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Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Hohenstein Elliott KA, Peterson C, Soundararajan A, Kan N, Nelson B, Spiering S, Mercola M, Bright GR - Stem Cells Int (2012)

Bottom Line: Standardization is critical for all future applications of stem cells and necessary to fully understand their potential.This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines.Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Intrexon Corporation, Cell Engineering Unit, 6620 Mesa Ridge Road, San Diego, CA 92121, USA.

ABSTRACT
Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

No MeSH data available.


Related in: MedlinePlus

Improved uniformity of stem cell cultures by laser-mediated passage. (a) Brightfield images of iPSC cultures 2 days after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Colonies are shown by red outline. Scale bar, 1 mm. (b) Alkaline phosphatase (AP) staining of iPSC colonies 1 day after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Scale bar, 1 mm. (c) Colony size of iPSC cultures on days 1 and 3 after laser-mediated passage (200 μm sections), StemPro EZPassage Disposable Stem Cell Passaging Tool (EZ Passage), manual passage using a pipette tip, collagenase treatment, or trypsin dissociation of cells. Longest diameter of each colony was manually measured using brightfield images (top, n = 20 colonies per data point). Number of cells per colony was manually counted using Hoechst stained cultures (bottom, n = 15 colonies per data point). Data are shown as scatter plot with red line indicating mean. The CV is shown in red text above each sample. Asterisks (*) indicate variances that are statistically significant when compared to laser-mediated passage using ANOVA, with a P ≤ 0.05 considered significant.
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fig2: Improved uniformity of stem cell cultures by laser-mediated passage. (a) Brightfield images of iPSC cultures 2 days after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Colonies are shown by red outline. Scale bar, 1 mm. (b) Alkaline phosphatase (AP) staining of iPSC colonies 1 day after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Scale bar, 1 mm. (c) Colony size of iPSC cultures on days 1 and 3 after laser-mediated passage (200 μm sections), StemPro EZPassage Disposable Stem Cell Passaging Tool (EZ Passage), manual passage using a pipette tip, collagenase treatment, or trypsin dissociation of cells. Longest diameter of each colony was manually measured using brightfield images (top, n = 20 colonies per data point). Number of cells per colony was manually counted using Hoechst stained cultures (bottom, n = 15 colonies per data point). Data are shown as scatter plot with red line indicating mean. The CV is shown in red text above each sample. Asterisks (*) indicate variances that are statistically significant when compared to laser-mediated passage using ANOVA, with a P ≤ 0.05 considered significant.

Mentions: Statistical analyses were performed using GraphPad Prism with a P value of ≤0.05 considered to be significant. One way analysis of variance (ANOVA) with Bartlett's test for equal variances was performed to evaluate resulting colony sizes generated after passage by five techniques (n = 20 colonies/sample, Figure 2(c)) and resulting EB sizes generated from laser-mediated, collagenase, and trypsin-passaged cells (n = 30 EB/sample, Figure 5(b)). Statistical analysis of QRT-PCR data (n = 3, Figures 4(c) and 5(d)) was performed using a two-tailed t-test.


Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential.

Hohenstein Elliott KA, Peterson C, Soundararajan A, Kan N, Nelson B, Spiering S, Mercola M, Bright GR - Stem Cells Int (2012)

Improved uniformity of stem cell cultures by laser-mediated passage. (a) Brightfield images of iPSC cultures 2 days after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Colonies are shown by red outline. Scale bar, 1 mm. (b) Alkaline phosphatase (AP) staining of iPSC colonies 1 day after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Scale bar, 1 mm. (c) Colony size of iPSC cultures on days 1 and 3 after laser-mediated passage (200 μm sections), StemPro EZPassage Disposable Stem Cell Passaging Tool (EZ Passage), manual passage using a pipette tip, collagenase treatment, or trypsin dissociation of cells. Longest diameter of each colony was manually measured using brightfield images (top, n = 20 colonies per data point). Number of cells per colony was manually counted using Hoechst stained cultures (bottom, n = 15 colonies per data point). Data are shown as scatter plot with red line indicating mean. The CV is shown in red text above each sample. Asterisks (*) indicate variances that are statistically significant when compared to laser-mediated passage using ANOVA, with a P ≤ 0.05 considered significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369526&req=5

fig2: Improved uniformity of stem cell cultures by laser-mediated passage. (a) Brightfield images of iPSC cultures 2 days after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Colonies are shown by red outline. Scale bar, 1 mm. (b) Alkaline phosphatase (AP) staining of iPSC colonies 1 day after laser-mediated passage (200 μm sections, left) or collagenase passage (right). Scale bar, 1 mm. (c) Colony size of iPSC cultures on days 1 and 3 after laser-mediated passage (200 μm sections), StemPro EZPassage Disposable Stem Cell Passaging Tool (EZ Passage), manual passage using a pipette tip, collagenase treatment, or trypsin dissociation of cells. Longest diameter of each colony was manually measured using brightfield images (top, n = 20 colonies per data point). Number of cells per colony was manually counted using Hoechst stained cultures (bottom, n = 15 colonies per data point). Data are shown as scatter plot with red line indicating mean. The CV is shown in red text above each sample. Asterisks (*) indicate variances that are statistically significant when compared to laser-mediated passage using ANOVA, with a P ≤ 0.05 considered significant.
Mentions: Statistical analyses were performed using GraphPad Prism with a P value of ≤0.05 considered to be significant. One way analysis of variance (ANOVA) with Bartlett's test for equal variances was performed to evaluate resulting colony sizes generated after passage by five techniques (n = 20 colonies/sample, Figure 2(c)) and resulting EB sizes generated from laser-mediated, collagenase, and trypsin-passaged cells (n = 30 EB/sample, Figure 5(b)). Statistical analysis of QRT-PCR data (n = 3, Figures 4(c) and 5(d)) was performed using a two-tailed t-test.

Bottom Line: Standardization is critical for all future applications of stem cells and necessary to fully understand their potential.This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines.Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Intrexon Corporation, Cell Engineering Unit, 6620 Mesa Ridge Road, San Diego, CA 92121, USA.

ABSTRACT
Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications.

No MeSH data available.


Related in: MedlinePlus