Fundamental cryobiology of cells from a bioengineered human corneal equivalent.

摘要

Technological advances in the bioengineering of human corneal equivalents establish the need for successful intermediate-term storage to facilitate their availability and use.;Successful cryopreservation of corneas has proven to be difficult, with much of the research based on empirical experimentation. It now seems that the ability to cryopreserve tissues, requires a more methodical approach that addresses the individual components of the tissue separately and subsequently bringing this knowledge together and applying it to the entire tissue. This thesis describes such an approach and provides a rational, scientific method for developing a cryopreservation protocol for a bioengineered human corneal equivalent.;Graded freezing studies of the human corneal endothelial monolayers showed that solution effects damage is not a significant cause of injury, but rapid cooling to -196°C is detrimental. Improved recovery and decreased detachment was seen after rapid cooling and storage at -80°C, indicating the need to reevaluate the final storage conditions for long-term preservation of corneas. Osmotic (membrane hydraulic conductivity; Lp, osmotically-inactive fraction, Arrhenius activation energy of Lp) and permeability parameters (membrane hydraulic conductivity, membrane cryoprotectant permeability; P s, reflection coeffiecient, and Arrhenius activation of Lp and Ps) of human corneal endothelial, keratocyte, and epithelial cells were determined. The osmotic and permeability values obtained for the constituent cells of the corneal equivalent show significant differences in their osmotic and low temperature responses, which has physiological consequences during freezing and thawing and makes clear the challenge of optimizing a cryopreservation strategy.;Knowledge of these parameters also allows theoretical prediction of addition and removal of cryoprotectants, response of cells to cooling at varying rates in different cryoprotectants and combinations of cryoprotectants. These simulations of the low temperature responses of the cells of the corneal equivalent, led to conclusions of mechanisms of cryoinjury, such as increased likelihood of intracellular ice formation during rapid cooling from the predictions of degree of supercooling, and cryoprotection, where low temperature simulations in the presence of cryoprotectants show a decrease in deleterious solute concentrations.;Combining knowledge from experimentation and mathematical modeling establishes a scientific and theoretical basis for the development of tissue cryopreservation protocols.