(770b) Engineering Xeno-Free Microcarriers with Recombinant Vitronectin, Albumin and UV Irradiation for Human Pluripotent Stem Cell Bioprocessing

The development of technologies for the expansion and directed differentiation of human pluripotent stem cells (hPSCs) [1] in large quantities under xeno-free conditions is a critical step toward enabling envisioned stem cell-based therapies. Stirred suspension microcarrier bioreactors afford great surface-to-volume ratio and scalability but their use in the expansion and differentiation of hPSCs require the engineering of xeno-free microcarriers and media. In this study, a simple and low-cost approach was established for generating microcarriers without animal-derived components.

Microcarriers were coated with recombinant vitronectin (VN) and/or human serum albumin (HSA) with or without UV illumination. The expression of genes and proteins was determined by quantitative PCR, flow cytometry, western blotting and immunostaining as described [2]. Fourier transform infrared (FTIR) spectroscopy, absorbance and ELISA methods were employed to characterize the surface of the engineered beads [3]. Directed hPSC differentiation regimens to definitive endoderm (DE), cardiac mesoderm (MS) and neuroectoderm (NE) were also reported [2, 4].

Polystyrene beads coated with VN, which is routinely used as a substrate for 2D culture of hPSCs resulted in 24.3±3.1% seeding efficiency of H9 human embryonic stem cells (hESCs) but the cells detached from the carriers and their concentration declined over 5 days of stirred suspension cultivation. The inclusion of HSA did not improve significantly the efficiency (27.8±1.3%, p=0.15) and growth of cells but increased their retention on the beads under agitation. However, UV irradiation resulted in enhanced seeding efficiency (30.5±1.6%, p=0.013) and retention while hPSCs grew 20.5±2-fold per passage over multiple successive passages and cell viability remained above 80%. More than 90% of the cells were SSEA4⁺ and OCT4⁺. After multi-passage culture, hPSCs formed embryoid bodies displaying tri-lineage specification markers. Similarly, hPSCs propagated on engineered microcarriers were coaxed to DE, MS or NE and expressed pertinent genes and proteins. The observed performance of this xeno-free system is comparable to that of cultures involving Matrigel-coated beads.

In conclusion, the microcarriers engineered in this study supported the growth and potential for multi-lineage specification of self-renewing hPSCs. Such culture modalities will be critical for the realization of the potential of stem cells in areas ranging from regenerative medicine to drug discovery.

 

Acknowledgements: Funding to EST is acknowledged from the National Institutes of Health (NHLBI R01-103709) and the National Science Foundation (CBET-1547785).

 

References:

1. Fan, Y., Wu, J., Ashok, P., Hsiung, M., Tzanakakis, E.S., Stem Cell Rev., 2015, 11:96-109.

2. Fan, Y., Hsiung, M., Cheng, C., Tzanakakis, E.S., Tissue Eng. Part A, 2014, 20:588-99.

3. Fan, Y., Zhang, F., Tzanakakis, E.S., ACS Biomaterials Science and Engineering, 2016.

4. Wu, J., Tzanakakis, E.S., PLoS One, 7:e50715, 2012.