Abstract
Clinical use of human pluripotent stem cells (hPSCs) is hampered by the technical limitations of their expansion. Here, we developed a chemically synthetic culture substrate for human pluripotent stem cell attachment and maintenance. The substrate comprises a hydrophobic polyvinyl butyral-based polymer (PVB) and a short peptide that enables easy and uniform coating of various types of cell culture ware. The coated ware exhibited thermotolerance, underwater stability and could be stored at room temperature. The substrate supported hPSC expansion in combination with most commercial culture media with an efficiency similar to that of commercial substrates. It supported not only the long-term expansion of examined iPS and ES cell lines with normal karyotypes during their undifferentiated state but also directed differentiation of three germ layers. This substrate resolves major concerns associated with currently used recombinant protein substrates and could be applied in large-scale automated manufacturing; it is suitable for affordable and stable production of clinical-grade hPSCs and hPSC-derived products.
Highlights
Clinical use of human pluripotent stem cells is hampered by the technical limitations of their expansion
Since most of the iPSCs were detached after the medium exchange on day 1, we supplemented the medium with ROCK inhibitor throughout the screening, which is known to support human pluripotent stem cells (hPSCs) survival[19] (Fig. 2A)
The difference in affinity between the cells and the resin surface showed that Polyvinyl butyral (PVB) is a better suitable base polymer than others including polyvinyl alcohol (PVA)[20,21,22,23,24], poly[poly monomethacrylate] (PEGMA)[25] and poly (N-isopropylacrylamide) (PNIPAM)[26], which are reported as a polymers applicable for hPSC culture
Summary
Clinical use of human pluripotent stem cells (hPSCs) is hampered by the technical limitations of their expansion. It supported the long-term expansion of examined iPS and ES cell lines with normal karyotypes during their undifferentiated state and directed differentiation of three germ layers This substrate resolves major concerns associated with currently used recombinant protein substrates and could be applied in large-scale automated manufacturing; it is suitable for affordable and stable production of clinicalgrade hPSCs and hPSC-derived products. Various xeno-free hPSC culture substrates have been developed and are commercially available, most of these require the inclusion of particular recombinant human extracellular matrix (ECM) proteins[5,6] like vitronectin, laminins, or their fragments[7,8,9] These substrates require a laborious and time consuming coating, are thermosensitive and expensive, hindering the cost-effective scale-up of hPSC production for clinical and industrial applications. Our results show that the modified PVB retained its characteristics on various containers, and showed great properties for long-term cell culture, proving that it might be suitable for large-scale and clinical grade hPSC production
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