CloneR™2

Defined supplement for improving survival of human ES and iPS cells in single-cell workflows
No options of this product are available.

Overview

Generate clonal human pluripotent stem cell (hPSC) lines that maintain their genomic integrity and downstream differentiation potential with this supplement. By using CloneR™2, you can increase the cloning efficiency and survival of human embryonic stem (ES) and induced pluripotent stem (iPS) cells under high-stress conditions, including seeding at low or high densities.

For your gene-editing workflows, add CloneR™2 to improve ES and iPS cell survival following electroporation and during clonal deposition (see data below). It also facilitates the survival and expansion of clonal lines generated by low-density seeding (<25 cells/cm2) or by FACS-sorting cells at 1 cell/well, without requiring single-cell adaptation. You can also use CloneR™2 supplement to increase survival of single cells plated at higher densities, including under stressful conditions such as the post-thaw recovery of ES and iPS cell lines and when creating monolayers ahead of downstream differentiation.

Defined and serum-free, CloneR™2 supplement is compatible for use with TeSR™ ES and iPS cell maintenance media, as well as your choice of cell culture matrix.
Advantages
⦁ More Colonies, Ready Sooner. Improved cloning efficiencies with clones ready for selection days sooner
⦁ Robust and Consistent Cloning. Similar high performance across culture systems and cell lines
⦁ Enhanced Survival. Increased plating efficiency at all densities and after high stress events such as electroporation or thawing
⦁ Straight to Single Cells. No single-cell passage adaptation phase required
Subtype
Supplements
Cell Type
Pluripotent Stem Cells
Species
Human
Application
Cell Culture
Brand
CloneR
Area of Interest
Cell Line Development, Disease Modeling, Stem Cell Biology
Formulation
Serum-Free

Scientific Resources

Educational Materials (5)

Brochure
Products for Human Pluripotent Stem Cells
Webinar
Improving Reproducibility of Your hPSC Research by Generating a High-Quality Cell Bank
21:53
Improving Reproducibility of Your hPSC Research by Generating a High-Quality Cell Bank
Webinar
Considerations for High-Efficiency Genome Editing of Human Pluripotent Stem Cells
50:01
Considerations for High-Efficiency Genome Editing of Human Pluripotent Stem Cells
Webinar
Facilitating hPSC Single Cell Seeding Workflows Using CloneR™2
12:49
Facilitating hPSC Single Cell Seeding Workflows Using CloneR™2
Webinar
Highly Efficient Single-Cell Human Pluripotent Stem Cell Cloning and Robust Cardiomyocyte Differentiation
48:54
Highly Efficient Single-Cell Human Pluripotent Stem Cell Cloning and Robust Cardiomyocyte Differentiation

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Three 10 cm dishes showing increased numbers of colonies from Y-27632 compound to CloneR™ to CloneR™2

Figure 1. CloneR™ and CloneR™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) CloneR™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 mTeSR™ Plus on Vitronectin XF™.

Images of colonies next to two graphs showing increased cloning efficiency and colony size in CloneR™2 across cell lines.

Figure 2. Use of CloneR™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 mTeSR™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 mTeSR™1 supplemented with CloneR™ or CloneR™2. mTeSR™1 supplemented with CloneR™2 increases (B) cloning efficiency and (C) colony size of hPSCs when compared with mTeSR™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.

Images of 96 well plates of cells with more colonies in CloneR™2 and summarized cloning efficiencies shown in a bar graph.

Figure 3. CloneR™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 mTeSR™ Plus supplemented with CloneR™2 demonstrate significantly improved (E) cloning efficiency and (B, D) colony size when compared to clones generated in mTeSR™ 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 CloneR™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.

Graph of seeding efficiency of multiple cell lines in Y-27632 compared to CloneR™2.

Figure 4. CloneR™2 Improves Seeding Efficiency at High Density

CloneR™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 mTeSR™ Plus supplemented with CloneR™2 or Y-27632. Cultures were analyzed 24 hours post-seeding. The use of CloneR™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).

Graph of increased expansion of hPSCs when plated in CloneR™2 compared to Y-27632

Figure 5. hPSCs Plated in CloneR™2 Show Increased Expansion

When used as a seeding supplement during single-cell passaging, CloneR™2 improves cell expansion when compared to using Y-27632. 3.0x10⁴ cells were seeded in 12-well plates coated with Corning® Matrigel® in mTeSR™ Plus supplemented with CloneR™2 or Y-27632. After 24 hours, the cultures were maintained in complete media (without a cloning supplement) and analyzed on day 5. CloneR™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).

Four graphs representing different cell lines for improved hPSC expansion in CloneR™2 following electroporation.

Figure 6. CloneR™2 Improves Recovery of hPSCs Following Electroporation

CloneR™2 can also be used as a survival supplement in gene-editing workflows that require electroporation. Four cell lines were electroporated, then plated in mTeSR™1 and mTeSR™ Plus containing Y-27632, CloneR™, or CloneR™2. Cultures were maintained in complete TeSR™ media (without cloning supplement) after 24 hours and analyzed after 5 days. In all 4 cell lines, (panels A-D) CloneR™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).

Bar graph showing improved post-thaw recovery of hPSCs in multiple cell lines when using CloneR™2

Figure 7. CloneR™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 CloneR™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 mTeSR™ Plus containing Y-27632 or CloneR™2 on Matrigel®. Cultures were maintained in complete mTeSR™ Plus (without cloning supplement) after 24 hours, and analyzed on day 4 or day 5. CloneR™2 improves the fold-expansion across all cell lines tested when compared to Y-27632, with at least 7 replicates (n) per cell line.

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