Avaliação de diferentes protocolos visando o estabelecimento de uma linhagem celular a partir de ovos e larvas de abelhas Apis mellifera africanizadas

Abstract

Honeybees are particularly important for humans and the environment, due to the fundamental role they play, through pollination. However, a significant decrease in colonies of the species Apis mellifera has been reported worldwide, caused by several factors, including viral infections. For the study and diagnosis of diseases caused by viruses, in vitro cell culture is used. Currently, there are only two cell lines of Apis mellifera, however, these lines are not commercially available, in addition these are permanently infected by the wing-deforming virus, which makes their use difficult, especially in research related to cell-pathogen interactions or between pathogens. The objective of this dissertation was to standardize techniques for the development of primary culture, easy and practical to use, with the aim of subsequent cell immortalization, aiming at the development of one or more cell lines of the species Apis mellifera. To obtain a continuous lineage from primary cultures, we describe five agents of cell immortalization. Currently, the cell immortalization techniques used in insect cell cultures are adapted to those use in mammalian cells. Each technique has advantages, limitations, practicalities, and genotypic and phenotypic implications. The choice of the immortalization technique is a fundamental and challenging step in the development of a cell line, since it should, allow long periods of cell viability, with a high number of passages, in addition to keeping the genotype and phenotype of the cells similar or identical to the original tissue. For the preparation of primary crops, Grace, Leibovitz and Schneider media were evaluated, with fetal bovine serum and antibiotics. Techniques for the maintenance and cryopreservation of cell cultures originating from eggs and bee larvae of Apis mellifera were also evaluated. For maintenance, trypsinization techniques, use of cell spreader and centrifugation of cell content were evaluated. Regarding cryopreservation, two different freezing solutions were evaluated. Through the described techniques, 79 cell cultures were developed, with up to 19 passages using the Grace medium, and with cell viability of up to 140 days with the Schneider medium. The use of larvae in primary culture, showed a higher proliferation rate, and cell longevity, when compared to the use of eggs. Among the evaluated media, the use of Grace medium in the elaboration of primary cultures presented higher rates of cell proliferation and replicability, proving to be a great option for the application of cell immortalization techniques. The use of trypsin during the maintenance of the cultures, demonstrated high cell death, being replaced by cell spreaders. The cryopreservation solution using 10% DMSO and 90% fetal bovine serum, provided the cryopreservation of cell cultures for eight months.