Cryopreservation and Post-thaw Characterization of Dissociated Human Islet Cells

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2022 Jan 26

PMID 35081145

Authors

Leah A Marquez-Curtis

Xiao-Qing Dai

Yan Hang

Jonathan Y Lam

James Lyon

Jocelyn E Manning Fox

Locksley E McGann

Patrick E MacDonald

Seung K Kim

Janet A W Elliott

Affiliations

Soon will be listed here.

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 Ca2+ 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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References

Ross-Rodriguez L, Elliott J, McGann L . Characterization of cryobiological responses in TF-1 cells using interrupted freezing procedures. Cryobiology. 2009; 60(2):106-16. DOI: 10.1016/j.cryobiol.2009.09.007. View

Saeedi P, Petersohn I, Salpea P, Malanda B, Karuranga S, Unwin N . Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the International Diabetes Federation Diabetes Atlas, 9 edition. Diabetes Res Clin Pract. 2019; 157:107843. DOI: 10.1016/j.diabres.2019.107843. View

Manning Fox J, Lyon J, Dai X, Wright R, Hayward J, van de Bunt M . Human islet function following 20 years of cryogenic biobanking. Diabetologia. 2015; 58(7):1503-12. PMC: 4472956. DOI: 10.1007/s00125-015-3598-4. View

Aldridge S, Teichmann S . Single cell transcriptomics comes of age. Nat Commun. 2020; 11(1):4307. PMC: 7453005. DOI: 10.1038/s41467-020-18158-5. View

von Mach M, Schlosser J, Weiland M, Feilen P, Ringel M, Hengstler J . Size of pancreatic islets of Langerhans: a key parameter for viability after cryopreservation. Acta Diabetol. 2003; 40(3):123-9. DOI: 10.1007/s00592-003-0100-4. View

Dobin A, Davis C, Schlesinger F, Drenkow J, Zaleski C, Jha S . STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2012; 29(1):15-21. PMC: 3530905. DOI: 10.1093/bioinformatics/bts635. View

Finak G, McDavid A, Yajima M, Deng J, Gersuk V, Shalek A . MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 2015; 16:278. PMC: 4676162. DOI: 10.1186/s13059-015-0844-5. View

Llufrio E, Wang L, Naser F, Patti G . Sorting cells alters their redox state and cellular metabolome. Redox Biol. 2018; 16:381-387. PMC: 5952879. DOI: 10.1016/j.redox.2018.03.004. View

Abazari A, Jomha N, Elliott J, McGann L . Cryopreservation of articular cartilage. Cryobiology. 2013; 66(3):201-9. DOI: 10.1016/j.cryobiol.2013.03.001. View

10.

Anders S, Pyl P, Huber W . HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics. 2014; 31(2):166-9. PMC: 4287950. DOI: 10.1093/bioinformatics/btu638. View

11.

Storr H, Kind B, Parfitt D, Chapple J, Lorenz M, Koehler K . Deficiency of ferritin heavy-chain nuclear import in triple a syndrome implies nuclear oxidative damage as the primary disease mechanism. Mol Endocrinol. 2009; 23(12):2086-94. PMC: 5419132. DOI: 10.1210/me.2009-0056. View

12.

Eskandari N, Marquez-Curtis L, McGann L, Elliott J . Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers. Cryobiology. 2018; 85:63-72. DOI: 10.1016/j.cryobiol.2018.10.001. View

13.

Baust J, Gao D, Baust J . Cryopreservation: An emerging paradigm change. Organogenesis. 2010; 5(3):90-6. PMC: 2781087. DOI: 10.4161/org.5.3.10021. View

14.

Baicu S, Taylor M . Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency. Cryobiology. 2002; 45(1):33-48. DOI: 10.1016/s0011-2240(02)00104-9. View

15.

Zhang W, Nilles T, Johnson J, Margolick J . The effect of cellular isolation and cryopreservation on the expression of markers identifying subsets of regulatory T cells. J Immunol Methods. 2016; 431:31-7. PMC: 4792753. DOI: 10.1016/j.jim.2016.02.004. View

16.

Picelli S, Faridani O, Bjorklund A, Winberg G, Sagasser S, Sandberg R . Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc. 2014; 9(1):171-81. DOI: 10.1038/nprot.2014.006. View

17.

Powell-Palm M, Zhang Y, Aruda J, Rubinsky B . Isochoric conditions enable high subfreezing temperature pancreatic islet preservation without osmotic cryoprotective agents. Cryobiology. 2019; 86:130-133. DOI: 10.1016/j.cryobiol.2019.01.003. View

18.

Yamanaka T, Tashima K, Takahashi R, Takashima S, Goto T, Hirabayashi M . Direct comparison of Cryotop vitrification and Bicell freezing on recovery of functional rat pancreatic islets. Cryobiology. 2016; 73(3):376-382. DOI: 10.1016/j.cryobiol.2016.09.003. View

19.

Nakayama-Iwatsuki K, Hirabayashi M, Hochi S . Fabrication of functional rat pseudo-islets after cryopreservation of pancreatic islets or dispersed islet cells. J Tissue Eng Regen Med. 2021; 15(7):686-696. DOI: 10.1002/term.3219. View

20.

Crisafuli F, Ramos E, Rocha M . Characterizing the interaction between DNA and GelRed fluorescent stain. Eur Biophys J. 2014; 44(1-2):1-7. DOI: 10.1007/s00249-014-0995-4. View