Fig. 3: Assessment of long-term expansion and stock banking potential for ISCs^3D-hIO cultured on XF-DISC.

a Schematic illustration of the long-term expansion capability and implementation of a stock banking system for ISCs^3D-hIO cultured on XF-DISC. b Theoretical growth curve of ISCs^3D-hIO cultured on a Matrigel-coated surface; XF-DISC and TCPS were estimated, and the cell numbers were quantified. c d Theoretical growth rate and experimentally determined growth rate after ISCs^3D-hIO were passaged for four passages on a Matrigel-coated surface and XF-DISC, respectively. e Morphologies of ISCs^3D-hIO on a Matrigel-coated surface and XF-DISC over the course of long-term expansion culture across 30 sequential passages. Scale bar: 1 mm (n = 3 samples/group). f Morphologies of ISCs^3D-hIO on a Matrigel-coated surface and XF-DISC after freezing and thawing cycles on Days 2 and 5 (n = 3 samples/group). Before freezing, the number of passages was 10. Scale bar: 500 µm. g Quantitative efficiency of viable cells after freezing and thawing. Data represent the mean ± SD (n = 3 biological samples), and a two-tailed t-test was applied to measure p values. h WST-1 assay of thawed ISCs^3D-hIO cultured for 5 days on a Matrigel-coated surface and XF-DISC. Data represent the mean ± SD (n = 3 biological samples), and a two-tailed t-test was applied to measure p values. i Quantitative efficiency of viable cells after the freezing and thawing of stocks stored for more than 3 years. Data represent the mean ± SD (n = 3 biological samples), and a two-tailed t-test was applied to measure p values. j WST-1 assay of thawed ISCs^3D-hIO cultured for 5 days on a Matrigel-coated surface and XF-DISC. Stocks were stored for more than 3 years. Data represent the mean ± SD (n = 3 biological samples), and a two-tailed t-test was applied to measure p values.