Protocol for Cryopreservation of Endothelial Monolayers.
Chondroitin sulfate
Cryopreservation
Dimethyl sulfoxide
Fluorescent microscopy
Hydroxyethyl starch
Interrupted slow cooling
Membrane integrity
Rinzl plastic
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2021
2021
Historique:
entrez:
16
8
2020
pubmed:
17
8
2020
medline:
13
3
2021
Statut:
ppublish
Résumé
One of the major challenges in the preservation of complex tissues is the cryosensitivity of the endothelium, the single layer of cells lining blood vessels, corneas, and other tissues. The increasing importance of endothelial monolayers in tissue-engineered constructs for transplantation and research warrants the need to develop protocols for the successful cryopreservation of cells in monolayers. In this chapter, we describe a recently published cryopreservation protocol that we developed based on examination of various factors that influence the post-thaw recovery of endothelial monolayers. To efficiently investigate cryopreservation protocol parameters, we employed an interrupted slow-cooling procedure (graded freezing) that allows dissecting loss of cell viability into contributions from slow-cooling injury and rapid-cooling injury. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a penetrating cryoprotectant (5% dimethyl sulfoxide) and a non-penetrating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled cooling at 0.2 °C/min or 1 °C/min, and removal of cryoprotectant immediately after thaw. The protocol has been validated for human umbilical vein and porcine corneal endothelial cell monolayers.
Identifiants
pubmed: 32797436
doi: 10.1007/978-1-0716-0783-1_30
doi:
Substances chimiques
Cryoprotective Agents
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
581-591Subventions
Organisme : CIHR
ID : MOP 133684
Pays : Canada
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