July 1, 2009 (Vol. 29, No. 13)

Alternative to Manual and Enzymatic Methods Seeks to Advance Propagation of ES and iPS Cells

Currently, large-scale culturing of embryonic and induced pluripotent stem (ES, iPS) cell lines for use in regenerative medicine and drug discovery applications is a major challenge in biomedical research and the pharmaceutical industry. To overcome this bottleneck, stem cell culture processes must become robust, reproducible, and capable of generating sufficient numbers of cells while still maintaining a stable stem cell phenotype.

Physical passage using specialized stem cell knives, razors, or pipettes to manually section stem cell colonies is widely accepted as the ideal method for propagation of human ES and iPS cell lines. Manual passage of stem cell lines does not involve the use of enzymes, and therefore is thought to better maintain genetic stability of human ES and iPS cells in long-term culture. 

Other benefits of manual passage of stem cell cultures include decreased cellular trauma and selective transfer of specific undifferentiated colonies. Scale-up of multiple stem cell lines using manual methods, however, is unattractive because of the high labor cost, tremendous time consumption, lack of automation, and variability of output associated with varying expertise. 

Due to the technical demands associated with manual passage, routine passage and expansion of human ES and iPS cell lines is often performed using enzymatic passage.  Enzymatic methods using collagenase or trypsin are useful for large-scale expansion and are well suited for automated platforms. Bulk passage using enzymatic methods, however, is highly problematic. Both collagenase and trypsin dissociation result in variable-sized colonies leading to significant inconsistency among cultures. In addition, enzymatic propagation results in significant cellular trauma that may be associated with increased rates of genetic instability. 

Cyntellect’s Stem Cell Manager powered by LEAP, a high-throughput, microplate cytometry system, is a series of stem cell management processes including automated physical passage of stem cells, automated embryoid body generation, and purification of specialized cell types derived from stem cells.

The Stem Cell Passage application automates physical passage of stem cell colonies using laser manipulation instead of a mechanical device, enabling a standardized, reproducible method for automated large-scale generation of uniform ES and iPS cultures.

The Stem Cell Passage application uses laser-mediated sectioning of ES and iPS cell cultures to create specific-sized clumps in a sterile closed multiwell plate environment. Clumps of defined size can then be transferred to new culture dishes by simple pipetting. Enzymes are not required for passage using the Stem Cell Passage application. Automated physical passage of ES and iPS cell cultures using the Stem Cell Passage application reduces the labor and time demands associated with manual passage techniques.

Emrbyonic Cultures

The Stem Cell Passage application is useful for routine passage of ES and iPS cell cultures growing in standard culture medium in well-plates on either feeder cells or substrates such as Matrigel®. Uniform and consistent colony sizes enable higher passage yields and more routine use of higher density plates (e.g., 384 well), supporting higher-throughput experimentation.

A series of laser pulses is used to systematically cut along specific grid lines resulting in stem cell clumps of defined size (Figure 1). Only cells along the section line are exposed to the laser light during sectioning. Following laser sectioning, clumps are rinsed off the plate by pipetting and then transferred to new cell culture vessels.  Feeder cells are nonproliferative, thus their inclusion in routine passage is not an issue since they will be diluted out in subsequent culture.

Figure 1. Laser-mediated sectioning of human iPS cell culture for passage

Reproducible Cultures

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). 

Figure 2. Laser-mediated sectioning of cultures at different X, Y dimensions. Relationship between culture section sizes and resulting colony sizes after one day (lower right)

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.

Figure 3. Confirmation of pluripotency after laser-mediated passage of human iPS cells

Kristi A. Hohenstein, Ph.D. ([email protected]), is a scientist, Anuradha Soundararajan is a research associate, and Gary Bright, Ph.D. ([email protected]), is senior director of applications development
at Cyntellect. Web: www.cyntellect.com.

Previous articleBioSante to Take Over Cell Genesys in a $38M All-Stock Deal
Next articleInvestigators Identify SNPs Associated with Predisposition to Schizophrenia