Monolayer Growth and Differentiated Function of Porcine and Rat Granulosa Cells Following Cryopreservation1

Abstract

Isolated granulosa cells from several species are routinely utilized in cell culture systems as a means to study the regulation of ovarian cell differentiation in vitro. To minimize trauma, the cells are generally utilized within a short time of ovarian excision. In this report we establish that freshly harvested porcine and rat granulosa cells can be preserved at sub-zero temperatures and express elements of endocrine-dependent growth and differentiated function in subsequent monolayer culture. Frozen/thawed porcine cells from 1–3 mm follicles of mature and immature animals exhibited follicle-stimulating hormone (FSH)-mediated iuteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor induction and FSH-stimulated progesterone secretion during a 9-day culture period. The extent of growth and functional responsiveness was critically dependent upon the serum concentration of the culture medium. A minimum of 5% serum was necessary for optimal growth, LH/hCG receptor induction, and steroidogenesis in porcine cells. Frozen/thawed granulosa cells from diethylstilbestrol (DES)-pnmed immature rats maintained in serum-free medium demonstrated significantly increased (P<O.001) LH/hCG receptor content and 200f-dihydroprogesterone secretion in the presence of FSH. These responses were inhibited by 3% serum. Platelet-derived growth factor, however, facilitated FSH-dependent LH/hCG receptor induction and progestin secretion in the presence of serum. The differentiated responses observed in frozen/thawed porcine and rat granulosa cells were remarkably similar to those of freshly harvested cells, indicating that cryopreservation maintained a significant degree of functional integrity. Granulosa cells can be stored for 6–7 weeks at −90°C without loss of viability or responsiveness. Longer term storage at this temperature, however, results in significantly decreased viability (P<0.O01) and severely impaired endocrine responsiveness. Cells preserved at −196°C consistently retained both cell viability (50%) and FSH responsiveness throughout 20–21 weeks, the maximum storage period tested. Cells initially frozen and stored at −90°C could be transferred to −196°C for extended storage provided the transfer was made during the useful period of −90°C storage. Cells stored at −90°C exhibited an apparent increase in [125I]iodo-hCG binding upon thawing. This was not observed in cells stored at −196°C for up to 5 weeks but may occur during longer term storage. The mechanism(s) involved is unknown but may be related to membrane perturbations secondary to freezing-induced changes in cell shape. This phenomenon did not appear to alter cellular responsiveness to gonadotropin during subsequent culture. These technical advances should benefit the fields of reproductive biology and cellular endocrinology by making more cell systems available to more laboratories