An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes.

Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.

Surface-Acoustic-Wave-Based Lab-on-Chip for Rapid Transport of Cryoprotectants across Cell Membrane for Cryopreservation with Significantly Improved Cell Viability.

Cryopreservation is essential to effectively extend the shelf life of delicate biomaterials while maintaining proper levels of cell functions. Cryopreservation requires a cryoprotective agent (CPA) to suppress intracellular ice formation during freezing, but it must be removed prior to clinical use due to its toxicity. Conventional multistep CPA loading and unloading approaches are time consuming, often creating osmotic shocks and causing mechanical injuries for biological samples. An efficient surface-acoustic-wave- (SAW-) based lab-on-a-chip (LoC) for fast loading and removal of CPAs is presented here. With the SAW-based multistep CPA loading/removal approach, high concentration (3 m) CPA can be successfully loaded and removed in less than 1 min. Results show that the technique causes the least harm to umbilical cord matrix mesenchymal stem cells as compared to conventional method, and an average of 24% higher cell recovery rate is achieved, while preserving the integrity and morphology of the cells. This device is the first of its kind to combine high loading/unloading efficiency, high cell viability, and high throughput into one LoC device, offering not only a more efficient and safer route for CPA loading and removal from cells, but also paving the way for other cryopreservation-dependent applications.

The application of convolution neural network based cell segmentation during cryopreservation.

For most of the cells, water permeability and plasma membrane properties play a vital role in the optimal protocol for successful cryopreservation. Measuring the water permeability of cells during subzero temperature is essential. So far, there is no perfect segmentation technique to be used for the image processing task on subzero temperature accurately. The ice formation and variable background during freezing posed a significant challenge for most of the conventional segmentation algorithms. Thus, a robust and accurate segmentation approach that can accurately extract cells from extracellular ice that surrounding the cell boundary is needed. Therefore, we propose a convolutional neural network (CNN) architecture similar to U-Net but differs from those conventionally used in computer vision to extract all the cell boundaries as they shrank in the engulfing ice. The images used was obtained from the cryo-stage microscope, and the data was validated using the Hausdorff distance, means ±â€¯standard deviation for different methods of segmentation result using the CNN model. The experimental results prove that the typical CNN model extracts cell borders contour from the background in its subzero state more coherent and effective as compared to other traditional segmentation approaches.

Controlled Release of Cryoprotectants by Near-Infrared Irradiation for Improved Cell Cryopreservation.

Cryopreservation is essential to store living cells and tissues for future use while maintaining the proper levels of cell functions. The use of cryoprotective agents (CPAs) to inhibit intracellular ice formation during cryopreservation is vital for cell survival, but the addition and removal of CPAs and ice recrystallization during rewarming will cause fatal injury to cells. The conventional CPA loading and unloading methods generate osmotic shocks and cause mechanical injury to biological samples, and the conventional method of rewarming using a water bath also leads to ice recrystallization and devitrification. A new CPA-loaded microparticle-based method for loading and photothermal rewarming under near-infrared (NIR) laser irradiation was proposed to overcome these difficulties. We have successfully achieved the controlled release of CPAs (2 M EG, 2 M PG, and 0.5 M trehalose) with a graphene oxide (GO, 0.04% w/v) core from a 1.5% (w/v) sodium alginate shell to the human umbilical vein endothelial cells (HUVECs) within 60 s using NIR laser irradiation (808 nm Lasever at 5000 mW/cm2) and successfully recovered the CPA-loaded cells with 0.04% (w/v) GO in 8-10 s using the same NIR irradiation. The results show that this method achieved 25% higher viability of HUVECs compared to the conventional method. In short, this study proposes a new approach for achieving controlled CPA loading to cells with a photothermal-induced strategy for cell cryopreservation.

A microfluidic approach for synchronous and nondestructive study of the permeability of multiple oocytes.

Investigation of oocyte membrane permeability plays a crucial role in fertility preservation, reproductive medicine, and reproductive pharmacology. However, the commonly used methods have disadvantages such as high time consumption, low efficiency, and cumbersome data processing. In addition, the developmental potential of oocytes after measurement has not been fully validated in previous studies. Moreover, oocytes can only maintain their best status in vitro within a very limited time. To address these limitations, we developed a novel multichannel microfluidic chip with newly designed micropillars that provide feasible and repeatable oocyte capture. The osmotic responses of three oocytes at different or the same cryoprotectant (CPA) concentrations were measured simultaneously, which greatly improved the measurement efficiency. Importantly, the CPA concentration dependence of mouse oocyte membrane permeability was found. Moreover, a neural network algorithm was employed to improve the efficiency and accuracy of data processing. Furthermore, analysis of fertilization and embryo transfer after perfusion indicated that the microfluidic approach does not damage the developmental potential of oocytes. In brief, we report a new method based on a multichannel microfluidic chip that enables synchronous and nondestructive measurement of the permeability of multiple oocytes.

Microencapsulation Facilitates Low-Cryoprotectant Vitrification of Human Umbilical Vein Endothelial Cells.

Vitrification has become one of the promising cryopreservation methods for biosamples including cells and tissues because the vitreous state reduces the damage of ice crystals to cells. However, besides extremely high cooling rates, routine vitrification protocols require a high concentration of penetrating cryoprotectants (pCPAs, ∼6-8 M), which is toxic for cells and brings trouble when removing pCPAs. Therefore, reducing the concentration of toxic pCPAs in vitrification remains a challenge, and advanced strategies are urgently needed. Hydrogel encapsulation has become one effective method to achieve low-cryoprotectant (CPA) concentration preservation of stem cells with rapid cooling, but there are very few related studies about endothelial cells (ECs). In this study, we achieved pCPA concentration (up to 3 M) vitrification by encapsulating human umbilical vein endothelial cells (HUVECs) into core-shell alginate hydrogel microcapsules. Alginate encapsulation increased HUVEC cryosurvival up to 80%, which is 60% improvement compared to control without encapsulation. Furthermore, two different sizes of capsules (diameter: ∼900 and 400 µm) were produced to explore the effects of microcapsule volume on the cell preservation results, and it was found that larger capsules (∼900 µm) have no significant effect on cell survival while improving encapsulation efficiency. This encapsulation method provides a new strategy for EC preservation and serves as an improvement to optimize the preservation of biosamples.

Sensing Cell Membrane Biophysical Properties for Detection of High Quality Human Oocytes.

Oocyte quality plays a crucial role in the early development and implantation of the embryos, and consequently has a profound impact on the accomplishment of assisted reproductive technology (ART). A simple and efficient method for detecting high-quality human oocytes is urgently needed. However, the clinically used morphological method is time-consuming, subjective, and inaccurate. To this end, we propose a practical and effective approach for detecting high-quality oocytes via on-chip measurement of the oocyte membrane permeability. We found that oocytes can be divided into two subpopulations (high-quality versus poor-quality oocytes) according to their membrane permeability differences, and as was further confirmed by subsequent in vitro fertilization (IVF) and development experiments (the blastocyst rates of high-quality and poor-quality oocytes were 60% and 0%, respectively). This approach shows great potentials in improving the success of ART, including both the fertilization and development rates, and thus it may have wide applications in the clinic.

Near-infrared laser mediated modulation of ice crystallization by two-dimensional nanosheets enables high-survival recovery of biological cells from cryogenic temperatures.

Two-dimensional (2D) graphene oxide (GO) and molybdenum disulfide (MoS2) nanosheets (NSs) have been widely used as photothermal agents and as potential carriers of antitumor drugs. Their spatial thermal effects have been extensively explored for use at physiological and hyperthermic temperatures (37 to 46 °C). Furthermore, the modulation of the spatial thermal distributions with these NSs may have even more profound applications in the microstructural control of biomaterials at cryogenic temperatures (-196 to 37 °C). These applications include bioinspired microfabrication via freezing, food and drug freeze-drying, and biomaterial cryopreservation. However, such thermal effects of NSs and their applications at cryogenic temperatures had never been fully explored. Therefore, in this study, we have utilized the near-infrared laser induced photothermal effects of GO and MoS2 NSs to suppress the ice nucleation and ice crystal growth during warming of the biosamples. Using this approach, biological cells subjected to fast cooling to a deeply frozen state (-196 °C) were successfully recovered with high survival rates and full biological functionality. Thus, we provide a NS based effective approach to control the crystallization behaviors of water during warming at cryogenic temperatures, as NSs may have wide applications in both materials science and bioengineering.