Single Cell Array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based Cell Classification and Selective Photorelease of Cells.

Single cell arrays provide an accurate classification of analyte cells through an image-based analysis of cellular phenotypes. Light-guided cell retrieval from a single cell array is a promising approach for the rapid and simple sorting of difficult to distinguish cells. In this study, we developed a single cell array enclosed with a photodegradable hydrogel in microwells to enable both comprehensive image-based single cell analysis and light-guided cell retrieval. In this system, individual cells became trapped in the microwells together with the photodegradable hydrogel at a high cell density on a chip regardless of cell type, adhesiveness, and motility. Fluorescence-stained model cells and vaccinated dendritic cells were identified by microscopic imaging and then selectively released through the light-induced degradation of the cell-embedding hydrogels. The target cells were selectively retrieved with a purity of >95% from the cell mixture through rapid photorelease, and the retrieved cells were confirmed to grow normally. Our results provide proof-of-principle that the photoresponsive microwell array serves as a versatile tool for image-based cell sorting in cellular researches and the manufacturing processes of high-performance cells.

A Single-Batch Fermentation System to Simulate Human Colonic Microbiota for High-Throughput Evaluation of Prebiotics.

We devised a single-batch fermentation system to simulate human colonic microbiota from fecal samples, enabling the complex mixture of microorganisms to achieve densities of up to 1011 cells/mL in 24 h. 16S rRNA gene sequence analysis of bacteria grown in the system revealed that representatives of the major phyla, including Bacteroidetes, Firmicutes, and Actinobacteria, as well as overall species diversity, were consistent with those of the original feces. On the earlier stages of fermentation (up to 9 h), trace mixtures of acetate, lactate, and succinate were detectable; on the later stages (after 24 h), larger amounts of acetate accumulated along with some of propionate and butyrate. These patterns were similar to those observed in the original feces. Thus, this system could serve as a simple model to simulate the diversity as well as the metabolism of human colonic microbiota. Supplementation of the system with several prebiotic oligosaccharides (including fructo-, galacto-, isomalto-, and xylo-oligosaccharides; lactulose; and lactosucrose) resulted in an increased population in genus Bifidobacterium, concomitant with significant increases in acetate production. The results suggested that this fermentation system may be useful for in vitro, pre-clinical evaluation of the effects of prebiotics prior to testing in humans.

Comparison of the Growth of Lactobacillus delbrueckii, L. paracasei and L. plantarum on Inulin in Co-culture Systems.

Lactobacillus delbrueckii TU-1, which apparently takes intact inulin into its cells and then degrades it intracellularly, was co-cultured in vitro with L. paracasei KTN-5, an extracellular inulin degrader; or L. plantarum 22A-3, a strain that is able to utilize fructose but not inulin; or both in order to prequalify inulin as a prebiotic agent in vivo. When L. delbrueckii TU-1 was co-cultured with L. paracasei KTN-5 on fructose or inulin, the growth of L. delbrueckii TU-1 on inulin was markedly higher than that of L. paracasei KTN-5, whereas the growth of L. delbrueckii TU-1 on fructose was much lower than that of L. paracasei KTN-5. These results suggest that L. delbrueckii TU-1 and L. paracasei KTN-5 were efficient at utilizing inulin and fructose, respectively. When L. plantarum 22A-3 was co-cultured with L. delbrueckii TU-1 on inulin, the growth of L. plantarum 22A-3 was enhanced by L. paracasei KTN-5 but not by L. delbrueckii TU-1, suggesting that the fructose moiety that L. paracasei KTN-5 released temporarily into the medium was "scavenged" by L. plantarum 22A-3. Thus, L. delbrueckii TU-1, L. paracasei KTN-5, and L. plantarum 22A-3 were then cultured altogether on inulin. The growth of L. delbrueckii TU-1 was unaffected but that of L. paracasei KTN-5 was markedly suppressed. This evidence suggests that prebiotic use of inulin supported the selective growth of intracellular inulin degraders such as L. delbrueckii rather than extracellular inulin degraders such as L. paracasei in the host microbiota.