Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates.
Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.
Quinn
M C J
MC
Department of Anatomy and Structural Biology, School of Medical Sciences, University of Otago, PO Box 913, Dunedin 9001, New Zealand.
McGregor
S B
SB
Stanton
J L
JL
Hessian
P A
PA
Gillett
W R
WR
Green
D P L
DP
D000328:Adult; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D018870:Endoplasmic Reticulum, Rough / Q000648:ultrastructure; D005260:Female; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D015571:Follicular Fluid / Q000166:cytology*; D006107:Granulosa Cells / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D008854:Microscopy, Electron; D008928:Mitochondria / Q000648:ultrastructure; D011962:Receptors, FSH / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005456:Fluorescent Dyes; D011962:Receptors, FSH; D016650:Fluorescein-5-isothiocyanate