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ES/iPS cell culture reagents

StemSure® series

StemSure® series consists of a group of products that can support the culture of ES and iPS cells. Quality control testing is performed on each lot of these products using the mouse ES cell D3 strain.

Product Information

Product Name Description
 StemSure® D-MEM
Basal medium optimized for mouse ES cell culture. Because it does not contain L-Glutamine, it requires supplementation with L-glutamine before use.
 StemSure® Serum Replacement
 (SSR)
A serum replacement for culturing ES and iPS cells. These stem cells can be stably cultured when this product is used as a substitute for serum.
 StemSure® 2-Mercaptoethanol
 Solution(2ME)
A reducing reagent necessary for culturing ES and iPS cells.
 StemSure® Monothioglycerol
 Solution(MTG)
A reducing reagent necessary for culturing ES and iPS cells. This is designed to replace 2ME.
 StemSure® Gelatin Solution
A reagent for coating cell culture vessel surfaces. It is used gelatin from porcine skin.
 StemSure® LIF
A factor essential for maintaining the undifferentiated ability of mouse ES cells.
 StemSure® Freezing Medium
Serum-free cell freezing medium suitable for cryopreserving various animal cells, including mouse ES cells.

Quality Control Testing

Product Name Sterility
Test
pH Osmolality Endotoxin
Test
Mycoplasma
Negative Test
Colony Formation Assay
or
Cell Proliferation Assay
ALP
Staining
Cell Viability
Test
 D-MEM
 SSR
 2ME
 MTG
 Gelatin
 LIF
 Freezing Medium

  
 ○:Performed, -:Not Performed

Culture of Mouse ES Cells D3 strain

Cell Morphology and ALP Staining

Mouse ES cells strain grown in the StemSure® series of culture reagents formed colonies with a shiny appearance that is typical of mouse ES cells. It was confirmed thar ALP staining was positive

00558_img02.jpg

Cell Growth Curve

Mouse ES cells D3 strain was cultured 14 passages in each culture media indicated in the figure (below). Subsequently, the cell proliferation potential was compared. We found that the StemSure® series of culture reagents can support the growth of mouse ES cells D3 strain as efficiently as Competitor A’s products.

00558_img03.jpg

< Media composition >
 StemSure® D-MEM + 15% SSR + 2mmol/l L-Glutamine + 1×MEM Non-essential
 Amino Acids + 0.1mmol/l StemSure® 2-Mercaptoethanol or 0.5mmol/l
 StemSure® Monothioglycerol + 1×Penicillin-Streptomycin +
 1,000units/ml StemSure® LIF

 (Cultured in 12-well plates coated with collagen)

Population Doubling Level

Subcultured using mouse ES cells D3 strain in easch culture media and examined the relationship between the number of culture days and cell population doubling level.. As shown in the figure (below), We found that the StemSure® series of culture reagents is equivalent to the population doubling level of Competitor A’s products..

00558_img04.jpg

< Media composition >

 StemSure® D-MEM + 15% SSR + 2mmol/l L-Glutamine + 1×MEM Non-essential

 Amino Acids + 0.1mmol/l StemSure® 2-Mercaptoethanol +

 1×Penicillin-Streptomycin + 1,000units/ml StemSure® LIF

 (Cultured in 12-well plates coated with collagen)

Expression of Undifferentiated Markers

Mouse ES cell D3 strain was cultured 8 passages in StemSure® series of culture media, and we confirmed that the undifferentiated markers (Nanog, Oct3/4, Sox2, and SSEA-1) were expressed in mouse ES cells D3 strain.

00558_img05.jpg

< Media composition >
 StemSure® D-MEM + 15% SSR + 2mmol/l L-Glutamine + 1×MEM Non-essential Amino Acids + 0.1mmol/l StemSure® 2-Mercaptoethanol
 + 1×Penicillin-Streptomycin + 1,000units/ml StemSure® LIF

Teratoma Formation

Mouse ES Cell D3 Strain was subcultured using StemSure® series. The cells were subcutaneously injected into immunodeficient mice. We subcutaneously verified that these teratomas contained multiple cell types from each of the major cell lineages, such as neural tissue (ectoderm), cartilage (mesoderm), and luminal structures lined with ciliated epithelium (endoderm).

00558_img06.jpg

< Media composition >
StemSure® D-MEM + 15% SSR + 2mmol/l L-Glutamine + 1×MEM Non-essential Amino Acids + 0.1mmol/l StemSure® 2-Mercaptoethanol
 + 1×Penicillin-Streptomycin + 1,000units/ml StemSure® LIF

Culture of human iPS Cells 201B7 Strain

Colony Morphology and ALP Staining

We confirmed that StemSure® Serum Replacement (SSR) can support the growth of human iPS cell 201BH7 strain. We also verified that ALP staining for these cells was positive.

00558_img07.jpg

<Media composition>
 D-MEM/Ham's F-12 + 20% SSR + 2mmol/l L-Glutamine +1×MEM Non-essential Amino
 Acids + 0.1mmol/l StemSure® 2-Mercaptoethanol + 1×Penicillin-Streptomycin + 5ng/ml  
 bFGF

Expression of Undeifferentiated Markers

Human iPS cells 201B7 strain were culured in the media containing SSR, we confirmed that the undifferentiated markers (Sox2, Oct3/4, SSEA-3, SSEA-4 and Tra-1-81) were expressed in human iPS cells 201B7 strain.

00558_img08.jpg

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