Three-dimensional culture of fresh and vitrified ovarian tissue in a novel tyramine substituted hyaluronan gel

Abstract

Follicle culture in matrices that allow 3-D growth may better mimic in vivo conditions by preserving follicle architecture and critical granulosa-oocyte interactions. This study explores methodology for culturing fresh and vitrified ovarian tissue. We further describe the application of a relatively new biomaterial for in vitro follicle maturation and the production of functional metaphase II oocytes. Mouse pre-antral follicle model for IVM and testing of a hyaluronan matrix. Ovaries from 12 to 14 day B6D2F1 pups were removed. Ovaries were vitrified using an ethylene glycol (EG)/DMSO protocol. Ovaries were equilibrated in chilled vitrification solutions (VS) containing 5, 10, 15 and finally 20% EG/DMSO in L-15 with 20% SSS. Ovaries were loaded on a nylon mesh and plunged in to a specially vented cryovial filled with LN2. Pre-antral follicles (FL) and ovarian follicle clusters were collected from fresh and vitrified-warmed ovaries either by mechanical isolation using needles or by enzymatic digestion in collagenase. The HA hydrogel (3 mg/ml) cross-linking was initiated by mixing with 0.03% hydrogen peroxide in a 25:1 ratio. HA gel was then quickly pipetted into the culture dish. Isolated preantral follicles or intact clusters with 4-6 follicles were rapidly seeded into the HA before gelling was completed. Follicle culture was performed in α-MEM supplemented with 5% FBS, 100 mIU/ml FSH, and ITS. Dishes were incubated at 37°C with 6% CO2. Final maturation was triggered with hCG (1.5 IU/ml) and EGF (5 ng/ml) after 12 days of growth. Outcome parameters monitored were follicle morphology, survival in culture, germinal vesicle breakdown, oocyte maturation and meiotic spindle retardance using Oosight Imaging system. Vitrified-warmed ovarian tissue was easily dissected into clusters with 4-5 intact follicles without exposure to enzyme. In contrast, enzymatic follicle isolation was more efficient and preferable for fresh tissue. With fresh ovaries, maturation rate in FL-clusters was similar to that observed with isolated follicles. Meiotic spindle retardance in ovulated oocytes from both culture types did not differ. The overall percent oocyte maturation to metaphase II was lower with vitrified tissue. HA is a natural matrix and supported follicle growth and oocyte maturation. The 3-D culture of FL-clusters provided a method to further mimic in vivo growth by preserving interfollicle connections and architecture. Functional competence of oocytes needs further assessment. Enzymatic treatment of vitrified-warmed ovaries for follicle isolation may not be the best approach due to tissue fragility.Table 1IVM summary data for fresh and vitrified tissueFrozenaFreshbFollicle - ClusterFollicle - IsolatedFollicle - ClusterFollicles observed during IVC69130154Antrum formationna51% (66/130)53% (81/154)Ovulated complexes after hCG93% (64/69)71% (92/130)66% (101/154)GVBD (MI)52% (33/64)30% (28/92)28% (28/101)Maturation to MII34% (22/64)59% (54/92)55% (56/101)Spindle Retardance (Range)na1.34- 3.641.62- 3.51aMechanically isolated follicle cluster. bEnzymatic isolation. Open table in a new tab