disease constitutes the most often cited indication for corneal
transplants. The global shortage of donor corneas has prompted
the alternative use of tissue-engineered grafts [3–6] or cell-based
injection therapies [7, 8], which requires the ex vivo expansion and
cryopreservation of corneal endothelial cells.
Endothelial cells, even in suspension, are susceptible to cryoinjury. For example, the routine procedure of cooling at 1
C/min in
the presence of 10% dimethyl sulfoxide leaves only about 66% of
human umbilical vein endothelial cells (HUVECs) and 76% of
porcine corneal endothelial cells viable [9–12]. We have developed
an interrupted slow-cooling procedure (graded freezing) that not
only identifies the occurrence and extent of cryoinjury but also
provides an efficient method for optimizing the degree of protection conferred by permeating and non-permeating cryoprotectants
(CPAs) [12–14]. In this experimental technique, the response of
cells is studied as a function of temperature throughout the cooling
protocol. At various temperatures along a slow-cooling profile
(e.g., 1
C/min), cells are either directly thawed and assessed for
membrane integrity to gain information about slow-cooling injury,
or plunged into liquid nitrogen and then thawed and assessed in
order to identify rapid-cooling injury. By iteratively using graded
freezing, we greatly improved cryopreservation of endothelial cells
in suspension using 5% dimethyl sulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as cryoprotectants that resulted in 94% postthaw membrane integrity relative to fresh control for HUVECs and
91% and 93% of fresh control for porcine and human corneal
endothelial cells, respectively [11, 12]. Furthermore, cryopreserved
endothelial cells were fully functional post-thaw [11, 12]. As a
permeating CPA, dimethyl sulfoxide colligatively reduces the
amount of ice formed at a given temperature and mitigates solute
effects injury [15]. Hydroxyethyl starch, a non-permeating CPA,
osmotically draws water out of the cells, thus mitigating damage
due to intracellular ice formation, and it also stabilizes the cell
membrane [16].
Cells in monolayers are extremely susceptible to intracellular ice
formation due to the presence of cell–cell and cell–matrix interactions [17]. Because the extracellular environment and the substrate
upon which cells are cultured experience volume excursions (contraction and expansion) during cryopreservation and thawing processes, we hypothesized that a substrate with a coefficient of linear
thermal expansion similar to that of ice will reduce mismatch strain
and prevent detachment of cells in monolayers from the surface
[18]. We subsequently found that such a substrate also has an effect
on viability of cells that remain attached [19]. Therefore, by studying the role of monolayer handling and CPA composition and using
Rinzl plastic (whose coefficient of thermal expansion is comparable
to that of ice) as a substrate, we developed a protocol to cryopreserve intact endothelial monolayers resulting in greater than 90%
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Leah A. Marquez-Curtis et al.
transplants. The global shortage of donor corneas has prompted
the alternative use of tissue-engineered grafts [3–6] or cell-based
injection therapies [7, 8], which requires the ex vivo expansion and
cryopreservation of corneal endothelial cells.
Endothelial cells, even in suspension, are susceptible to cryoinjury. For example, the routine procedure of cooling at 1
C/min in
the presence of 10% dimethyl sulfoxide leaves only about 66% of
human umbilical vein endothelial cells (HUVECs) and 76% of
porcine corneal endothelial cells viable [9–12]. We have developed
an interrupted slow-cooling procedure (graded freezing) that not
only identifies the occurrence and extent of cryoinjury but also
provides an efficient method for optimizing the degree of protection conferred by permeating and non-permeating cryoprotectants
(CPAs) [12–14]. In this experimental technique, the response of
cells is studied as a function of temperature throughout the cooling
protocol. At various temperatures along a slow-cooling profile
(e.g., 1
C/min), cells are either directly thawed and assessed for
membrane integrity to gain information about slow-cooling injury,
or plunged into liquid nitrogen and then thawed and assessed in
order to identify rapid-cooling injury. By iteratively using graded
freezing, we greatly improved cryopreservation of endothelial cells
in suspension using 5% dimethyl sulfoxide (DMSO) and 6% hydroxyethyl starch (HES) as cryoprotectants that resulted in 94% postthaw membrane integrity relative to fresh control for HUVECs and
91% and 93% of fresh control for porcine and human corneal
endothelial cells, respectively [11, 12]. Furthermore, cryopreserved
endothelial cells were fully functional post-thaw [11, 12]. As a
permeating CPA, dimethyl sulfoxide colligatively reduces the
amount of ice formed at a given temperature and mitigates solute
effects injury [15]. Hydroxyethyl starch, a non-permeating CPA,
osmotically draws water out of the cells, thus mitigating damage
due to intracellular ice formation, and it also stabilizes the cell
membrane [16].
Cells in monolayers are extremely susceptible to intracellular ice
formation due to the presence of cell–cell and cell–matrix interactions [17]. Because the extracellular environment and the substrate
upon which cells are cultured experience volume excursions (contraction and expansion) during cryopreservation and thawing processes, we hypothesized that a substrate with a coefficient of linear
thermal expansion similar to that of ice will reduce mismatch strain
and prevent detachment of cells in monolayers from the surface
[18]. We subsequently found that such a substrate also has an effect
on viability of cells that remain attached [19]. Therefore, by studying the role of monolayer handling and CPA composition and using
Rinzl plastic (whose coefficient of thermal expansion is comparable
to that of ice) as a substrate, we developed a protocol to cryopreserve intact endothelial monolayers resulting in greater than 90%
582
Leah A. Marquez-Curtis et al.