Cell Morphology and Growth Under Daily vs. Skip- Day Feeding

To assess the impact of daily vs. skip-day feeding regimens, brightfield images of PSXi011-D and PSXi013-E iPSC lines were taken at Day 1 and Day 4 of passage 4 (P4) (Figure 2A). At Day 1, cells in both conditions demonstrated robust attachment and characteristic hiPSC colony morphology, with no visible differences in cell density or confluency. By Day 4, cultures in both conditions had expanded significantly, forming tightly packed colonies. No major morphological differences were observed between the daily and skip-day conditions, indicating that both feeding schedules effectively supported hiPSC growth and maintenance.

Cell Viability, Expansion, and Doubling Time Analysis

To quantitatively compare growth performance across conditions, cell viability, fold expansion, cell density at harvest, and doubling time were measured using three passages as biological replicates (Figure 2B).

• Cell Viability at Harvest: Both PSXi011-D and PSXi013-E exhibited high post-harvest viability, exceeding 90% in both daily and skip-day conditions, with no significant differences between groups.
• Fold Expansion: On average, both cell lines achieved an ≥8- fold expansion per passage, meeting expectations for robust hPSC culture. No statistically significant differences were observed between daily and skip-day conditions.
• Cell Density at Harvest: By the end of each passage, cell densities remained comparable across conditions, indicating that reduced feeding frequency did not negatively impact proliferation rates.
• Doubling Time: Both conditions maintained an average doubling time of 18-24 hours, in line with standard hPSC growth expectations.

Overall, these results confirm that the defined reagent paradigm, incorporating HiDef-B8 and Ready-CEPT, supports reproducible iPSC expansion while enabling flexible feeding schedules. The skip-day feeding approach successfully sustained hPSC growth without compromising cell viability, expansion efficiency, or genomic stability, providing a potential strategy to reduce handling time and media consumption in routine culture.

Cryopreservation and Post-Thaw Recovery

To assess the effectiveness of the combined reagent paradigm for cryopreservation and recovery, cells were frozen at P5 using PluriFreeze cryomedia and thawed under standard conditions. Key post-thaw metrics, including cell viability and live cell recovery, were measured across both daily and skip-day feeding conditions (Figure 3).

Viability at thaw remained high across conditions, with both PSXi011-D and PSXi013-E maintaining ≥80% post-thaw viability, exceeding the 70% viability threshold (blue dotted line). This indicates that the defined workflow supports high survival rates upon thawing, regardless of the media change frequency leading up to cryopreservation. Live cell recovery, calculated as viable cells counted at thaw relative to live cells loaded at freezing, remained above 60% for all conditions, exceeding the 50% live cell recovery threshold (blue dotted line). For PSXi011-D, both daily and skip-day conditions maintained recovery rates above 70%, indicating that the reduced feeding schedule did not negatively impact post-thaw recovery. For PSXi013-E, while recovery was slightly lower in the skip-day condition, it remained within an acceptable range for hPSC cryopreservation.

These results demonstrate that the HiDef-B8 and Ready-CEPT paradigm provides a robust and reproducible approach for hPSC cryopreservation, ensuring high viability and reliable post-thaw recovery across both cell lines. The slight differences in live cell recovery between conditions suggest that feeding frequency before cryopreservation may influence post-thaw outcomes for some cell lines, emphasizing the importance of optimizing culture conditions based on specific experimental needs.

Pluripotency Marker Expression and Genomic Stability

To confirm the retention of pluripotency following cryopreservation and recovery, flow cytometry analysis was performed on PSXi011-D and PSXi013-E iPSCs four days post- thaw (Figure 4A). Both cell lines maintained high protein expression levels of key pluripotency markers, including OCT4, SOX2, NANOG, and SSEA-4, across both daily and skip-day feeding conditions. The percentage of positive cells remained above the expected threshold (dotted line), indicating that the combined reagent paradigm preserves the stem cell phenotype post-thaw with no detectable loss of pluripotency markers.

To assess genomic stability, copy number variation (CNV) analysis was performed on PSXi013-E cultured under both daily and skip-day conditions (Figure 4B). The results showed normal copy number values across all chromosomes, with no detectable gains or losses. These findings confirm that the HiDef-B8 and Ready-CEPT workflow maintains genomic integrity, supporting its reliability for sustained hPSC culture and banking.