Improved Procedures for Human Granulosa Cell Isolation and Long-term in Vitro Expansion

Granulosa cells represent an essential part of the ovary's somatic compartment that maintains oocyte development and maturation and also provides the hormonal environment necessary to coordinate uterine receptivity with ovulation and, subsequently, to sustain early pregnancy. Primarily due to their major role in the processes of steroidogenesis and folliculogenesis, the study of granulosa cells is likely to be essential for comprehending and elucidating the cellular and molecular mechanisms underlying ovarian development and functions in both physiological and pathological conditions. Density gradient centrifugation is the primary method for the isolation of these cells from follicular fluid. However, this technique is time-consuming and in demand of additional reagents, experience, and dexterity. Furthermore, the limited half-life and proliferative potential of human granulosa cells put additional challenges for their study, making the conventional approaches for cultivation less applicable. Our aim was to develop dependable and effective protocols for the isolation and long-term cultivation of primary granulosa cells while preserving their functional and morphological characteristics. Human granulosa cells were isolated from follicular fluid by centrifugation and treatment with hyaluronidase without using density gradient centrifugation and/or red blood cell lysis buffer procedures. Steroidogenesis, growth dynamics, and the population doubling time of granulosa cells cultivated in a newly designed expansion medium, were determined. The medium consisted of DMEM/F12 supplemented with 5% human serum and 33% human follicular fluid. The isolation technique described in this study ensures the generation of granulosa cells with a very high level of purity. Granulosa cells cultivated in the newly developed expansion medium actively grew without changing morphologically and without losing the ability to produce estradiol and progesterone. Moreover, in this particular expansion medium, granulosa cells demonstrated a significantly increased proliferation rate, with a much shorter population doubling time compared to the control cultures grown in medium containing fetal bovine serum. Here we present a simple, fast, and cost-effective procedure for human granulosa cell isolation, combined with a reliable approach for their rapid growth and long-term cultivation based on a newly formulated expansion medium. Together, these two methods provide a comprehensive system for generating granulosa cell cultures, enabling their extensive study for the purposes of reproductive biology and medicine.