AN ALTERNATIVE METHOD FOR ISOLATING HIGH-PURITY BOVINE LUTEAL CELLS: CENTRIFUGATION IN PERCOLL DISCONTINUOUS DENSITY GRADIENT (PDDG)

MARURI, A.; BEZENZETTE, GM.; TEPLITZ, G.; LOMBARDO, DM.

Ciudad Autónoma de Buenos Aires

Jornada; V Jornadas de Jóvenes Investigadores; 2015

Secretaría de Ciencias y Técnica - Escuela de Graduados (FVET-UBA)

Introduction: corpus luteum (CL) is a transient endocrine gland, established by residual follicular wall cells (granulosa and theca cells) following ovulation. The main function of CL is the production of progesterone, a hormone which regulates various reproductive functions. There are at least four distinct morphological cell types: large luteal cells, small luteal cells, capillary endothelial cells, and fibroblast cells. The aim of this study was to evaluate the efficacy of a PDDG, to purify luteal cells. Materials and methods: the bovine CL from early luteal phase was treated by mechanical and enzymatic digestion to obtain a primary culture. Once the cells were confluent, were trypsinized and centrifuged in a PDDG (10%, 20%, 30%, 35%, 37.5% and 40%). The purified cells, called passage 1 (P1), were identified by immunocytochemistry for 3β-hydroxysteroid dehydrogenase. Results: we found 100% purity in Percoll density layers 5 and 6 clearly higher than standard technique without selection with Percoll that only reached 80.6%±7.02 purity. Conclusion: this method offers a cost effective alternative to isolate luteal cells in high-purity to be able to use as in vitro model in various assays.