Claudin-1) and the transporter protein MRP2 was markedly induced by using 0.3 mg/ml Matrigel. Thus, high-performance three-dimensional tissues fabricated by this method are applicable to increasing the efficiency of drug screening and to regenerative medicine.

Improved Oxygen Supply to Multicellular Spheroids Using A Gas-permeable Plate and Embedded Hydrogel Beads.

Culture systems for three-dimensional tissues, such as multicellular spheroids, are indispensable for high-throughput screening of primary or patient-derived xenograft (PDX)-expanded cancer tissues. Oxygen supply to the center of such spheroids is particularly critical for maintaining cellular functions as well as avoiding the development of a necrotic core. In this study, we evaluated two methods to enhance oxygen supply: (1) using a culture plate with a gas-permeable polydimethylsiloxane (PDMS) membrane on the bottom, and; (2) embedding hydrogel beads in the spheroids. Culturing spheroids on PDMS increased cell growth and affected glucose/lactate metabolism and CYP3A4 mRNA expression and subsequent enzyme activity. The spheroids, comprised of 5000 Hep G2 cells and 5000 20 µm-diameter hydrogel beads, did not develop a necrotic core for nine days when cultured on a gas-permeable sheet. In contrast, central necrosis in spheroids lacking hydrogel beads was observed after day 3 of culture, even when using PDMS. These results indicate that the combination of gas-permeable culture equipment and embedded hydrogel beads improves culture 3D spheroids produced from primary or PDX-expanded tumor cells.

Generation of Hepatic Tissue Structures Using Multicellular Spheroid Culture.

The hepatic functions of the hepatocytes in multicellular spheroid (MCS) are lower than those in the liver. One of the causes is that conventional hepatic MCSs do not reproduce liver-specific microstructures such as hepatic cord. It is necessary to design the inner structure of hepatic MCSs mimicking a structural feature of hepatic cord to represent further hepatic functions. Here we introduce a unique method to engineer the microarchitectures in the MCSs by formation of void spaces or filling of extracellular matrices (ECMs).

Triple quadrupole mass spectrometry comparative DNA adductomics of Hep G2 cells following exposure to safrole.

A DNA adduct screening pipeline was constructed to apply triple quadrupole mass spectrometry comparative DNA adductomics to investigate the effects of the naturally-occurring plant constituent, safrole (4-allyl-1,2-methylenedioxybenzene), on human hepatoma cells, Hep G2. DNA from Hep G2 cells that were exposed to or not exposed to safrole were digested to 2'-deoxynucleosides and analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) whereby the neutral loss of 2'-deoxyribose was targeted by monitoring the [M+H]+ > [M+H - 116]+ transition over a defined range. Comparative analyses through construction of DNA adductome maps revealed numerous putative DNA adduct candidates. Targeted product ion scan investigations allowed for detailed fragmentation ion analyses and the identities of at least five bulky alkylated adducts of 2'-deoxyguanosine and 2'-deoxyadenosine with molar masses greater than 400 Da each were proposed. All adducts were derived from safrole exposure and pathways to explain the occurrence of these adducts in Hep G2 cells through metabolism of safrole are discussed. This study demonstrates the potential utility of constructing triple quadrupole MS comparative DNA adductomics pipelines to screen chemicals for DNA adducts by using human cell lines.