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A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells

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ABSTRACT

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

No MeSH data available.


Medium containing thermostable bFGF supports every-other-day supplementation of hPSC cultures.hPSCs (WA09 hESC line) were seeded at 2 x 105 cells per well in a 6-well tissue culture treated coated with L7™ hPSC matrix in the defined hPSC medium. Three days post-seeding, the medium of metabolically active hPSC cultures was replaced with freshly supplemented basal medium containing either bFGF or TS bFGF. Twenty four and 48 hours post-addition, a sample of the medium was taken and the concentration of bFGF present determined by ELISA. A) bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3). B) TS bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3).
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pone.0161229.g002: Medium containing thermostable bFGF supports every-other-day supplementation of hPSC cultures.hPSCs (WA09 hESC line) were seeded at 2 x 105 cells per well in a 6-well tissue culture treated coated with L7™ hPSC matrix in the defined hPSC medium. Three days post-seeding, the medium of metabolically active hPSC cultures was replaced with freshly supplemented basal medium containing either bFGF or TS bFGF. Twenty four and 48 hours post-addition, a sample of the medium was taken and the concentration of bFGF present determined by ELISA. A) bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3). B) TS bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3).

Mentions: To further optimize this hPSC medium formulation (i.e. L7 formulation), we examined the stability of bFGF present in mitotically active cultures of hPSCs, one and two days post-addition in comparison with a thermo-stable version of bFGF (TS bFGF) (Fig 2A). In both the absence and presence of stabilizing concentrations of porcine-derived heparin, native bFGF concentrations significantly declined 24 hours post-addition and continued to fall to a very minimal level after 48 hours (about %95 decay). In contrast, the level of TS bFGF declined to about 60% and 32% of the initial concentration after 24 and 48 hrs, respectively (Fig 2B). The starting concentration of TS bFGF in this experiment was different from bFGF concentration on day 0, however the concentration was chosen to support a minimum bFGF requirement identified in growth cytokine evaluation studies (data not shown). The quantity of TS bFGF present after 48 hours was comparable to the levels of native bFGF observed 24 hours post-supplementation with daily medium changes. Importantly, TS bFGF was stable in the hPSC medium in the absence of heparin, and therefore heparin could be eliminated from the medium, resulting in the final defined, xeno-free hPSC medium formulation (Table 1).


A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells
Medium containing thermostable bFGF supports every-other-day supplementation of hPSC cultures.hPSCs (WA09 hESC line) were seeded at 2 x 105 cells per well in a 6-well tissue culture treated coated with L7™ hPSC matrix in the defined hPSC medium. Three days post-seeding, the medium of metabolically active hPSC cultures was replaced with freshly supplemented basal medium containing either bFGF or TS bFGF. Twenty four and 48 hours post-addition, a sample of the medium was taken and the concentration of bFGF present determined by ELISA. A) bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3). B) TS bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016087&req=5

pone.0161229.g002: Medium containing thermostable bFGF supports every-other-day supplementation of hPSC cultures.hPSCs (WA09 hESC line) were seeded at 2 x 105 cells per well in a 6-well tissue culture treated coated with L7™ hPSC matrix in the defined hPSC medium. Three days post-seeding, the medium of metabolically active hPSC cultures was replaced with freshly supplemented basal medium containing either bFGF or TS bFGF. Twenty four and 48 hours post-addition, a sample of the medium was taken and the concentration of bFGF present determined by ELISA. A) bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3). B) TS bFGF concentrations present in the hPSC medium at the time of addition, 24 hours- and 48 hours-post addition (n = 3).
Mentions: To further optimize this hPSC medium formulation (i.e. L7 formulation), we examined the stability of bFGF present in mitotically active cultures of hPSCs, one and two days post-addition in comparison with a thermo-stable version of bFGF (TS bFGF) (Fig 2A). In both the absence and presence of stabilizing concentrations of porcine-derived heparin, native bFGF concentrations significantly declined 24 hours post-addition and continued to fall to a very minimal level after 48 hours (about %95 decay). In contrast, the level of TS bFGF declined to about 60% and 32% of the initial concentration after 24 and 48 hrs, respectively (Fig 2B). The starting concentration of TS bFGF in this experiment was different from bFGF concentration on day 0, however the concentration was chosen to support a minimum bFGF requirement identified in growth cytokine evaluation studies (data not shown). The quantity of TS bFGF present after 48 hours was comparable to the levels of native bFGF observed 24 hours post-supplementation with daily medium changes. Importantly, TS bFGF was stable in the hPSC medium in the absence of heparin, and therefore heparin could be eliminated from the medium, resulting in the final defined, xeno-free hPSC medium formulation (Table 1).

View Article: PubMed Central - PubMed

ABSTRACT

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

No MeSH data available.