Passage of human ES and iPS cell lines using laser-mediated sectioning allows for greater control over stem cell cultures, improving standardization of passage procedures and resulting in uniform, reproducible cultures with higher yield. Compared to typical enzymatic methods, laser-mediated sectioning results in a significantly narrower size distribution of resulting stem cell colonies (Figure 1).
Collagenase- and trypsin-passaged human iPS cell cultures resulted in highly variable colony sizes (mean ± SD, 124.3 ± 85.7 and 82.2 ± 72.9 cells/colony, respectively), as determined by manual counting of Hoechst-stained colonies. In contrast, cultures passaged using the Stem Cell Passage application resulted in significantly more uniform distribution of colonies (77.1 ± 17.3 cells/colony).
The size of the resulting colonies can be specifically controlled by selection of the clump size within the Stem Cell Passage application. Human iPS cell cultures sectioned into 80, 120, 160, 200, and 240 µm square clumps result in progressively larger sized colonies as determined by counting of Hoechst-stained colonies (Figure 2).
Maintenance of Phenotype
Human ES and iPS cell cultures expanded using the Stem Cell Passage application maintained a normal morphology and growth rate, demonstrating continued self-renewal. In addition, laser-passaged cultures continued to express characteristic pluripotentency markers including Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 (Figure 3), and were capable of forming well-differentiated embryoid bodies that differentiated into all three primary germ layers (as determined by immunocytochemical and QRT-PCR analyses), and maintained a stable karyotype.
Propagation of ES and iPS cell lines using laser-mediated sectioning is performed in a sterile, closed environment. Scale-up of multiple stem cell lines using the Stem Cell Passage application is attractive due to reduced labor cost, improved standardization, superior consistency of stem cell cultures, and lack of enzymatic dissociation. The application also reduces the skill threshold for generation of consistent high quality ES and iPS cultures.
In summary, the Stem Cell Passage application on LEAP combines the ease of enzymatic methods and the genetic stability of manual passage, allowing efficient, automated propagation of ES and iPS cell cultures. Passage using laser-mediated sectioning of human ES and iPS cells maintains a stable stem cell phenotype.