three dimensionally culture of sheep preantral follicles in a thiolated hyaluronic acid hydrogel

Abstract three dimensional (3d) culture of ovarian follicles is an alternative option to preserve fertility for patients who can not benefit from ovarian tissue cryopreservation. testing of novel biomaterials for the 3d culture of follicles from large animals may optimize culture conditions that ultimately can be used for human follicles. in this research, we synthesized a thiolated hyaluronic acid hydrogel (ha-sh) and compared its performance for ovarian follicle development with the routinely used alginate hydrogel. follicles encapsulated in ha-sh hydrogel had a higher survival rate than those encapsulated in alginate. also, the increase in follicle diameter after 17-day culture in hydrogels was higher in the ha-sh group. the present study results show that ha-sh hydrogel is a promising hydrogel for follicle culture. introduction among the fertility preservation options for females, ovarian tissue cryopreservation (otc) is the only option for prepubertal girls. otc does not require ovarian stimulation and sexual maturity [1]. ovarian tissue autografting following otc carries the risk of reseeding malignant cells for patients with systemic diseases such as leukemia [2]. in vitro growth and maturation of ovarian follicles is the alternative strategy to avoid the reintroduction of cancer cells and preserve fertility. both two dimensional (2d) and three dimensional (3d) culture systems are applied for in vitro culture of follicles. two-dimensionally cultured follicles attach to the culture surface and spread out, resulting in the non-physiological condition. 3d culture of follicles via encapsulating them in hydrogels preserves their structure and provides them a niche for better growth and maturation [3]. the hydrogels tested range from synthetic polymers to natural ones and bioresponsive hydrogels [4]. this report describes the first application of novel thiolated hyaluronan (ha-sh) hydrogel for the culture of isolated sheep preantral follicles. we synthesized ha-sh through chemically modification of ha and then obtained a hydrogel through self-crosslinking of free thiol groups. the hydrogel was applied for encapsulation of sheep preantral follicles to investigate its applicable capacity for supporting in vitro culture of ovarian follicles. experimental to prepare ha-sh, 50 mg of ha was dissolved in 10 ml of deionized water. then n-hydroxysuccinimide (nhs) (28.75 mg, 2 mmol) and 1-ethyl-3-(3 dimethyllaminopropyl) carbodiimide hydrochloride (edci) (71.8 mg, 3 mmol) were added under slow stirring at room temperature. the ph was adjusted to 5.5. the solution was incubated for 2 h, ph 5.3. at the completion of the incubation, cysteamine hydrochloride (14.25 mg, 1 mmol) was added, the ph readjusted to 5.5, and incubated overnight, under the same conditions. the solution was then transferred to dialysis membranes and dialyzed against dilute hcl solution (ph = 3.5) containing 1% nacl for 24 h and against dilute hcl solution (ph = 3.5) for the next 24 h, and then freeze dried. all the steps of the synthesis process were performed under nitrogen. the degree of thiolation was assessed using h nmr. for follicle encapsulation, the lyophilized ha-sh was dissolved in hepes modified m199 at a concentration of 5 mg/ml. subsequently, isolated preantral follicles were loaded into 5 µl droplets of the solution and transferred to a 37°c incubator to make a hydrogel. after 1 h incubation, the encapsulated follicles were cultured in medium 199 supplemented with 2% fetal bovine serum, 10 µg/ml fsh, 1% its (10 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml selenium; invitrogen) and 50µg/ml activin a for 17 days. as a control, follicles were encapsulated in 1% alginate. follicle viability and growth were assessed during and at the end of the culture period. results and discussion thiol substitution as the reaction approval was confirmed using h nmr. ha-sh presented typical h nmr peaks around 2.5 ppm compared to the spectrum of native ha (figure 1). according to the h nmr spectra, degree of thiol substitution for ha-sh was determined as 23%.  the potential of ha-sh hydrogel to support in vitro follicle development was evaluated through 17-day follicle culture. a low degeneration rate was recorded for ha-sh encapsulated follicles compared to those encapsulated in alginate (figure 2a). although the diameter of embedded follicles in both hydrogels increased over the culture, it was significantly higher in the ha group (133figure 2b). the higher growth of the ha-sh encapsulated follicles may be due to the hydrogel degradation over time. disulfide bond in ha-sh hydrogel could be degraded gradually by gsh produced by follicles [5] and provided enough space for follicle growth compared to alginate which does not degrade. on the other hand, decreasing the rigidity produce a dynamic environment that better mimics the environment within the ovary [6]. conclusions as described, the ha-sh hydrogel can support follicle growth and development for 17 days of in vitro culture. gradually degradation of this bioresponsive hydrogel provides suitable rigidity and space for the follicles over time. besides, self-crosslinking and transparency make it a desirable hydrogel for follicles in vitro culture.