Limits...
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

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.


Differences observed in the stability of distinct hPSC medium formulations.Four hPSC medium formulations were found to support the maintenance of hPSC lines in culture. To evaluate the stability of each formulation, both freshly supplemented medium and medium that was supplemented and then stored at 4°C for 14 days prior to addition to the hPSC lines, were compared. In this representative example using WA09 cells, all formulations sustained high expression levels of pluripotency markers when the medium was used within seven days after supplementation with bFGF and other factors. However, only the medium containing the L7 supplement formulation was able to sustain robust expression of the hPSC markers with aged medium (14–21 days) post supplementation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5016087&req=5

pone.0161229.g001: Differences observed in the stability of distinct hPSC medium formulations.Four hPSC medium formulations were found to support the maintenance of hPSC lines in culture. To evaluate the stability of each formulation, both freshly supplemented medium and medium that was supplemented and then stored at 4°C for 14 days prior to addition to the hPSC lines, were compared. In this representative example using WA09 cells, all formulations sustained high expression levels of pluripotency markers when the medium was used within seven days after supplementation with bFGF and other factors. However, only the medium containing the L7 supplement formulation was able to sustain robust expression of the hPSC markers with aged medium (14–21 days) post supplementation.

Mentions: To develop a defined, xeno-free hPSC medium, basal medium components and supplements were systematically evaluated using a step-by-step approach and different EOD experiments explained in the Materials and Methods section to identify medium formulations that supported hPSC survival, proliferation and self-renewal during routine cultivation. Initially, components of the basal medium and key supplements were evaluated independently on three hESC lines: WA07, WA09 and WA14. These hESC lines were independently cultivated for ten passages and the medium evaluated for its ability to support cell attachment to hESC-qualified matrigel, maintain normal hPSC cell morphology, promote cell growth and deter spontaneous differentiation. Four formulations were found to be superior based on these selection criteria (data not shown). To evaluate the combined stability of their components, each formulation was then aged for two weeks at 4°C and then directly compared to its freshly prepared counterpart (Fig 1). While all four media formulations were initially similar in their ability to promote cell growth, prevent spontaneous differentiation and express markers of pluripotency, only L7 formulation was found to maintain these criteria two weeks post-supplementation.


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
Differences observed in the stability of distinct hPSC medium formulations.Four hPSC medium formulations were found to support the maintenance of hPSC lines in culture. To evaluate the stability of each formulation, both freshly supplemented medium and medium that was supplemented and then stored at 4°C for 14 days prior to addition to the hPSC lines, were compared. In this representative example using WA09 cells, all formulations sustained high expression levels of pluripotency markers when the medium was used within seven days after supplementation with bFGF and other factors. However, only the medium containing the L7 supplement formulation was able to sustain robust expression of the hPSC markers with aged medium (14–21 days) post supplementation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016087&req=5

pone.0161229.g001: Differences observed in the stability of distinct hPSC medium formulations.Four hPSC medium formulations were found to support the maintenance of hPSC lines in culture. To evaluate the stability of each formulation, both freshly supplemented medium and medium that was supplemented and then stored at 4°C for 14 days prior to addition to the hPSC lines, were compared. In this representative example using WA09 cells, all formulations sustained high expression levels of pluripotency markers when the medium was used within seven days after supplementation with bFGF and other factors. However, only the medium containing the L7 supplement formulation was able to sustain robust expression of the hPSC markers with aged medium (14–21 days) post supplementation.
Mentions: To develop a defined, xeno-free hPSC medium, basal medium components and supplements were systematically evaluated using a step-by-step approach and different EOD experiments explained in the Materials and Methods section to identify medium formulations that supported hPSC survival, proliferation and self-renewal during routine cultivation. Initially, components of the basal medium and key supplements were evaluated independently on three hESC lines: WA07, WA09 and WA14. These hESC lines were independently cultivated for ten passages and the medium evaluated for its ability to support cell attachment to hESC-qualified matrigel, maintain normal hPSC cell morphology, promote cell growth and deter spontaneous differentiation. Four formulations were found to be superior based on these selection criteria (data not shown). To evaluate the combined stability of their components, each formulation was then aged for two weeks at 4°C and then directly compared to its freshly prepared counterpart (Fig 1). While all four media formulations were initially similar in their ability to promote cell growth, prevent spontaneous differentiation and express markers of pluripotency, only L7 formulation was found to maintain these criteria two weeks post-supplementation.

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.