Increased Cell Survival of Human Primary Conjunctival Stem Cells in Dimethyl Sulfoxide-Based Cryopreservation Media

Arianne J. H. van Velthoven, Marina Bertolin, Vanessa Barbaro, Mireille M. J. P. E. Sthijns, Rudy M. M. A. Nuijts, Vanessa L. S. LaPointe, Mor M. Dickman*, Stefano Ferrari

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

2 Citations (Web of Science)


Glycerol and dimethyl sulfoxide (DMSO) are widely used cryoprotectants for freezing human cell cultures. During the manufacturing process of ocular stem cell-based autographs, ex vivo cultivated ocular cells are cryopreserved for quality control purposes in accordance with regulatory requirements. The efficiency of the cryopreservation methods is limited by their effect on cell survival and quality. We compared two cryopreservation reagents, glycerol and DMSO, for their influence on the survival and quality of human primary conjunctival cultures. We found increased cell viability after cryopreservation in DMSO compared to cryopreservation in glycerol. The clonogenic and proliferative capacity was unaffected by the cryopreservation reagents, as shown by the colony forming efficiency and cumulative cell doubling. Importantly, the percentage of p63 alpha- and keratin 19 (K19)-positive cells following cryopreservation in DMSO or glycerol was comparable. Taken together, our results demonstrate that cryopreservation in DMSO improves cell survival compared to cryopreservation in glycerol, with no subsequent effect on cell proliferative-, clonogenic-, or differentiation capacity. Therefore, we advise the use of a 10% DMSO-based cryopreservation medium for the cryopreservation of human primary conjunctival cells, as it will improve the number of cells available for the manufacturing of conjunctival stem cell-based autografts for clinical use.

Original languageEnglish
Pages (from-to)67-72
Number of pages6
JournalBiopreservation and Biobanking
Issue number1
Early online date13 Nov 2020
Publication statusPublished - 1 Feb 2021


  • ocular surface epithelium
  • conjunctiva
  • cell-based therapy
  • cryopreservation
  • regeneration

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