Fig. 3. Differentiation and physiological characters of ORN precursor spheroids. (A) Fluorescence image of odor-activated ORNs. (B) Increase fluorescence levels of calcium indicator upon odorant mixture stimulation. (C) Net-odor evoked intracellular calcium concentration depending on the ORN precursor spheroids cultivation time (n=47 in day 3, n=58 in day 5, n=39 in day 10). (D) Response rate of ORNs depending on spheroid cultivation time (n=60 in day 3, n=97 in day 5, n=89 in day 10). (E) Top left is a whole window of confocal microscope software and white circles indicate cells of interest. Right three figures are magnified images. Bottom graphs show the change of fluorescence intensity upon odorant stimulation. Buffer solution, 100 µM IBMP, 100 µM IVA, and 100 µM citralva were treated at 50 sec, 245 sec, 500 sec, respectively. X axis is 50 sec and y axis is 10% change of fluorescence intensity. Scale bar in the left figure is 200 µm and in the right three figures is 40 µm. (F) Schematic illustration of cryopreservation of ORN spheroids, thawing, and differentiations. Five days cultivated ORN spheroids were cryopreserved for 35 days and thawed on the laminin coated plate for 72 hours for differentiation. (G) Net odor-evoked intracellular calcium concentration of the differentiated ORNs from cryopreserved ORN spheroids upon odorant mixture stimulation. (H) Response rate of the differentiated ORNs from cryopreserved ORN spheroids upon odorant mixture stimulation (number of experiments=8, total monitored cell number=75, *<0.05). Data were obtained from three separate animal culture experiments.
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