Copper grid

To control the size, zeta potential, and stability of liposomes, dynamic and electrophoretic light scattering was used. The measurements were carried out on a Zetasizer Nano ZS device (Malvern Instruments Ltd., Worcestershire, UK) at 25 °C. For measurements, all solutions were diluted with Milli-Q water to 2 mM (toward PC). All characteristics of the device and research methods are described in [36 (link)].
The size and morphology of the liposomes were confirmed by transmission electron microscopy using a Hitachi HT7700 Exalens microscope (Hitachi High-Technologies Corporation, Tokyo, Japan). For the experiment, fresh dispersions of liposomes were prepared, the concentration of which was carefully selected to achieve an acceptable number of aggregates in the field of view without the formation of a thick film on the grid, i.e., 5 μM. Sample was dispersed on 300 mesh 3 mm copper grid (Ted Pella) with continuous carbon-formvar support films and dried at room temperature. The images were acquired at an accelerating voltage of 100 kV. The calculation of the diameter of the aggregates was carried out using ImageJ software (Version number is 1.53t).

Vitrification was performed as previously described by Cha et al. [24 (link)]. Ethylene glycol (EG, 102466, Sigma-Aldrich) and dimethyl sulfoxide (DMSO, D2650, Sigma-Aldrich) were used as cryoprotectants in the vitrification solution. Oocytes were equilibrated in PBS based media containing 7.5% EG, 7.5% DMSO, and 20% FBS for 2.5 min, and then transferred to media containing 15% EG, 15% DMSO, and 0.5 M sucrose (Fisher Scientific, Fair Lawn, USA) for 20 s. Equilibrated oocytes (20 to 25) were loaded onto a copper grid (Ted Pella Inc., Redding, USA) and dipped directly into liquid nitrogen (LN₂). Vitrified oocytes were stored in a LN₂ tank for 2–4 weeks. For the warming procedure, the grid was taken out from the LN₂ tank and serially incubated in 20% FBS containing PBS with descending concentrations of sucrose (0.5, 0.25, 0.125, 0 M) for 2.5 min each. The vitrified-warmed oocytes were washed in Quinn’s Advantage medium containing HEPES and 20% FBS. Washed oocytes were cultured in M16 media (M7292, Sigma-Aldrich) at 37 °C, in 5% CO₂ for 1 or 3 h. For Necrostatin-1 (Nec1, N9037, Sigma-Aldrich) supplementation, 1 μM of Nec1 [25 (link)] was added to the final vitrification solution (15% EG, 15% DMSO, and 0.5 M sucrose). Oocytes without any marked morphological deformation and discoloration under an inverted microscope were considered as survived ones and used for further analysis.

Alginate solution was prepared by adding 0.02 g of lyophilized alginate to 1 mL of deionized water, along with 0.25 M mannitol and 10 mM HEPES. Reproductive organs were washed in DPBS, immersed in the alginate solution for about 30 s, transferred to the CaCl₂ solution, and allowed to stand for approximately 1 min for encapsulation. We loaded cryoprotective agents (CPAs) into them in two steps. This was performed by first incubating encapsulated reproductive organs (RO) with a pre-equilibrium solution made of 10% dimethyl sulfoxide (DMSO), and 10% ethylene glycol (EG) for 10 min. Next, the organs were transferred into 500 μL vitrification solution made of 20% DMSO and 20% EG for 2 min and put on a copper grid (Ted Pella, Redding, CA, USA). The RO-laden copper grid was then plunged into liquid nitrogen and held there for at least 1 min and thawed sequentially for 3 min in 0.5 M, 0.25 M, 0.125 M, and 0 M sucrose. After thawing, the cells were isolated from chopped the tissues with 1 mL syringe, and measured cell viability using live/dead and nuclear staining with fluorescein diacetate (FDA, live cells in green), propidium iodide (PI; dead cells in red) (2 μg/1 mL) and hoechst 33258 (nuclear in blue) (1 μg/1 mL), respectively. The same procedure was carried out for the control group without RO.