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Figure 1. CloneR?3 Improves Clonal Generation Following Single-Cell Deposition of hPSCs

Single-cell deposition is the gold standard for clonal derivation, but is typically limited by low efficiency. Four hPSC lines (H1, H9, SCTi003-A, and WLS-1C) were seeded using FACS-based cell seeding to deposit 1 cell per well of a 96-well plate. Clones generated in TeSR?-AOF supplemented with CloneR?3 demonstrate significantly improved cloning efficiency when compared to clones generated with Rho kinase inhibitor (Y-27632) or CEPT. Across four cell lines tested, CloneR?3 had an average cloning efficiency of 29.6 ¡À 11.8%. In contrast, Y-27632 and CEPT had average cloning efficiencies of 9.6 ¡À 8.5% and 11.6 ¡À 8.3%, respectively, with 7 biological replicates per cell line on plates precoated with LN-521. A two-tailed unpaired t-test was conducted for each cell line comparison where * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.

Figure 2. CloneR?3 Improves Cloning Efficiency Compared to Y-27632

Four hPSC lines (H1, H9, SCTi003-A, and WLS-1C) were seeded at clonal density (50 cells/cm?) in TeSR?-AOF supplemented with Y-27632 or CloneR?3 on Vitronectin XF?. (A) Supplementation with CloneR?3 results in increased cloning efficiency of hPSCs when compared to Y-27632. (B) Representative whole-well images of two hPSC lines (H9 and WLS-1C) on Day 10 after seeding show the increase in the number of clones observed per well when supplemented with CloneR?3. A two-tailed unpaired t-test was conducted for each cell line comparison where * denotes p < 0.05.

Figure 3. CloneR?3 Is Compatible with Single-Cell Seeding of hPSCs

Three hPSC lines (H1, H9, and SCTi003-A) were seeded as single cells at 9 x 10? to 14 x 10? cells/cm? on Laminin-521 in TeSR?-AOF supplemented with Y-27632, CEPT, or CloneR?3 for the first 24 hours. After five days in culture, cells were harvested and counted. In the H9 and SCTi003-A cell lines, supplementation with CloneR?3 results in a trend toward improved fold expansion when compared with Y-27632 or CEPT. Each point represents one biological replicate averaged from two technical replicates. Bars represent the mean ¡À SD from the average of three biological replicates.

Figure 4. CloneR?3 Improves Cell Yields During the Transition from 2D to 3D Culture

The transition from static 2D culture to 3D suspension culture is a bioprocessing bottleneck that can be stressful for hPSCs and may result in lower expansion on the first passage after seeding. Four hPSC lines (H1, SCTi003-A, SCTi004-A, and STiPS-M001) maintained in 2D culture conditions were dissociated to small clumps using ReLeSR? and seeded at a density of 5 x 10? cells/mL on an orbital shaker (2.5 cm orbital diameter) at 70 rpm. Across four cell lines, hPSCs seeded into TeSR?-AOF 3D supplemented with CloneR?3 exhibited cell yields 1.58-fold higher than Y-27632-supplemented cells after four days in culture. Each point represents one biological replicate averaged from two technical replicates. Bars represent the mean ¡À SD from the average of three biological replicates. A two-tailed unpaired t-test was conducted for each cell line comparison where * denotes p < 0.05.

Figure 5. CloneR?3 Improves Yields of High-Quality hPSCs During Routine 3D Culture

Four hPSC lines (H9, STiPS-M001, SCTi003-A, and SCTi004-A) adapted for one passage to 3D suspension culture were dissociated into small clumps with GCDR, seeded at a density of 5 x 10? cells/mL in TeSR?-AOF 3D supplemented with CloneR?3 or Y-27632 and clump passaged with GCDR every 3 to 4 days for 5 passages. (A) Cells supplemented with CloneR?3 demonstrate higher cell yields after four days in culture when compared to cells supplemented with Y-27632. Each point represents one biological replicate averaged from two technical replicates. Bars represent the mean ¡À SD from the average of three biological replicates. A two-tailed unpaired t-test was conducted for each cell line comparison where * denotes p < 0.05 and ** denotes p < 0.01. (B) Images of SCTi003-A and SCTi004-A aggregates supplemented with CloneR?3 and Y-27632 show typical morphology indicative of high-quality, healthy hPSCs. (C) Aggregates were then dissociated to single cells, stained for the undifferentiated markers TRA-1-60 and OCT4, and analyzed by flow cytometry. Cultures supplemented with CloneR?3 and Y-27632 maintained high expression (>90%) of TRA-1-60 and OCT4. Bars represent the mean ¡À SD from the average of two technical replicates.

Figure 6. CloneR?3 Improves Cell Yields Following Single-Cell Passaging of 3D Aggregate Cultures

Passaging 3D hPSC aggregates as single cells can be more stressful than traditional clump passaging, especially when culturing more sensitive cell lines or seeding at lower densities. Aggregates from four hPSC lines (H9, STiPS-M001, SCTi003-A, and SCTi004-A) were dissociated to single cells using GCDR and re-seeded at a cell density of 5 x 10? cells/mL. Single-cell seeded hPSC aggregates cultured in TeSR?-AOF 3D supplemented with CloneR?3 demonstrate higher cell yields after four days in culture when compared to cells seeded in TeSR?-AOF 3D supplemented with Y-27632. Across the four cell lines tested, Y-27632 and CloneR?3 had an average cell yield of 5.7 x 10? ¡À 19.3%, and 7.4 x 10? ¡À 11.8%, respectively. Each point represents one biological replicate averaged from two technical replicates. Bars represent the mean ¡À SD from the average of three biological replicates. A two-tailed unpaired t-test was conducted for each cell line comparison where ** denotes p < 0.01.

Figure 7. CloneR?3 Improves Post-Thaw Recovery of Cryopreserved Single Cells in 3D Suspension Culture

Thawing cryopreserved hPSCs is a stressful event that typically results in significant cell loss and reduced post-thaw expansion. SCTi003-A and SCTi004-A aggregates that were cryopreserved as single cells were seeded into TeSR?-AOF 3D supplemented with CloneR?3 or Y-27632. Thawed single cells were seeded at a cell density of 1 x 10? cells/mL into non-tissue culture-treated six-well plates on an orbital shaker. Cryopreserved single cells supplemented with CloneR?3 demonstrate higher post-thaw recovery after four days in culture when compared to cryopreserved single cells supplemented with Y-27632. Across both cell lines tested, Y-27632 and CloneR?3 had an average cell yield of 4.1 x 10? ¡À 10.5%, and 7.3 x 10? ¡À 3.6%, respectively. Each point represents one biological replicate averaged from two technical replicates. Bars represent the mean ¡À SD from the average of two biological replicates.