Three dimensional culture of fresh and vitrified mouse pre-antral follicles in a hyaluronan-based hydrogel: a preliminary investigation of a novel biomaterial for in vitro follicle maturation

Abstract

BackgroundFolliculogenesis within the ovary requires interaction between somatic cell components and the oocyte. Maintenance of 3-dimensional (3-D) architecture and granulosa-oocyte interaction may be critical for successful in vitro maturation of follicles. Testing of novel biomaterials for the 3-D culture of follicles may ultimately lead to a culture model that can support the longer in vitro culture intervals needed for in vitro maturation of human oocytes from ovarian tissue biopsies.MethodsA novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. The HA was prepared at concentrations from 2 to 5 mg/ml. HA hydrogel was also formulated and tested with matrix proteins (ECM). Enzymatically isolated pre-antral follicles from the ovaries of 10–12 day SJL pups were divided amongst control (CT) and HA treatments. The growth of both fresh and vitrified follicles was assessed after encapsulation in the hydrogel. The basal culture medium was MEM alpha supplemented with FSH, LH, ITS and 5% FBS. Maturation was triggered by addition of hCG and EGF after in vitro culture (IVC). Outcome parameters monitored were follicle morphology, survival after IVC, antrum formation, GVBD and MII formation. Differences between treatments were analyzed.ResultsHA and ECM-HA encapsulated follicles looked healthy and maintained their 3-D architecture during IVC. In control cultures, the follicles flattened and granulosa:oocyte connections appeared fragile. Estradiol secretion per follicle was significantly higher by Day 12 in ECM-HA compared to HA or CT (4119, 703 and 1080 pg/ml, respectively). HA and ECM-HA cultured follicles had similar survival rates (62% and 54%, respectively), percent GV breakdown (96–97%), MII formation (47–48%) and oocyte diameters at the end of IVC. Control cultures differed significantly in percent GVBD (85%) and MII formation (67%) . Vitrified-warmed follicles encapsulated in HA had an oocyte maturation rate to MII of 54% as compared to 57% in non-embedded follicles.ConclusionsInitial testing of this new and unique HA-based hydrogel was quite promising. The ease of follicle encapsulation in HA, its optical transparency and ability to be molded combined with its support of follicle growth, estradiol secretion and resumption of meiosis make this HA-hydrogel particularly attractive as model for 3-D ovarian follicle culture.

Three-dimensional systems for in vitro follicular culture: overview of alginate-based matrices

“…Recently, a novel tyramine-based hyaluronan (HA) hydrogel was tested for its biocompatibility with ovarian follicles. In this study, the HA hydrogel supported follicle growth, oestradiol secretion and resumption of meiosis (Desai et al 2012). The glycosaminoglycan HA is primarily found in the connective tissue matrix and plays an important role in a variety of biological processes, such as maintenance of the matrix structure, homeostasis of the extracellular space and interactions with other components of the matrix, thus forming a network of macromolecules that interacts with the surrounding cells (Laurent and Fraser 1992).…”

Towards a 3D culture of mouse ovarian follicles

“…Moreover, HA can be easily dissolved in water (HA hydrogel) and, when needed, it is quickly degraded with the help of enzymes (e.g., hyaluronidase) (Isayeva et al, 2010). Once HA is turned into a hydrogel, its optical transparency allows the observation of the cultured cells inside; also, its physical features enable this material to be molded into many different and functional shapes, thus permitting the operator to adjust the culture conditions to specific cell types and to obtain a gel with the pursued characteristics of morphology, stiffness and bioactivity (Desai et al, 2012). It is for its bioactivity that a number of researchers prefer HA hydrogel to inert synthetic biomaterials such as PEG .…”

“…As for the in vitro culture of ovarian follicles, a recent novel method describes, with promising results, the culture of mouse fresh or vitrified preantral follicles embedded in a 3D scaffold appropriately shaped to form a cylindrical bead (Desai et al, 2012). Table 3 gives a summary of the most critical steps of the procedures used for the preparation of this matrix and the culture of follicles.…”

Chondrocyte Cultivation in Hyaluronan-Tyramine Cross-Linked Hydrogel

“…This versatility is reflected in literature proposing possible diverse applications of this HA-TA derivative [14][15][16].…”