CHICK EMBRYONIC KIDNEY CELL ISOLATION AND CRYOPRESERVATION

Abstract

Chicken kidney cells are important sources of primary cultures for virus study as well as vaccine production. The preparation of primary culture requires expertise as the risk of contamination is often very high. Yields of primary culture preparation are an important factor, especially when cells are used in vaccine production. The ability to cryopreservc primary cells is another important factor that needs to be taken into consideration since large stock must be available for urgent situations. In this study, kidneys of eighteen day old chick embryo were isolated and cold trypsin procedure was used to prepare the primary culture. Cold trypsin tissue dispersion is suitable for embryonic tissue and it is less arduous than the common warm trypsin procedure. Cells were seeded at 106 cell/ml and maintained in BME supplemented with S - 10% FBS, 0,3% tryptose phosphate broth and 1 % antibiotic-antimycotic at 38.SoC with Hepes as the main buffer system. Confluent monolayer was consistently obtained after 3 - 4 days. Yields for cold trypsin method were consistently 2 to 3 fold higher than those of warm trypsin method. Recovery rates after 24 h in culture were comparable to that of warm trypsin (74 - 78%). Initial results for in situ cryopreservation showed that it was possible to preserve chick embryonic kidney cells at -86°C while attached to culture substrates. The cryopreservation procedure does not require slow cooling step that is usually required in standard cryopreservation procedure. Chick embryonic kidney' cells,prepared by this method are also permissive to influenza virus infection, as cytopathic effects typically appear two days after inoculation