Currently, human embryonic stem cells (hESCs) are cultured on feeder cells or complex mixtures of proteins extracted from mouse tumors. To allow commercialization of hESC-derived therapeutic cells, culture methods are required that are robust and scalable and that use chemically defined, xeno-free materials.
Corning Life Sciences has manufactured products to enable cell culture research and production for over 100 years. Corning Synthemax Surface is a novel synthetic surface that permits consistent long-term self renewal of multiple hESC lines in defined, xeno-free media, and differentiation of cells to functional cardiomyocytes.
Synthemax Surface is a synthetic surface composed of RGD-containing short peptides covalently immobilized on acrylate coating to mimic the natural cell environment. The surface is manufactured under current cGMP using a proprietary surface coating technology and defined, xeno-free materials, providing lot-to-lot consistency.
Culture vessels with Synthemax Surface are ready to use and do not require any additional preparation by the end user. Synthemax Surface is terminally sterilized by gamma irradiation, eliminating the risk of pathogen contamination associated with biological surfaces. Unlike biological surfaces that require special storage conditions and have a limited shelf life, Synthemax Surface is stable at room temperature for at least two years.
Results
The ability of Synthemax Surface to support long-term self renewal of hESCs was evaluated by serial passaging of H7 hESCs on Synthemax Surface in defined, xeno-free medium (X-VIVO™ 10 basal medium supplemented with 80 ng/mL hrbFGF and 0.5 ng/mL hrTGF-β(X-VIVO10+GF medium)).
As shown in Figure 1A, Synthemax Surface supported a stable proliferation rate of H7 hESCs (doubling time of 41 ± 5 hours) for 12 serial passages. Importantly, cells retained normal karyotype at the end of 12 passages (Figure 1B).
hESC-specific phenotypic markers, such as Oct4 and SSEA4, were evaluated qualitatively by indirect immunofluorescence staining (Figure 2A,B) and quantitatively by flow cytometry (Figure 2C,D). After ten passages on Synthemax Surface in X-VIVO10+GF medium, H7 hESCs retained high levels of both markers, suggesting that they maintained their undifferentiated status.
These results demonstrate that H7 hESC can be successfully propagated on Synthemax Surface for multiple passages in xeno-free, defined medium, while retaining important hESC characteristics, such as stable proliferation rate, phenotypic marker profile, and normal karyotype. Similar results were demonstrated with other hESC lines (H1, H9, BG01v) and defined media conditions (mTeSR®1, STEMPRO® hESC SFM, and NutriStem™).
Pluripotency, the ability to differentiate into cells of all three germ layers, is a fundamental property of hESCs. This property is a critical parameter when evaluating new culture conditions for hESCs.
As shown in Figure 3A, H7 hESCs maintained on Synthemax Surface for ten serial passages were able to differentiate into cells of all three germ layers when injected into immunodeficient mice, confirming their pluripotent status. These results demonstrate that H7 hESCs can be successfully propagated on Synthemax Surface for multiple passages in defined, xeno-free medium with a stable proliferation rate and normal karyotype, while retaining hESC-specific phenotypic marker expression and pluripotency.
For therapeutic applications of hESCs, it is highly desirable to have defined culture conditions for both the expansion and the differentiation phases of therapeutic cell production.
To demonstrate this, H7 hESCs cultured on Synthemax Surface for ten passages were differentiated to cardiomyocytes on the same surface using a directed differentiation protocol. As demonstrated by immunofluorescence staining in Figure 3B, Synthemax Surface supports differentiation of H7 hESCs into cardiomyocytes expressing specific markers—α-actinin and Nkx2.5.
Conclusions
Synthemax Surface is a nonbiological synthetic surface that mimics the natural cell environment. It is GMP manufactured with xeno-free materials and doesn’t require additional coating by the end users.
Synthemax Surface supports long-term multipassage self renewal of hESCs in defined, xeno-free media with more consistent proliferation rate compared to biological substrates.
After multipassage culture on Synthemax Surface, hESCs retain pluripotency and the ability to differentiate to functional cardiomyocytes.
Zara Melkoumian, Ph.D. (melkoumiz@corning.com), is manager, cell biology at Corning Life Sciences. Ralph Brandenberger (RBrandenberger@Geron.com) is director, process sciences at Geron. The authors wish to acknowledge Jennifer L. Weber, Paula Dolley-Sonneville, David M. Weber, Andrei G. Fadeev, and Yue Zhou of Corning, and Jiwei Yang and Catherine Priest of Geron.