The Effect of Polyvinylpyrrolidone and the Cooling Rate during Corneal Cryopreservation

Abstract

The effect upon endothelial cell survival of (a) PVP and (b) the cooling rate was investigated during the cryopreservation of rabbit corneas with 3 mol/liter dimethyl suffoxide (Me2SO) dissolved in a hyperkalemic buffer vehicle solution that we designated CPTES; this solution was designed specifically to restrict deleterious ionic imbalances and cell swelling during hypothermic procedures. Polyvinylpyrrolidone (PVP) was used as the colloid and the corneas were cooled at 0.03, 0.1, 1, 25, or 125°C/min, using the minimum amount of extracellular solution. Electron microscopy as well as staining with fluorescein diacetate and ethidium bromide (FDA/EB) was used to assess cellular integrity. To reduce osmotic stress, steps for the serial equilibration of the cryoprotectant additives (CPAs) were based upon calculations that predict endothelial volume during CPA exchange. A toxicity study showed that at 0°C all the CPA equilibration protocols were well tolerated; for example, FDA/EB staining indicated that 97% intact cells were retained following direct transfer to 3 mol/liter Me2SO in CPTES to which 40% w/v PVP had been added as an osmotic buffer. However, less than 20% of cells were intact by FDA/EB staining with all corneas frozen at rates > 1°C/min regardless of which equilibration protocol was employed, nor were there any intact cells when 3 mol/liter Me2SO in CPTES was used alone at the lowest cooling rate. At intermediate cooling rates viability was improved: the highest mean survival of 81% was obtained using 3 mol/liter Me2SO in CPTES plus 40% PVP. Electron microscopy showed that detachment of the endothelial layer often occurred, but least damage was evident following exposure to 3 mol/liter Me2SO in CPTES plus 40% PVP and cooling at 1°C/min. No thawed cornea could maintain normal control of hydration immediately upon return to isotonic medium. The results show that, with these cryopreservation protocols, loss of cell integrity occurs at cooling rates greater than 1°C/min, whereas at lower rates higher survival of individual cells was achieved, but cellular adhesion to the basement membrane was impaired.