Methods for Expansion of Three-Dimensional Cultures of Human Embryonic Stem Cells Using a Thermoresponsive Polymer

Xiaoli Chen, Linda Harkness, Zhongfan Jia, Andrew Prowse, Michael J. Monteiro, Peter P. Gray

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.

Original languageEnglish
Pages (from-to)146-157
Number of pages12
JournalTissue Engineering - Part C: Methods
Volume24
Issue number3
DOIs
Publication statusPublished - Mar 2018
Externally publishedYes

Keywords

  • hPSC expansion
  • human pluripotent stem cells
  • suspension cultures
  • thermoresponsive nanobridges

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