Fig. 4

Characterization of clinically available iCEC products. a Scheme of the cell production process for clinical use. b Viability of cryopreserved iCEC products postthawing in several commercial cryopreservation reagents. Cell viability was measured by the AOPI system. c Immunofluorescence staining showing that the iCEC products expressed ZO-1 and Na⁺ K⁺ ATPase. Nuclei were stained with DAPI. Cells were analyzed after 5 days of further iCECs differentiation postthawing the day 24 iCEC products. The reagents used for each cell freezing are shown at the bottom. Scale bars: 50 μm. d Quantification of the positive rate of ZO-1/Na⁺ K⁺ ATPase and N-cadherin cells in cryopreserved iCECs after flow cytometry analysis. e The viability of postthawing iCEC products was measured at 0 h and in cells placed on ice for 6 h before and after simulated injection. These lines represent 3 independent experiments. f Immunofluorescence staining of ZO-1 and Na⁺ K⁺ ATPase for iCEC products, which were cultured for 5 days from e. Cells were on ice for 6 h prior to stimulation injection. Nuclei were stained with DAPI. Scale bars: 50 μm