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Figure 1. CloneR™ and ����DzԱ��™2 Supplements Improve Cloning Efficiency and Colony Size

hPSCs display a considerable increase in cloning efficiency when cloned using (B) CloneR™ compared to using (A) Y-27632 compound. (C) ����DzԱ��™2 further improves cloning efficiency and increases colony size when compared to either Y-27632 compound or CloneR™. Shown are examples of H9 hESCs in 10-cm dishes, plated at 200 cells per dish (~4 cells/cm²) in m�ձ𳧸�™ Plus on Vitronectin XF™.

Figure 2. Use of ����DzԱ��™2 Enables Improved Cloning Efficiency and Larger Colonies When Compared to Use of CloneR™

(A) Representative images of 200 cells (H9 cell line) in 12-well plates grown in m�ձ𳧸�™1 on Vitronectin XF™ at day 8 after seeding. Three hES (H1, H7 and H9) and 5 hiPS (WLS-1C, STiPS-F016, STiPS-M001, STiPS-R038 and STiPS-B004) cell lines were seeded at clonal density (50 cells/cm²) on Vitronectin XF™, in m�ձ𳧸�™1 supplemented with CloneR™ or ����DzԱ��™2. m�ձ𳧸�™1 supplemented with ����DzԱ��™2 increases (B) cloning efficiency and (C) colony size of hPSCs when compared with m�ձ𳧸�™1 supplemented with CloneR™. Each data point in (B) represents an average of 3 technical replicates, with at least 7 biological replicates (n) per cell line.

Figure 3. ����DzԱ��™2 Improves Clonal Generation Following Single Cell Deposition

Single-cell deposition is the gold standard of cloning; it relies on a FACS-based method that typically results in low cloning efficiency. Clones generated in m�ձ𳧸�™ Plus supplemented with ����DzԱ��™2 demonstrate significantly improved (E) cloning efficiency and (B, D) colony size when compared to clones generated in m�ձ𳧸�™ Plus supplemented with CloneR™. (A, C) Representative images of H9 hESCs cultured for eight days plated on Vitronectin XF™. Across four cell lines tested, CloneR™ and ����DzԱ��™2 had an average cloning efficiency of 21.2 ± 5.6% and 38.6 ± 6.0%, respectively, with at least 3 biological replicates per cell line. * denotes p < 0.05; *** denotes p < 0.001 by unpaired t-tests.

Figure 4. ����DzԱ��™2 Improves Seeding Efficiency at High Density

����DzԱ��™2 improves single-cell seeding efficiency when used as a supplement in media for the first 24 hours of culture, compared to using Y-27632 as a supplement. 5.0x10⁵ cells were seeded in 12-well plates coated with Corning® Matrigel® in m�ձ𳧸�™ Plus supplemented with ����DzԱ��™2 or Y-27632. Cultures were analyzed 24 hours post-seeding. The use of ����DzԱ��™2 resulted in an average seeding efficiency of 98.2 ± 12.8% compared to the use of Y-27632, which resulted in an average seeding efficiency of 81.9 ± 15.8%, across all cell lines (n = 3 replicates per line).

Figure 5. hPSCs Plated in ����DzԱ��™2 Show Increased Expansion

When used as a seeding supplement during single-cell passaging, ����DzԱ��™2 improves cell expansion when compared to using Y-27632. 3.0x10⁴ cells were seeded in 12-well plates coated with Corning® Matrigel® in m�ձ𳧸�™ Plus supplemented with ����DzԱ��™2 or Y-27632. After 24 hours, the cultures were maintained in complete media (without a cloning supplement) and analyzed on day 5. ����DzԱ��™2 resulted in an average expansion of 49.1 ± 10.4 compared to Y-27632, which resulted in a lower average expansion of 21.1 ± 8.2, across all cell lines (n = 3 replicates per line).

Figure 6. ����DzԱ��™2 Improves Recovery of hPSCs Following Electroporation

����DzԱ��™2 can also be used as a survival supplement in gene-editing workflows that require electroporation. Four cell lines were electroporated, then plated in m�ձ𳧸�™1 and m�ձ𳧸�™ Plus containing Y-27632, CloneR™, or ����DzԱ��™2. Cultures were maintained in complete �ձ𳧸�™ media (without cloning supplement) after 24 hours and analyzed after 5 days. In all 4 cell lines, (panels A-D) ����DzԱ��™2 dramatically improved cell survival and expansion when used as a supplement in the first 24 hours immediately following electroporation compared to both Y-27632 and CloneR™ (n = 2 replicates per cell line).

Figure 7. ����DzԱ��™2 Improves Post-Thaw Recovery of hPSCs

Thawing cryopreserved cells can result in low expansion or loss of the culture within the first passage. Using ����DzԱ��™2 as a seeding supplement within the first 24 hours of thawing cells ameliorates this effect, improving post-thaw recovery of hPSCs. Three cell lines were frozen as single cells, then thawed into m�ձ𳧸�™ Plus containing Y-27632 or ����DzԱ��™2 on Matrigel®. Cultures were maintained in complete m�ձ𳧸�™ Plus (without cloning supplement) after 24 hours, and analyzed on day 4 or day 5. ����DzԱ��™2 improves the fold-expansion across all cell lines tested when compared to Y-27632, with at least 7 replicates (n) per cell line.