Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in h...
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.
Human embryonic stem (ES) cells are a potential source of cells for developmental studies and for a variety of applications in transplantation therapies and drug discovery. However, human ES cells are difficult to culture and maintain at a large scale, which is one of the most serious obstacles in human ES cell research. Culture of human ES cells on MEF cells after disassociation with accutase has previously been demonstrated by other research groups. Here, we confirmed that human ES cells (H9) can maintain stem cell properties when the cells are passaged as single cells under a feeder-free culture condition. Accutase-dissociated human ES cells showed normal karyotype, stem cell marker expression, and morphology. We prepared frozen stocks during the culture period, thawed two of the human ES cell stocks, and analyzed the cells after culture with the same method. Although the cells revealed normal expression of stem cell marker genes, they had abnormal karyotypes. Therefore, we suggest that accutase-dissociated single cells can be usefully expanded in a feeder-free condition but chromosomal modification should be considered in the culture after freeze-thawing.
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제안 방법
, 2005). To confirm the karyotypes of human ES cells, we analyzed the chromosomal status of ES cells using the GTG-banding method and monitored whether translocations and/or aneuploidies are found (Fig. 1D). After 20 enzymatic passages using accutase, the human ES cells had normal karyotype.
We selected two of the stocks (frozen at passage 11 and passage 24, respectively) and cultured them on Matrigel-coated plates after usual thawing. We cultured the stock frozen at passage 11 for another 3 passages and performed a FACS analysis to assess expression of ES cell markers (Batch 1) (Fig. 4A). We cultured the other stock (passage 24) for another 14 passages and checked ES cell marker expression (Batch 2) (Fig.
대상 데이터
H9 human ES cells (WiCell Research Institute, Madison, USA) were maintained on MEF cells in an ES medium containing 80% DMEM/F12 (Gibco, Carlasbad, USA), 20% KnockOut serum replacement medium (Gibco), 1 mM L-glutamine (Sigma-Aldrich, St. Louis, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich), 1% nonessential amino acids (Gibco), and 4 ng/ml human basic fibroblast growth factor (bFGF).
성능/효과
2B). These results reveal that TrypLE may be used as an alternative to accutase in the culture of human ES cell H9 after single cell dissociation.
참고문헌 (25)
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