Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells

Abstract

Ice-free cryopreservation, known as vitrification, is an appealing approach for banking of adherent cells and tissues because it prevents dissociation and morphological damage that may result from ice crystal formation. However, current vitrification methods are often limited by the cytotoxicity of the concentrated cryoprotective agent (CPA) solutions that are required to suppress ice formation. Recently, we described a mathematical strategy for identifying minimally toxic CPA equilibration procedures based on the minimization of a toxicity cost function. Here we provide direct experimental support for the feasibility of these methods when applied to adherent endothelial cells. We first developed a concentration- and temperature-dependent toxicity cost function by exposing the cells to a range of glycerol concentrations at 21°C and 37°C, and fitting the resulting viability data to a first order cell death model. This cost function was then numerically minimized in our state constrained optimization routine to determine addition and removal procedures for 17 molal (mol/kg water) glycerol solutions. Using these predicted optimal procedures, we obtained 81% recovery after exposure to vitrification solutions, as well as successful vitrification with the relatively slow cooling and warming rates of 50°C/min and 130°C/min. In comparison, conventional multistep CPA equilibration procedures resulted in much lower cell yields of about 10%. Our results demonstrate the potential for rational design of minimally toxic vitrification procedures and pave the way for extension of our optimization approach to other adherent cell types as well as more complex systems such as tissues and organs.

A Mathematical Framework for Developing Freezing Protocols in the Cryopreservation of Cells

“…Exterior ice boundary determining cell damage, and hence optimizing operating conditions, such as the cooling rate. Benson and colleagues have investigated optimal control problems for CPA equilibration, introducing the concept of a CPA toxicity cost function that should be minimized [6,7,9,13,14]. We consider a similar toxicity cost function, and add a new cost function to characterize intracellular ice formation, in order to estimate cell damage as a function of cooling rate.…”

Microfluidics for cryopreservation

“…Except for osmotic injuries, CPA toxicity is also a dominant factor for successful cryopreservation of living systems by both freezing and vitrification (Benson et al, 2012; Cordeiro et al, 2015; Davidson et al, 2015; Fahy, 1981, 1986a, 2010; Fahy and Karow, 1977; Fahy et al, 1990; Fahy et al, 1984; Fahy et al, 2004a, b; Fahy et al, 2004c). Significant effort has been devoted to the study on CPA toxicity neutralization, especially in the direction of organ vitrification (Fahy, 1981, 1986b, 1987, 1994; Fahy et al, 1985; Fahy et al, 2004c; Khirabadi et al, 1988).…”

“…Benson et al pioneered a toxicity cost function (it reflects the cumulative damage caused by toxicity) based mathematical optimization of the procedures for CPA equilibration (addition and removal), the predictions on human oocytes yield significantly less toxicity than conventional stepwise procedures (Benson et al, 2012; Davidson et al, 2014). Its extension form was further successfully used for the design of CPA equilibration for adherent endothelial cells (Davidson et al, 2015). Compared with programmed slow freezing, vitrification/low-CPA vitrification requires comparatively more CPA; therefore CPA addition and removal processes are more time consuming and costly (Choi et al, 2015a; Choi et al, 2015b; Huang et al, 2015; Wang et al, 2016).…”

Surface‐Acoustic‐Wave‐Based Lab‐on‐Chip for Rapid Transport of Cryoprotectants across Cell Membrane for Cryopreservation with Significantly Improved Cell Viability

“…Researchers studied human umbilical vein endothelial cells (HUVECs) in a self‐made microperfusion chamber, measured their cryobiological properties, and optimized the removal of 1.5 m DMSO at 25 °C . Benson et al and Davidson et al optimized cryoprotectant equilibration using a piecewise‐constant procedure, reduced toxicity, and osmotic shock for oocyte cryopreservation . Microfluidics has also been explored for CPA loading and unloading.…”