Iva Popović

IN VITRO CULTIVATION OF CANINE LIMBAL TRANSPLANT

Limbal epithelial stem cells are the ultimate source of regeneration of the entire corneal epithelium under both normal and injured conditions. The corneal epithelium plays a crucial role in homeostasis and integrity of the eye. To maintain the integrity of the ocular surface, corneal epithelial cells must be balanced by stem cells, located at the limbus. The limbus is the crossing area between the cornea and sclera, 1 mm in width, and together with conjunctival epithelium plays an important role in regenerating the cornea after traumatic injuries. The aim of this study was to evaluate the culturing patterns of canine limbal stem cells and to optimize growing conditions of these cell cultures in order to develop a reliable biomedical model intended for studying the potentials of allografts/xenografts originated from canine tissues. Canine stem cell equivalents have potentials in reparative/regenerative veterinary medicine.

In vitro cultivation of porcine limbal stem cells

Abstract Similarly as in other organ structures stem cells are present in cornea residing basal epithelial layer termed palisade of Voight. A growing interest in allografts and xenografts implies a thorough study of regenerative potentials of these cells, as well as a clear description of their patterns in in vitro tissue cultures to be grafted. Recently we have developed a simple method for cultivation of porcine corneal epithelial stem cells obtained by biopsy from the limbal region. Eight enucleated eyes were obtained from four slaughtered pigs. 5 mm2 samples of limbal epithelium were taken by keratotomy method. The primary cultures of these cells showed phenotypic and morphometric characteristics of porcine corneal epithelial cells following May-Grunwald-Giemsa staining. After 5d of sowing they reached 80% of confluence. With the «Night & Day» lenses a total confluence was achieved 5d earlier in comparison to the cells that were grown in the secondary cultures. Accordingly, the use of porcine limbal stem cells has potentials in veterinary medicine (as novel approach in reparative/regenerative medicine of pets, horses and selected breeds), but also are in accordance with the extensive studies on the potential use of xenografts, mainly swine tissues/organs, in humans.

Qualitative and quantitative parameters of swine cell immunity

Flow cytometry is a modern analytic method for qualitative and quantitive analyses of procaryotic and eukaryotic cells. Classical analytical methods examine one characteristic as an average of an examined cell population. Flow cytometry simultaneously measures several biochemical and physical parameters of every single cell in a heterogenic cell sample. Flow cytometry is routinely used all over the world in professional work of chemical laboratories in veterinarian medicine. For that purpose on Veterinary faculty of University in Zagreb, machine for flow cytometry was standardized for use in veterinarian medicine, especially for immunophenotypification of haemopoetic cells of domestic animals. In this manuscript we showed the method of standardisation on a model of swine, on samples of peripheral blood and for immunophenotypification of CD45+, CD3+ and CD21+ cells.