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

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

Authors 

Tzanakakis, E. - Presenter, Tufts University
Fan, Y., Tufts University
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.