Chapter 30
Protocol for Cryopreservation of Endothelial Monolayers
Leah A. Marquez-Curtis, Nasim Eskandari, Locksley E. McGann,
and Janet A. W. Elliott
Abstract
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.
Key words Cryopreservation, Dimethyl sulfoxide, Hydroxyethyl starch, Chondroitin sulfate, Interrupted slow cooling, Membrane integrity, Fluorescent microscopy, Rinzl plastic
1 Introduction
A monolayer of endothelial cells (also called the endothelium)
constitutes the inner lining of blood vessels, lymphatic vessels,
and other tissues, including the cornea. The endothelium was
initially perceived as a mere diffusion barrier between the blood
and surrounding tissues but is now recognized as a key player in
controlling blood fluidity, platelet aggregation, and leukocyte trafficking and in the regulation of inflammation and angiogenesis
[1]. In the cornea, the endothelial cell monolayer acts as a barrier
and pump to restrict movement of water into the stroma; excess
water imbibed by the stroma causes swelling of the cornea, resulting in blurred vision and eventual blindness [2]. Age-dependent
deterioration of the corneal endothelium or its impairment by
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_30, © Springer Science+Business Media, LLC, part of Springer Nature 2021
581
Protocol for Cryopreservation of Endothelial Monolayers
Leah A. Marquez-Curtis, Nasim Eskandari, Locksley E. McGann,
and Janet A. W. Elliott
Abstract
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.
Key words Cryopreservation, Dimethyl sulfoxide, Hydroxyethyl starch, Chondroitin sulfate, Interrupted slow cooling, Membrane integrity, Fluorescent microscopy, Rinzl plastic
1 Introduction
A monolayer of endothelial cells (also called the endothelium)
constitutes the inner lining of blood vessels, lymphatic vessels,
and other tissues, including the cornea. The endothelium was
initially perceived as a mere diffusion barrier between the blood
and surrounding tissues but is now recognized as a key player in
controlling blood fluidity, platelet aggregation, and leukocyte trafficking and in the regulation of inflammation and angiogenesis
[1]. In the cornea, the endothelial cell monolayer acts as a barrier
and pump to restrict movement of water into the stroma; excess
water imbibed by the stroma causes swelling of the cornea, resulting in blurred vision and eventual blindness [2]. Age-dependent
deterioration of the corneal endothelium or its impairment by
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_30, © Springer Science+Business Media, LLC, part of Springer Nature 2021
581