Journal: Nature Communications

Article Title: Large scale and integrated platform for digital mass culture of anchorage dependent cells

doi: 10.1038/s41467-019-12777-3

Figure Lengend Snippet: Real-time monitoring of various cells cultured on a single-layer CCP. a Photographic image of four single-layer CCPs in an incubator, controlled wirelessly by a single laptop. b Screenshot images of the user interactive software for each mode of monitoring and stimulations: impedance sensing (top left), pH/K + sensing (bottom left), electrical stimulation (top right), and temperature sensing/thermal stimulation (bottom right). c Monitored data (impedance, temperature, pH, and K + ) for cultures of four types of cells (hDFB human dermal fibroblasts, hMSC human mesenchymal stem cell, C2C12 mouse myoblast, HL-1 mouse cardiac muscle cell; mean). In the C2C12 culture, the growth medium was changed to the differentiation medium on day 5 (arrow). d – g Impedance mappings of hDFB ( d ), hMSC ( e ), C2C12 ( f ), and HL-1 ( g ). The blue color indicates the lowest impedance, whereas red color indicates the impedance in maximum. h Growths of four types of cells as determined by the WST-8 assay. The absorbance is increased as the number of cells is increased. i Fluorescent microscopic images of F-actin-stained C2C12 cells during proliferation (top, scale bar: 10 μm) and myosin heavy chain staining of C2C12 cells showing myotube formation of the cells during differentiation (bottom, scale bar: 50 μm). j Expression of the muscle-specific gene for myogenin (red line) and MyoD (blue line) in C2C12 cells ( n = 3, mean ± s.d., myogenin compared to day 0, * P < 0.05, ** P < 0.01; MyoD compared to day 0 # P < 0.05, ## P < 0.01, ANOVA). k Fluorescent microscopic images of F-actinstained HL-1 during proliferation (top, scale bar: 40 μm) and connexin 43 (Cx43) expression of the cells during differentiation (bottom, scale bar: 30 μm)

Article Snippet: C2C12 myoblasts (C2C12; CRL-1772, ATCC), hDFB (PCS-201-012, ATCC), HL-1 (SCC065, Merck), and hMSC (PT-2501, Lonza) were used for the experiments.

Techniques: Cell Culture, Software, Staining, Expressing

Journal: Nature Communications

Article Title: Large scale and integrated platform for digital mass culture of anchorage dependent cells

doi: 10.1038/s41467-019-12777-3

Figure Lengend Snippet: Applications of multilayerd LISCCPs. a Photographs showing a multilayered LISCCP used for in vitro drug toxicity testing of drugs and cosmetics. LISCCP is divided into two single-layer CCPs and one triple-layered CCP to conduct three different tests. Scale bar: 5 cm. b Testing to evaluate the effects of 7CZ NP-mediated ROS scavenging on HL-1 using single-layer CCPs. The impedance mappings of HL-1 culture along with its corresponding live (green)/dead (red) cell images are shown for the treatment of H 2 O 2 +7CZ NPs (top) and H 2 O 2 only (bottom). Scale bar: 500 μm. c Triple-layered impedance mapping of HL-1 treated with H 2 O 2 at one corner and 7CZ NPs at the opposite corner (left) and their live/dead cell images (right). Scale bar: 500 μm. d Viability of hDFB cultured on each layer of LISCCP at t = 0 ( n = 8, mean ± s.d., ns, not significant, ANOVA). e Impedance mappings and live/dead cell images of hDFB treated with phosphate-buffered saline (PBS), H 2 O 2 , or one of the surfactants (ALS ammonium lauryl sulfate, BAC benzalkonium chloride, Tween TWEEN 60). Scale bar: 100 μm. f Viability and number of hMSC cultured on LISCCP for three passages (cell number, n = 8, * P < 0.001 versus passage 1, # P < 0.01 versus passage 2, ANOVA with Bonferroni’s post-test; cell viability, n = 4, Box: median; 25th to 75th percentiles, Whisker: min to max, ns, not significant, ANOVA). Large expansion of cells shows the utilization of LISCCP in large-scale cell manufacturing for cell therapy. g Caspase-3 gene expression of hMSC cultured on LISCCP for three passages ( n = 3, ns, not significant, ANOVA with Bonferroni’s post-test). h Staining images of h MSC differentiated into osteogenic (left, Alizarin red O staining) and adipogenic (right, Oil red O staining) lineage cells. Red indicates osteogenic or adipogenic lineage cells. Scale bar: 50 μm. i Impedance monitoring of hMSC cultures that were induced to undergo osteogenic and adipogenic differentiation by medium change on day 7 (arrow) when the cells were 90% confluent

Article Snippet: C2C12 myoblasts (C2C12; CRL-1772, ATCC), hDFB (PCS-201-012, ATCC), HL-1 (SCC065, Merck), and hMSC (PT-2501, Lonza) were used for the experiments.

Techniques: In Vitro, Cell Culture, Saline, Whisker Assay, Gene Expression, Staining