The generation of stem cell–derived β-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human β-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries.

Article Highlights
  • Human stem cell–derived β-like cells (sBCs) are a vital tool for the field of diabetes research but are technically difficult to produce and challenging to maintain in culture.

  • We describe a new cost-effective and simple approach to cryopreserving differentiated sBCs.

  • Frozen and thawed (F/T) sBCs are more uniform, display an enriched β-cell fraction, and have improved function while depleting SOX9+ progenitors.

  • F/T sBCs can successfully engraft and display stimulated insulin release in the absence of any cystic structures.

  • Cryopreservation will enable more researchers to use sBCs for complex experiments to investigate human diabetes pathogenesis.

This article contains supplementary material online at https://doi.org/10.2337/figshare.26364676.

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