Biostation ct

hESC (line H9, WiCell) was expanded and differentiated as previously described 21; however, EBs were not transferred to attachment culture conditions as done previously, but were maintained in 3D culture throughout differentiation using bacteriological dishes and low attachment six‐well plates (Corning, New York, USA). For IGF‐1 treatment, cultures were differentiated in ventral neural induction media (VNIM) media 21 supplemented with recombinant human IGF‐1 (5 ng/ml, Sigma‐Aldrich, San Louis, USA) until day 37, then in basal knockout serum‐free media 14 with 10 ng/ml IGF‐1 until day 90 (Fig. 1Aii). Control cultures were differentiated in parallel in the absence of IGF‐1 (Fig. 1Ai). Time‐lapse capture of culture morphology was achieved using a BioStation CT (Nikon Corporation, Tokyo, Japan).

Cells were plated at approximately 25% confluence on collagen IV or fibronectin coated (24) well plates. Time lapse microscopy was performed using a BioStation CT (Nikon) and the change in cell number after 48 hours was determined by manually counting cells per vision field. Numerical data presented is the average of at least three biological replicates +/- the standard deviation. Statistical analysis of attachment was performed using the Student's t-test method. Statistical significance was determined if the two-sided p value of the test was < 0.05.

The human cervical cancer cell line HeLa was obtained from Dr. Masao Kawakita at The Tokyo Metropolitan Institute of Medical Science (Rinshoken) (Tokyo, Japan) [20 (link)] on October 7, 2009. The cells were grown in soft agar in accordance with an assay protocol, a detailed description of which is shown in S1 Table. HeLa cells in 0.35% agarose medium were seeded on a solid layer of 0.7% agarose medium in a 6-well culture plate and incubated for 1 day. Seeding density was kept sufficiently low (500 cells/well) to prevent spheroids from touching each other to analyze the individual growth process of each spheroid via time-lapse imaging of its growth. After 5 mL/well of the growth medium was added, the cells were incubated for 14 days from day 2–15 in a BioStation CT (Nikon, Tokyo, Japan) composed of a transport unit for plate transportation within the incubation area and an observation unit for automated imaging. During this period, the transport unit individually conveys the plates from the storage rack to the observation stage in accordance with configured schedules. Spheroid images were captured automatically in each well at 3 h intervals with a phase-contrast microscope at 10× magnification at each time point. Fig 2 shows an example of time-lapse spheroid growth.

Regarding the image acquisition, MSCs were seeded to 6-well plates (Corning Incorporated, NY, USA) at 2000 cells/cm² (N = 3 wells per each lot) and cultured for 6 days before performing the T-cell proliferation inhibitory potency measurement. Phase-contrast microscopic images of cells were acquired using a BioStation CT (Nikon Corporation) employing a previously reported method of mesenchymal stem cell image acquisition, and tiled images (8 × 8 tiling covering 16 mm², 1000 × 1000 pixels²/well) were acquired at 4× magnification. Time intervals were set to 6 h, which began at 12 h following seeding till 138 h. In addition, images containing high noise of debris were eliminated manually.

Cell migrating ability of IGF-1R silenced pancreatic cancer cells were measured using the scratch assay. Briefly, cells were seeded in 6-well plates at a density of 3.5×10⁵ cells/well for IGF-1R transfection. At this density, PANC-1 and HPAC cells reached monolayer confluency after 48×h. A straight wound or scratch was then gently created in the cell monolayers with a sterile pipette tip. Cells detached by the scratch were washed twice with PBS and cultures were then supplemented with fresh medium and monitored for 96 h at 37°C using the Biostation CT (Nikon Instruments Inc. Melville, NY, USA) for continuous observation. The Biostation was automatically programmed to capture photographs at 2 h intervals up to 96 h. Migration images were captured and documented at different time points using NIS-Element AR software.

Cell migration was evaluated using scratch assay as described in our previous study [41 (link)]. HPAC and MIAPaCa-2 cells were seeded on a 6 well plate at a density of 8 × 10⁵ cells/well and 6 × 10⁵ cells/well for PANC-1. Cells were cultured at 37°C in 5% CO₂ until monolayer confluency was reached. A scratch was made with a sterile pipette tip and washed twice with PBS and fresh media was added. Cells were treated with 15μM gedunin or vehicle (Dimethyl Sulfoxide).
HPAC cells were further studied using 1μg/ml of rhShh, 15μM gedunin, 5μM GANT-61, and combinations such as 15μM gedunin with 1μg/ml rhShh and 5μM GANT-61 with 1μg/ml rhShh in order to understand the involvement of sonic hedgehog signaling in pancreatic cancer cell migration. Images were taken at 2h intervals for a time period of 72h at 37°C using the Biostation CT (Nikon Instruments Inc. Melville, NY, USA). The distance migrated by the cells was calculated using NIS-Element AR software (24809702).