Automated stem cell passage powered by LEAP physical passage of ES and iPS cell colonies

IntroductionDownload as PDF

Passaging of stem cells is typically performed using enzymatic and/or physical techniques. Enzymatic techniques, such as using collagenase or trypsin, are the most widely used and allow rapid passaging for large-scale expansion, but result in variable-sized colonies and significant cellular trauma which may be associated with increased rates of genetic instability (Mitalipova et al. 2005). In contrast, physical passage of stem cells without enzymes is thought to better maintain genetic stability in long-term culture. Benefits of physical passaging include generation of similar sized clumps, decreased cellular trauma, and selective transfer of specific colonies (Oh et al. 2005; Joannides et al. 2006). In addition, physical passaging is essential for production of new GMP-quality, clinical-grade stem cell lines (Thomson 2007). However, manual passaging of stem cell colonies is labor-, cost-, and time-intensive, and therefore not suitable for large scale culture.

The Automated Stem Cell Passage application, a component of the Stem Cell Manager, on the LEAP™ system, automates physical passaging of stem cells using laser manipulation instead of a mechanical device, thereby enabling large scale production of stem cell cultures.

Approach & Results

Human iPS and ES cells (Kan and Mercola 2009) were passaged using the Automated Stem Cell Passage Application and Stem Cell Purification Kit. Colonies were cultured in normal growth medium on MEFs in a 96-well plate and were passaged every 4-5 days. The Automated Stem Cell Passage Application first imaged the entire well with fluorescence and/or brightfield illumination identifying colonies to be passaged (Fig. 1A) based on image segmentation. The edges of the colonies were identified and the laser used to separate the edge of the colony from the surrounding culture (Fig. 1B). Next, the colonies were systematically sectioned into defined sizes using the laser (Fig. 1C, D). Sectioned colonies were rinsed off the plate by gentle pipetting (Fig. 1E) and transferred to new cell culture vessels. Well-defined, uniform colony formation resulted (Fig. 1F).

When it is not necessary to select specific colonies, whole wells can be sectioned without specifically identifying the colonies. This provides a faster approach for large scale passage.

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  Fig. 1. Human iPS cell cultures passaged in 96W plate using the Automated Stem Cell Passage Application. (A) Colonies were identified by brightfield (or fluorescence) imaging,(B) isolated by laser sectioning along border, (C) sectioned into clumps of defined size (whole well image in D), and (E) removed by pipetting. (F) This application resulted in uniform colony formation throughout the well (day 3; colonies were manually outlined for greater visibility).

Laser-mediated sectioning of stem cell cultures was performed at several sizes. Resulting cultures were stained with Hoechst one day after laser-mediated passage and counted manually to determine the number of cells per colony. Increasing section size resulted in progressively larger colony sizes one day after passage (Fig. 2).

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  Fig. 2. Relationship between clump size and resulting colony size

Laser-mediated sectioning of stem cell cultures can be limited to individual identified colonies or to all colonies within the culture. Specific colonies can be identified by specific markers (surface or expressed), size, and/or morphology. Given the propensity of cells around the border of stem cell colonies to spontaneously differentiate, the user can selectively trim a certain proportion of the edge cells during processing to remove undesired cells.

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  Fig. 3. Laser-mediated passage of iPS cell cultures results in more uniform colony formation as compared with enzymatic methods.

Laser-mediated sectioning of stem cell colonies resulted in a significantly more uniform size range of resulting stem cell colonies as compared with typical enzymatic methods of passage (Fig. 3). Stem cell cultures were passaged by each technique, plated onto fresh MEFs, stained with Hoechst one day after passage and counted manually to determine the size distribution of resulting colonies. Collagenase- and trypsin-passaged iPS cell cultures resulted in variable sized colonies (mean + SD, 40.9 ± 28.0 (range, ~5-90) and 31.9 ± 22.1 (range, <5-70) cells/colony, respectively). In contrast cultures passaged by laser-mediated sectioning into 80 µm pieces resulted in significantly more uniform colonies containing 35.3 ± 5.4 (range, ~20-40) cells/colony (Fig. 3).

The Automated Stem Cell Passage application has been used for propagation of cultures maintained in both KODMEM and DMEM/F-12 based medium growing on either feeder cells or Matrigel™. Cultures maintained normal morphology and growth rate and demonstrated continued self-renewal. In addition, cultures maintained by laser-mediated passage continued to express characteristic stem cell markers including Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81 (Fig. 4) and were capable of forming well-differentiated embryoid bodies expressing markers of all three primary germ layers.

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  Fig. 4. Human iPS cell cultures passaged using the Automated Stem Cell Passage application maintain stem cell properties. These cells express Oct4, Sox2, Nanog, SSEA4, TRA1-60, and TRA1-81.

Conclusion

The Automated Stem Cell Passage application on LEAP provides an efficient, automated physical passaging capability generating uniform-sized undifferentiated colonies of stem cells. The ability to select a specific population of stem cell colonies and choose specific regions of colonies to be passaged ensures propagation of well-defined, undifferentiated stem cells only. Human stem cells successfully propagated using laser-mediated passage continued to maintain their unique stem cell properties. The Automated Stem Cell Passage application provides a reproducible method for propagation of high-quality, large-scale ES and iPS cell cultures.

References

1. Mitalipova et al, Nat Biotechnol. 2005;23:19-20.
2. Kan and Mercola. Human iPS cell lines. In prep. 2009.
3. Oh et al, Stem Cells 2005;23:605.
4. Joannides et al, Stem Cells 2006;24:230.
5. Thomson, Trends Biotechnol. 2007;25:224