There is a great demand for blood and stem cells in clinic. It is difficult to achieve high throughput and to increase the cooling rate at the same time during vitrification. In this paper, a micro-droplet spray system with a container collection device was fabricated, and HepG2 cells were sprayed by this system for high-throughput vitrification. First, the container collection device and a cryo-paper were used to receive micro-droplets in the spray vitrification system. The results showed that the cell survival rate and 24h adhesion rate in container collection vitrification group were significantly higher than those in cryo-paper collection group. Second, HepG2 cells were sprayed and vitrified at increased cell density, and it was found that the results of micro-droplet spray vitrification did not change significantly. Finally, micro-droplet spray vitrification is compared with slow freezing. Cell processing capacity in the vitrification group increased, meanwhile, the cell survival rate and 24h adhesion rate in the vitrification group were significantly higher than those in slow freezing group. The results indicated that the micro-droplet spray vitrification system with container collection device designed in this paper can achieve high-throughput cell vitrification, which is of great significance for mass preservation of small cells.
Dimethyl sulfoxide (Me2SO) supplemented with fetal bovine serum (FBS) is a widely used cryoprotectant combination. However, high concentration of Me2SO is toxic to cells, and FBS presents problems related to diseases such as bovine spongiform encephalopathy and viral infections. Silk protein is a kind of natural macromolecule fiber protein with good biocompatibility and hydrophilicity. The aim of this paper is to analyze the cryoprotective mechanism of silk protein as cryoprotectant. Firstly, differential scanning calorimetry (DSC) was used to measure the thermal hysteresis activity (THA) of silk protein. The THA of 10 mg/mL sericin protein was 0.96°C, and the THA of 10% (V/V) fibroin protein was 1.15°C. Then the ice recrystallization inhibition (IRI) of silk protein-PBS solution was observed with cryomicroscope. The cold stage was set at − 7°C, after 40 minutes’ incubation, the mean grain size rate (MGSR) of sericin protein and fibroin protein were 28.99% and 3.18%, respectively, which were calculated relative to phosphate buffer saline (PBS) control. It is indicated that sericin and silk fibroin have certain effects of inhibiting recrystallization of ice crystals. Finally, the structure and physicochemical properties of silk protein were analyzed by Fourier transform infrared spectroscopy (FTIR). The results showed that the content of the random coil was 75.62% and the β-sheet structure was 24.38% in the secondary of sericin protein. The content of the β-sheet structure was 56.68%, followed by random coil structure 22.38%, and α-helix 16.84% in the secondary of fibroin protein. The above analysis demonstrates the feasibility of silk fibroin as a cryoprotectant, and provides a new idea for the selection of cryoprotectants in the future.
In order to reduce osmotic damage and chemical toxicity of cryoprotectants (CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group (98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.