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Cited by 26Pancreatic function and diabetesDiabetes and associated disordersReproductive Biology and Fertility

Cryopreservation and post-thaw characterization of dissociated human islet cells

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Marquez-Curtis, Leah A., Dai, Xiao-Qing, Hang, Yan, Lam, Jonathan Y., Lyon, James, Manning Fox, Jocelyn E., McGann, Locksley E., MacDonald, Patrick E. +2 more·2022·PLOS ONE
DOI 10.1371/journal.pone.0263005PMC PMC8791532
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Abstract

The objective of this study is to optimize the cryopreservation of dissociated islet cells and obtain functional cells that can be used in single-cell transcriptome studies on the pathology and treatment of diabetes. Using an iterative graded freezing approach we obtained viable cells after cooling in 10% dimethyl sulfoxide and 6% hydroxyethyl starch at 1°C/min to –40°C, storage in liquid nitrogen, rapid thaw, and removal of cryoprotectants by serial dilution. The expression of epithelial cell adhesion molecule declined immediately after thaw, but recovered after overnight incubation, while that of an endocrine cell marker (HPi2) remained high after cryopreservation. Patch-clamp electrophysiology revealed differences in channel activities and exocytosis of various islet cell types; however, exocytotic responses, and the biophysical properties of voltage-gated Na+and Ca 2+channels, are sustained after cryopreservation. Single-cell RNA sequencing indicates that overall transcriptome and crucial exocytosis genes are comparable between fresh and cryopreserved dispersed human islet cells. Thus, we report an optimized procedure for cryopreserving dispersed islet cells that maintained their membrane integrity, along with their molecular and functional phenotypes. Our findings will not only provide a ready source of cells for investigating cellular mechanisms in diabetes but also for bio-engineering pseudo-islets and islet sheets for modeling studies and potential transplant applications.

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  • Better viability and post-thaw function has been achieved by dispersing islets, freezing them as single cells, and then reaggregating them into clusters upon thaw.

    “Better viability and post-thaw function has been achieved by dispersing islets, freezing them as single cells, and then reaggregating them into clusters upon thaw.186,187”

    From Developments in stem cell-derived islet replacement therapy for treating type 1 diabetes (2023) · cites this paper as [187]

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Authors
Marquez-Curtis, L. A., Dai, X.-Q., Hang, Y., Lam, J. Y., Lyon, J., Manning Fox, J. E., McGann, L. E., MacDonald, P. E., Kim, S. K., & Elliott, J. A. W.
Affiliations
  • University of Alberta (CA)
  • Stanford Medicine (US)
Corresponding author
Elliott, Janet A. W.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2022-01-26
Volume
17
Issue
1
Pages
e0263005
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0263005
PMC
PMC8791532
OpenAlex
W4210522117
Cited by
26
References
69
Citations by year
2026: 5 · 2025: 8 · 2024: 6 · 2023: 5 · 2022: 2
FWCI
5.02
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Canada Research Chairs
  • Silicon Valley Community Foundation (#2017-174469)
  • National Institutes of Health (R01 DK120447, R01 DK107507, R01 DK126482, U01 DK123716)
  • Stanford Islet Research Core in the Stanford Diabetes Research Center (P30 DK116074)
  • Juvenile Diabetes Research Foundation United States of America (5-COE-2019-860-S-B, 3-PDF-2014-196-A-N)
  • Stanford Translational Research & Applied Medicine
  • H. L. Snyder Medical Foundation
  • Elser Trust
  • Steven and Michele Kirsch Foundation
  • Gift from S. and K. Skeff
Topics
  • Health Sciences › Medicine › Surgery › Pancreatic function and diabetes
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Diabetes and associated disorders
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Reproductive Biology and Fertility
Keywords
islet cryopreservation, dimethyl sulfoxide, hydroxyethyl starch, post-thaw cell viability, exocytosis, voltage-gated ion channels, single-cell transcriptomics, diabetes mellitus, islet transplantation, plasma membrane integrity
MeSH terms
RNA-Seq, Adolescent, Adult, Aged, Antigens, Differentiation, Cryoprotective Agents, Female, Humans, Islets of Langerhans, Male, Middle Aged, Calcium Channels, Sodium Channels, Cryopreservation, Islets of Langerhans Transplantation, Single-Cell Analysis

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