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    Tissue Engineering

    (K-426) Characterization of Microgels for a Microporous Annealed Particle Hydrogel for Human Ovary Tissue Encapsulation

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row K - Poster # 426
    Introduction:: Primary ovarian insufficiency (POI) is a common consequence in individuals with ovaries who undergo anticancer treatments. Due to a decrease in ovarian follicle numbers patients with POI experience loss of ovarian endocrine function and many other negative side effects such as: infertility, anxiety, depression, hypothyroidism, cardiovascular disease, and osteoporosis. Current treatments for POI include hormone replacement therapy (HRT) and oocyte and embryo conservation. However, HRT supplies static levels of estradiol and progesterone that do not mimic the dynamic levels of hormones produced by the native ovaries, and oocyte or embryo conservation are not available for pediatric cancer patients treated before puberty. Ovarian tissue cryopreservation followed with auto transplantation (OTCT) was recently approved in the clinic and it is the only option that could restore ovarian endocrine function and fertility in patients (1). Unfortunately, 50% to 90% of ovarian follicles are lost in the transplantation process due to delayed revascularization and ischemic injury to the tissue (2, 3). Microporous annealed particle (MAP) hydrogels have been shown to facilitate cellular infiltration which could promote graft revascularization. We hypothesized that encapsulation of human ovarian tissue in a hydrogel will facilitate graft revascularization and attenuate ischemic injury.  To aid in cell adhesion, microgels were modified with the integrin binding peptide, RGD. In this report we investigated how the addition of RGD affected microgel swelling to inform future material testing and microporosity characterization of the MAP hydrogel.  

    Materials and Methods:: Droplet microfluidics techniques were used to produce microgels. Step emulsification microfluidic devices were made of PDMS and plasma bonded to glass slides (4). A mixture of 10:1 (w:w) PDMS base to curing agent was poured over a silicon wafer master mold. The PDMS mixture was degassed and placed in the oven overnight at 80 °C. The next day the cured PDMS devices were removed from the mold and cleaned using isopropanol and ethanol to be bonded to the glass slides. A 10% w/v 8-arm poly(ethylene-glycol)-maleimide (PEG-MAL) mixed with a 3-functional degradable crosslinking peptide (GCYKNSGCYKNSCG) and the 1mM RGD solution was flown through the microfluidic device concurrently with a fluorinated oil to produce microgels. The produced microgels were then washed in aqueous buffer to remove the oil from the microgels. Characterization of the microgels was done using ImageJ and MATLAB software. The microgels were imaged on the first day while in oil and after washing while in aqueous HEPES buffer and then daily for 6 consecutive days. The diameter of microgels was recorded and analyzed for each condition.

    Results, Conclusions, and Discussions:: We hypothesized that microgels modified with RGD would swell more than the controls without the RGD because RGD occupies some of the cross-linking sites potentially decreasing the crosslinking density of the microgels. Analysis of microgel diameter plots for RGD vs control microgels of the same day showed no difference in trends (Figure 1 A-E). The diameters of the microgels with and without RGD were 65.182 microns and 68.338 microns, respectively, after washing in HEPES. To further compare the diameters of the control microgels versus the RGD group, the average diameter for each group was plotted against one another for the duration of the observation period (7 days). The diameters of the microgels with and without RGD remained consistent across the 7 days, ranging from 60 to 75 microns in diameter with no significant difference between the average diameter of the control group compared to the RGD group. We concluded that substitution with 1 mM RGD had no significant effect on crosslinking density of the hydrogels. With this preliminary data, we also concluded that future extended material testing and characterization of the MAP hydrogel porosity as well as an in vitro degradation assay to investigate long-term material properties of the microgels can be performed on control microgels that do not include RGD.  

    Acknowledgements (Optional): :

    References (Optional): :

    1. Khattak, Hajra et al. “Fresh and cryopreserved ovarian tissue transplantation for preserving reproductive and endocrine function: a systematic review and individual patient data meta- analysis.” Human reproduction update vol. 28,3 (2022): 400-416. doi:10.1093/humupd/dmac003 




    2. Silber, S. J., DeRosa, M., Goldsmith, S., Fan, Y., Castleman, L., & Melnick, J. (2018). Cryopreservation and transplantation of ovarian tissue: results from one center in the USA. Journal of assisted reproduction and genetics, 35(12), 2205–2213.  




    https://doi.org/10.1007/s10815-018-1315-1 




    3. Dolmans M-M, von Wolff M, Poirot C, et al. Transplantation of cryopreserved ovarian tissue in a series of 285 women: a review of five leading European centers. Fertil Steril. 2021;115(5):1102- 1115. doi:10.1016/j.fertnstert.2021.03.008  




    4. de Rutte, Joseph Michael, et al. “Scalable High‐Throughput Production of Modular Microgels for in Situ Assembly of Microporous Tissue Scaffolds.” Advanced Functional Materials, vol. 29, no. 25, 18 Mar. 2019, p. 1900071, https://doi.org/10.1002/adfm.201900071. Accessed 6 Oct. 2021.