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.

Cell membrane permeability coefficients determined by single-step osmotic shift are not applicable for optimization of multi-step addition of cryoprotective agents: As revealed by HepG2 cells.

HepG2 cells have a number of research applications and cryopreservation of these cells would improve supply and thus facilitate the study. Development of effective cryopreservation protocols relies on knowledges of the fundamental mass transport characteristics of HepG2 cell membrane. Currently, the permeability parameters estimated from single-step addition are routinely used to predict the osmotic responses of the cells in multistep protocols, as well as used for prediction of optimal cooling rates. However, the reasonability of this approach has not been rigorously studied. Here we measured the hydraulic conductivity (Lp) and the permeability coefficient (Ps) of HepG2 cells in the absence/presence of dimethyl sulfoxide (Me2SO) at various temperatures with single and multistep addition of Me2SO. We found that the permeability yielded via one-step addition of the Me2SO cannot exactly predict the volume change of the cells when the CPA was added in multiple steps.

Effects of trehalose vitrification and artificial oocyte activation on the development competence of human immature oocytes.

Sucrose and trehalose are conventional cryoprotectant additives for oocytes and embryos. Ethanol can artificially enhance activation of inseminated mature oocytes. This study aims to investigate whether artificial oocyte activation (AOA) with ethanol can promote the development competence of in vitro matured oocytes. A total of 810 human immature oocytes, obtained from 325 patients undergoing normal stimulated oocyte retrieval cycles, were in vitro maturated (IVM) either immediately after collection (Fresh group n = 291)) or after being vitrified as immature oocytes (Vitrified group n = 519). These groups were arbitrarily assigned. All fresh and vitrified oocytes which matured after a period of IVM then underwent intra-cytoplasmic sperm injection (ICSI). Half an hour following ICSI, they were either activated by 7% ethanol (AOA group) or left untreated (Non-AOA group). Fertilization, cleavage rate, blastocyst quality and aneuploidy rate were then evaluated. High-quality blastocysts were only obtained in both the fresh and vitrified groups which had undergone AOA after ICSI. Trehalose vitrification slightly, but not significantly, increased the formation rates of high-quality embryos (21.7% VS 15.4%, P > 0.05) and blastocysts (15.7% VS 7.69%, P > 0.05)) when compared with sucrose vitrification. Aneuploidy was observed in 12 of 24 (50%) of the AOA derived high quality blastocysts. High-quality blastocysts only developed from fresh or vitrified immature oocytes if the ICSI was followed by AOA. This information may be important for human immature oocytes commonly retrieved in normal stimulation cycles and may be particularly important for certain patient groups, such as cancer patients. AOA with an appropriate concentration of ethanol can enhance the developmental competence of embryos.

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.

The Unusual Properties of Polytetrafluoroethylene Enable Massive-Volume Vitrification of Stem Cells with Low-Concentration Cryoprotectants.

Injectable stem cell-hydrogel constructs hold great potential for regenerative medicine and cell-based therapies. However, their clinical application is still challenging due to their short shelf-life at ambient temperature and the time-consuming fabrication procedure. Banking the constructs at cryogenic temperature may offer the possibility of "off-the-shelf" availability to end-users. However, ice formation during the cryopreservation process may compromise the construct quality and cell viability. Vitrification, cooling biological samples without apparent ice formation, has been explored to resolve the challenge. However, contemporary vitrification methods are limited to very small volume (up to ~0.25 ml) and/or need highly toxic and high concentration (up to ~8 M) of permeable cryoprotectants (pCPAs). Here, we show that polytetrafluoroethylene (PTFE, best known as Teflon for making non-stick cookware) capillary is flexible and unusually stable at a cryogenic temperature. By using the PTFE capillary as a flexible cryopreservation vessel together with alginate hydrogel microencapsulation and Fe3O4 nanoparticle-mediated nanowarming to suppress ice formation, massive-volume (10 ml) vitrification of cell-alginate hydrogel constructs with a low concentration (~2.5 M) of pCPA can be achieved. This may greatly facilitate the use of stem cell-based constructs for tissue regeneration and cell based therapies in the clinic.

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.

Dual Suppression Effect of Magnetic Induction Heating and Microencapsulation on Ice Crystallization Enables Low-Cryoprotectant Vitrification of Stem Cell-Alginate Hydrogel Constructs.

Stem cells microencapsulated in hydrogel as stem cell-hydrogel constructs have wide applications in the burgeoning cell-based medicine. Due to their short shelf life at ambient temperature, long-term storage or banking of the constructs is essential to the "off-the-shelf" ready availability needed for their widespread applications. As a high-efficiency, easy-to-operate, low-toxicity, and low-cost method for long-term storage of the constructs, low-cryoprotectant (CPA) vitrification has attracted tremendous attention recently. However, we found many cells in the stem cell-alginate constructs (∼500 µm in diameter) could not attach to the substrate post low-CPA vitrification with ∼2 M penetrating CPAs. To address this problem, we introduced nanowarming via magnetic induction heating (MIH) of Fe3O4 nanoparticles to minimize recrystallization and devitrification during the warming step of the low-CPA vitrification procedure. Our results indicate that high-quality stem cell-alginate hydrogel constructs with an intact microstructure, high immediate cell survival (>80%), and greatly improved attachment efficiency (by nearly three times, 68% versus 24%) of the encapsulated cells could be obtained post-cryopreservation with nanowarming. Moreover, the cells encapsulated in the cell-hydrogel constructs post-cryopreservation maintained normal proliferation under 3D culture and retained intact biological function of multilineage differentiation. This novel low-CPA vitrification approach for cell cryopreservation enabled by the combined use of alginate hydrogel microencapsulation and Fe3O4 nanoparticles-mediated nanowarming may be valuable in facilitating the widespread application of stem cells 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.

Enhanced killing of HepG2 during cryosurgery with Fe3O4-nanoparticle improved intracellular ice formation and cell dehydration.

Cryosurgery is a minimally invasive treatment that utilize extreme low temperatures to destroy abnormal tissues. The clinical monitoring methods for cryosurgery are almost based on the visualization of the iceball. However, for a normal cryosurgery process, the effective killing region is always smaller than the iceball. As a result, the end of the cryosurgery process can only be judged by the surgeons according to their experience. The subjective judgement is one of the main reasons for poor estimation of tumor ablation, and it sparks high probability of recurrence and metastasis associate with cryosurgery. Being different from the previous optimization studies, we develop a novel approach with the aid of nanoparticles to enlarge the effective killing region of entire iceball, and thus it greatly decrease the difficulty of precise judgement of the cryosurgery only by applying the common clinical imaging methods. To verify this approach, both the experiments on a tissue-scale phantom with embedded living HepG2 cells in agarose and on a cell-scale cryo-microscopic freeze-thaw stage are performed. The results indicate that the introduction of the self-synthesized Fe3O4 nanoparticles significantly improved cell killing in the cryosurgery and the range of killing is extended to the entire iceball. The potential mechanism is further revealed by the cryo-microscopic experiments, which verifies the presence of Fe3O4 nanoparticles can significantly enhance the probability of intracellular ice formation and the cell dehydration during freezing hence it promote precise killing of the cells. These findings may further promote the widespread clinical application of modern cryosurgery.

Measurement of Thermal Conductivities of Two Cryoprotective Agent Solutions for Vitreous Cryopreservation of Organs at the Temperature Range of 77 K-300 K Using a Thermal Sensor Made of Microscale Enamel Copper Wire.

Biobanking of organs by cryopreservation is an enabling technology for organ transplantation. Compared with the conventional slow freezing method, vitreous cryopreservation has been regarded to be a more promising approach for long-term storage of organs. The major challenges to vitrification are devitrification and recrystallization during the warming process, and high concentrations of cryoprotective agents (CPAs) induced metabolic and osmotic injuries. For a theoretical model based optimization of vitrification, thermal properties of CPA solutions are indispensable. In this study, the thermal conductivities of M22 and vitrification solution containing ethylene glycol and dimethyl sulfoxide (two commonly used vitrification solutions) were measured using a self-made microscaled hot probe with enameled copper wire at the temperature range of 77 K-300 K. The data obtained by this study will further enrich knowledge of the thermal properties for CPA solutions at low temperatures, as is of primary importance for optimization of vitrification.

Vitreous Cryopreservation of Human Umbilical Vein Endothelial Cells with Low Concentration of Cryoprotective Agents for Vascular Tissue Engineering.

Cryopreservation of human umbilical vein endothelial cells (HUVECs) is important to tissue engineering applications and the study of the role of endothelial cells in cardiovascular and cerebrovascular diseases. The traditional methods for cryopreservation by vitrification (cooling samples to a cryogenic temperature without apparent freezing) using high concentration of cryoprotective agents (CPAs) and slow freezing are suboptimal due to the severe toxicity of high concentration of CPAs and ice formation-induced cryoinjuries, respectively. In this study, we developed a method to cryopreserve HUVECs by vitrification with low concentration of CPAs. This is achieved by optimizing the CPAs and using highly thermally conductive quartz capillary (QC) to contain samples for vitrification. The latter minimizes the thermal mass to create ultra-fast cooling/warming rates. Our data demonstrate that HUVECs can be vitrified in the QC using 1.4 mol/L ethylene glycol and 1.1 mol/L dimethyl sulfoxide with more than 90% viability. Moreover, this method significantly improves the attachment efficiency of the cryopreserved HUVECs. The attached cells post-cryopreservation proliferate similarly to fresh cells. Therefore, this study may provide an effective vitrification technique to bank HUVECs for vascular tissue engineering and other applications.