Development of a Cryopreservation Procedure for a 3D Tissue Culture Model

Background: The eye irritation test, developed by Draize et. al. in 1944, is used to study toxicological chemical substances the eye irritation test. In order to produce toxicologically-normalized results as well as to circumvent moral and ethical concerns, a Hemicornea cell culture model was developed intending to replace the Draize test in the future. In order make the model available for a wider use, transportation and storage facilities are required. Cryopreservation is useful here. This study deals with the stroma equivalent, an essential part of Hemicornea model. The individual components of the stroma equivalent-namely, collagen and HCK-Ca Cells, as well as their interaction-were examined with regard to the cryopreservation and optimized. First, the isolated collagen of rat tail with different compositions of collagen was checked on its suitability for use in the stroma equivalent. In these experiments a dependency between NaHCO3 and the consistency of the collagen mixture could be identified. HCK-Ca cells-yet another component of the Hemicornea Model-were frozen with the anti-freezing media 10% (v/v) DMSO, Biofreeze and 10% (v/v) DMSO + 40% (v/v) HES at the cooling rates of 10 K/min, 5 K/min, 1 K/min and 0.2 K/min. The highest number of intact cell membranes and the best re-cultivation efficiency was obtained with the anti-freeze media 10% (v/v) DMSO and the cooling rate of 10 K/min. In addition to this, cultivated stroma equivalent were cryopreserved with the above freezing media and cooling rates. The appearance of cracks was observed during the production of stroma equivalent. After cryopreservation and subsequent thawing of these stroma equivalent, an increased number of viable cells in the area of intact collagen layers and an increased number of vital cells in the region of defective collagen layers was observed (with the fluorescence microscope). Finally, a construction was developed which will enable a directional solidification of the Hemicornea models.