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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

Stem cell colony size was controlled by section size using laser-mediated passage. (a) Brightfield images of human iPSC (top, middle) and ESC (bottom) cultures cut into 100–250 μm sections. Scale bar, 1 mm. (b) Colony size over time following propagation of 100–250 μm iPSC sections (top) or ESC sections (bottom) by laser-mediated passage. Number of cells per colony were manually counted using Hoechst stained cultures (left, n = 15 colonies per data point). Longest diameter of each colony was manually measured using brightfield images (right, n = 15 colonies per data point). Data are shown as mean + s.d.
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fig1: Stem cell colony size was controlled by section size using laser-mediated passage. (a) Brightfield images of human iPSC (top, middle) and ESC (bottom) cultures cut into 100–250 μm sections. Scale bar, 1 mm. (b) Colony size over time following propagation of 100–250 μm iPSC sections (top) or ESC sections (bottom) by laser-mediated passage. Number of cells per colony were manually counted using Hoechst stained cultures (left, n = 15 colonies per data point). Longest diameter of each colony was manually measured using brightfield images (right, n = 15 colonies per data point). Data are shown as mean + s.d.

Mentions: To determine the impact of section size on resulting colony size, stem cell cultures were cut into square cell sections ranging from 75 to 300 μm in size and transferred to new culture dishes by gentle pipetting (Figure 1(a), hiPSCs (top, middle), hESCs (bottom)). Sections below 75 μm contained very few cells (<8 cells/section), whereas 300 μm sections were too large to easily remove from the plate by gentle pipetting alone. Stem cell colony size and number of cells per colony were assessed by brightfield imaging and fluorescent staining of nuclei, respectively, after processing cultures into 100–250 μm sections (Figure 1(b)). Human iPSC cultures sectioned into 100, 150, 200, and 250 μm sizes resulted in sections containing 12, 25, 47, and 68 cells, respectively (Figure 1(b), top). Three days after passage human iPSC colonies measured 306, 367, 493, and 693 μm in diameter with 62, 119, 184, and 283 cells per colony, respectively. Similar results were obtained with all iPSC cell lines (data not shown) and with human ESCs (Figure 1(b), bottom) using the same laser processing conditions.


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)

Stem cell colony size was controlled by section size using laser-mediated passage. (a) Brightfield images of human iPSC (top, middle) and ESC (bottom) cultures cut into 100–250 μm sections. Scale bar, 1 mm. (b) Colony size over time following propagation of 100–250 μm iPSC sections (top) or ESC sections (bottom) by laser-mediated passage. Number of cells per colony were manually counted using Hoechst stained cultures (left, n = 15 colonies per data point). Longest diameter of each colony was manually measured using brightfield images (right, n = 15 colonies per data point). Data are shown as mean + s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369526&req=5

fig1: Stem cell colony size was controlled by section size using laser-mediated passage. (a) Brightfield images of human iPSC (top, middle) and ESC (bottom) cultures cut into 100–250 μm sections. Scale bar, 1 mm. (b) Colony size over time following propagation of 100–250 μm iPSC sections (top) or ESC sections (bottom) by laser-mediated passage. Number of cells per colony were manually counted using Hoechst stained cultures (left, n = 15 colonies per data point). Longest diameter of each colony was manually measured using brightfield images (right, n = 15 colonies per data point). Data are shown as mean + s.d.
Mentions: To determine the impact of section size on resulting colony size, stem cell cultures were cut into square cell sections ranging from 75 to 300 μm in size and transferred to new culture dishes by gentle pipetting (Figure 1(a), hiPSCs (top, middle), hESCs (bottom)). Sections below 75 μm contained very few cells (<8 cells/section), whereas 300 μm sections were too large to easily remove from the plate by gentle pipetting alone. Stem cell colony size and number of cells per colony were assessed by brightfield imaging and fluorescent staining of nuclei, respectively, after processing cultures into 100–250 μm sections (Figure 1(b)). Human iPSC cultures sectioned into 100, 150, 200, and 250 μm sizes resulted in sections containing 12, 25, 47, and 68 cells, respectively (Figure 1(b), top). Three days after passage human iPSC colonies measured 306, 367, 493, and 693 μm in diameter with 62, 119, 184, and 283 cells per colony, respectively. Similar results were obtained with all iPSC cell lines (data not shown) and with human ESCs (Figure 1(b), bottom) using the same laser processing conditions.

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