Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps.
OBJECTIVE
To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production.


METHODS
Contamination of GC by white blood cells was assessed by Wright's stain and immunohistochemistry. Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes [PML]); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes).


RESULTS
Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4%. Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes.


CONCLUSIONS
These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented.
Department of Obstetrics and Gynecology, University of Chicago, Illinois.
M W
MW
Beckmann
D
D
Polacek
L
L
Seung
J R
JR
Schreiber
D001773:Blood Cells; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D005260:Female; D006107:Granulosa Cells / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes; D011374:Progesterone / Q000096:biosynthesis; D012462:Saline Solution, Hypertonic / Q000494:pharmacology
D006063:Chorionic Gonadotropin; D012462:Saline Solution, Hypertonic; D011374:Progesterone