Measurement of Cryoprotectant Permeability in Adherent Endothelial Cells and Applications to Cryopreservation

Abstract

Vitrification is a promising approach for cryopreservation of adherent cells because it allows complete avoidance of ice formation. However, high cryoprotectant (CPA) concentrations are required to prevent freezing, and exposure to high CPA concentrations increases the risk of osmotic and toxic damage. Although cell membrane transport modeling can be used for rational design of CPA equilibration procedures, the necessary permeability data is extremely scarce for adherent cells. This study validates a method for in situ measurement of water and CPA permeability in adherent cells based on the fluorescence quenching of intracellular calcein. Permeability parameters for endothelial monolayers were measured during exposure to four common cryoprotectants (dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol) at temperatures of 4, 21, and 37 °C. Propylene glycol exhibited the highest permeability and gycerol the lowest. The data was fit using an Arrhenius model, yielding activation energies ranging from 45 to 61 kJ/mol for water transport and 84 to 99 kJ/mol for CPA transport. These permeability parameters will facilitate the development of mathematically-optimized CPA equilibration procedures for vitrification of adherent endothelial cells. Our results establish calcein fluorescence quenching as an effective method for measurement of CPA permeability in adherent cells.

Appendix

We described the relationship between fluorescence intensity and cell volume using a modified Stern–Volmer model. The Stern–Volmer relationship describes the change in fluorescence intensity due to physical interaction between a fluorophore and quenching molecule as follows:

$$ F = \frac{{F^{*} }}{1 + KQ} $$

(A1)

where F is the fluorescence intensity of the fluorophore in the presence of quenching interactions, F* is the fluorescence intensity in the absence of quenching, K is the quenching constant, and Q is the quencher concentration. Previous studies suggest that a portion of intracellular calcein molecules are not free to interact with quencher molecules because of binding or compartmentalization within the cell.8,19 Modifications to the Stern–Volmer relationship have been described previously that account for accessible and inaccessible fluorophores, where it is assumed that any change in fluorescence intensity is due to the physical interaction of quenchers with accessible fluorophores only and the fluorescence intensity of inaccessible fluorophores remains constant.27,44 Similarly, we can divide the intracellular calcein molecules into two groups, one of which is free to interact with intracellular quencher molecules, and one that does not interact with the quenchers. The calcein molecules that are free to interact with quenchers yield a fluorescence intensity described by Eq. (A1), whereas non-interacting fluorophores give rise to a constant intensity, resulting in the following equation for the total fluorescence:

$$ F = \frac{{F^{*} }}{1 + KQ} + F_{\text{B}} $$

(A2)

where F _B is the fluorescence intensity due to non-interacting calcein molecules (i.e., the portion of the fluorescence that is not sensitive to quencher concentration). The quencher concentration can be expressed in terms of the cell volume by assuming that the endogenous quencher molecules (e.g., intracellular proteins45) are membrane impermeable and trapped within the cell. This assumption results in the following expression for the quencher concentration:

$$ Q = \frac{{Q_{0} V_{\text{w0}} }}{{V_{\text{w}} + V_{\text{CPA}} }} $$

(A3)

where Q ₀ is the quencher concentration under isotonic conditions. Substituting Eqs. (A3) into (A2) and normalizing by the fluorescence intensity under isotonic conditions (F ₀) results in:

$$ \overline{F} \equiv \frac{F}{{F_{0} }} = \left( {\frac{{\overline{V} + \beta \alpha }}{{\overline{V} + \alpha }}} \right)\left( {\frac{1 + \alpha }{1 + \beta \alpha }} \right) $$

(5)

where $\alpha \equiv KQ_{0}$ and $\beta \equiv \frac{{F_{\text{B}} }}{{F^{*} + F_{\text{B}} }}$. The parameter α describes the extent of quenching that occurs under isotonic conditions, and β is the fraction of total fluorophores that is insensitive to quencher concentration and therefore insensitive to cell volume.