In situ monitoring of loading and elution processes of cryoprotectants at the single-cell level with Raman spectroscopy.

BACKGROUND: The analysis of cell membrane permeability plays a crucial role in improving the procedures of cell cryopreservation, which will affect the specific parameter settings in loading, removal and cooling processes. However, existing studies have mostly focused on deriving permeability parameters through osmotic theoretical models and cell volume response analysis, and there is still a lack of the direct experimental evidence and analysis at the single-cell level regarding the migration of cryoprotectants. RESULTS: In this work, a side perfusion microfluidics chips combined with Raman spectroscopy system was built to monitor in situ the Raman spectroscopy of extracellular and intracellular solution during loading and elution process with different cryoprotectant solution systems (single and dual component). And it was found that loading a high concentration cryoprotectant solution system through a single elution cycle may result in significant residual protective agent, which can be mitigated by employing a multi-component formula but multiple elution operations are still necessary. Furthermore, the collected spectral signals were marked and analyzed to was perform preliminary relative quantitative analysis. The results showed that the intracellular concentration changes can be accurately quantified by the Raman spectrum and are closely related to the extracellular solution concentration changes. SIGNIFICANCE AND NOVELTY: By using the method of small flow perfusion (≤20 µL/min) in the side microfluidic chip after the gravity sedimentation of cells, the continuous loading and elution process of different cryoprotectants on chip and the spectral acquisition can be realized. The intracellular and extracellular concentrations can be quantified in situ based on the ratio of spectral peak intensities. These results indicate that spectroscopic analysis can be used to effectively monitor intracellular cryoprotectant residues.

Accurate Temperature Reconstruction in Radiofrequency Ablation for Atherosclerotic Plaques Based on Inverse Heat Transfer Analysis.

Radio frequency ablation has emerged as a widely accepted treatment for atherosclerotic plaques. However, monitoring the temperature field distribution in the blood vessel wall during this procedure presents challenges. This limitation increases the risk of endothelial cell damage and inflammatory responses, potentially leading to lumen restenosis. The aim of this study is to accurately reconstruct the transient temperature distribution by solving a stochastic heat transfer model with uncertain parameters using an inverse heat transfer algorithm and temperature measurement data. The nonlinear least squares optimization method, Levenberg-Marquardt (LM), was employed to solve the inverse heat transfer problem for parameter estimation. Then, to improve the convergence of the algorithm and reduce the computational resources, a method of parameter sensitivity analysis was proposed to select parameters mainly affecting the temperature field. Furthermore, the robustness and accuracy of the algorithm were verified by introducing random noise to the temperature measurements. Despite the high level of temperature measurement noise (ξ = 5%) and larger initial guess deviation, the parameter estimation results remained closely aligned with the actual values, with an overall ERMS consistently below 0.05. The absolute errors between the reconstruction temperature at the measurement points TC1, TC2, and TC3, and the actual temperature, remained within 0.33 °C, 2.4 °C, and 1.17 °C, respectively. The Levenberg-Marquardt algorithm employed in this study proficiently tackled the ill-posed issue of inversion process and obtained a strong consistency between the reconstructed temperature the actual temperature.

Cryopreservation of organoids: Strategies, innovation, and future prospects.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Optimization of Annealing and Metal Films Radiofrequency Heating Procedures for Vitrified Umbilical Arteries.

Vitrification is well known for its application in the cryopreservation of blood vessels, which will address the supply-demand imbalance in vascular grafts for the treatment of cardiovascular disease. Thermal stress damage and devitrification injury in umbilical arteries (UAs) require attention and resolution during the vitrification and rewarming process. In this study, we validated several cooling annealing protocols with temperatures (-130 to -100 °C) and annealing duration durations (10-20 s). Among these, the umbilical artery subjected to annealing at -110 °C for 10 s exhibited the most favorable glass transition and retained 93% of its elastic modulus (0.625 ± 0.030 MPa) compared to the fresh group. Extended annealing temperatures and durations can effectively reduce thermal stress damage, leading to improved mechanical properties by minimizing temperature gradients during cooling. Furthermore, three metal radiofrequency methods were utilized for rewarming, including the use of additional metal films and different magnetic field strengths (20, 25 kA/m). Metal radiofrequency (adding an extra metal film for cryoprotectants rewarming, 20 kA/m) achieved faster and more uniform rewarming, preserving the extracellular matrix (ECM), collagen fibers, and elastic fibers without significant differences compared to the fresh group (P < 0.05). Moreover, its preservation of the biomechanical properties of blood vessels was better than that of water bath heating. Theoretical analysis supports these findings, indicating that radiofrequency heating (RFH) with metal films reduces temperature gradients and thermal stresses during arterial rewarming. RFH contributes to the cryopreservation and clinical application of large-lumen biomaterials, overcoming challenges associated with vascular vitrification and rewarming.

Optimization of Deep Eutectic Solvents Enables Green and Efficient Cryopreservation.

Cryopreservation presents significant opportunities for biomedical applications including cell therapy, tissue engineering, and assisted reproduction. Dimethyl sulfoxide (DMSO), the most commonly used cryoprotectant (CPA), can be added to cells to prevent cryogenic damage. However, the toxicity of cryoprotectants restrains its further development in many areas with safety concerns such as clinical treatment. Therefore, the development of low-toxicity cryoprotectants is essential for medical research. This work reports deep eutectic solvents (DES) as naturally biocompatible osmoprotectants for green and efficient cryopreservation of human umbilical cord mesenchymal stem cells (HuMSC), which may be an ideal alternative to DMSO. The six types of DESs were explored for thermal properties, toxicity, and permeability in cells. Raman spectroscopy and viscosity studies showed that DES exhibited an improved hydrogen-bonding system as the temperature decreased. By optimizing the freezing process (cooling rate, incubation time, and loading procedure) of DES, the viability of mouse embryonic fibroblast cells (NIH-3T3) after thawing was significantly improved. The HuMSC were successfully preserved with no significant difference (p > 0.05) in cell viability (94.65%) after thawing compared with DMSO, which preserved the cell differentiation function and improved the cell proliferation rate. The mechanism of DES in cryopreservation was investigated, and it was found that DES could bind water molecules and effectively inhibit the growth of ice crystals during ice recrystallization, reducing mechanical damage to cells. This study highlights the excellent performance of DES as a low-toxicity CPA for stem cell preservation, which may be a significant advance for future clinical cell therapy.

Self-assembly of tamarind seed polysaccharide via enzymatic depolymerization and degalactosylation enhanced ice recrystallization inhibition activity.

Polysaccharides are becoming potential candidates for developing food-grade cryoprotectants due to their extensive accessibility and health-promoting effects. However, unremarkable ice recrystallization inhibition (IRI) activity and high viscosity limit their practical applications in some systems. Our previous study found a galactoxyloglucan polysaccharide from tamarind seed (TSP) showing moderate IRI activity. Herein, the enhancement of the IRI performance of TSP via enzymatic depolymerization and degalactosylation-induced self-assembly was reported. TSP was depolymerized and subsequently removed ∼40 % Gal, which induced the formation of supramolecular rod-like fiber self-assembles and exhibited a severalfold enhancement of IRI. Ice shaping assay did not show obvious faceting of ice crystals, indicating that both depolymerized and self-assembled TSP showed very weak binding to ice. Molecular dynamics simulation confirmed the absence of molecular complementarity with ice. Further, it highlighted that degalactosylation did not cause significant changes in local hydration properties of TSP from the view of a single oligomer. The inconsistency between molecular simulation and macroscopic IRI effect proposed that the formation of unique supramolecular self-assemblies may be a key requirement for enhancing IRI activity. The findings of this study provided a new opportunity to enhance the applied potential of natural polysaccharides in food cryoprotection.

Investigation of Rapid Rewarming Chips for Cryopreservation by Joule Heating.

Rapid and uniform rewarming is critical to cryopreservation. Current rapid rewarming methods require complex physical field application devices (such as lasers or radio frequencies) and the addition of nanoparticles as heating media. These complex devices and nanoparticles limit the promotion of the rapid rewarming method and pose potential biosafety concerns. In this work, a joule heating-based rapid electric heating chip (EHC) was designed for cryopreservation. Uniform and rapid rewarming of biological samples in different volumes can be achieved through simple operations. EHC loaded with 0.28 mL of CPA solution can achieve a rewarming rate of 3.2 × 105 °C/min (2.8 mL with 2.3 × 103 °C/min), approximately 2 orders of magnitude greater than the rewarming rates observed with an equal capacity straw when combined with laser nanowarming or magnetic induction heating. In addition, the degree of supercooling can be significantly reduced without manual nucleation during the cooling of the EHC. Subsequently, the results of cryopreservation validation of cells and spheroids showed that the cell viability and spheroid structural integrity were significantly improved after cryopreservation. The viability of human lung adenocarcinoma (A549) cells postcryopreservation was 97.2%, which was significantly higher than 93% in the cryogenic vials (CV) group. Similar results were seen in human mesenchymal stem cells (MSCs), with 93.18% cell survival in the EHC group, significantly higher than 86.83% in the CV group, and cells in the EHC group were also significantly better than those in the CV group for further apoptosis and necrosis assays. This work provides an efficient rewarming protocol for the cryopreservation of biological samples, significantly improving the quantity and quality of cells and spheroids postcryopreservation.

A study on the relationship between the crystallization characteristics of quenched droplets and the effect of cell cryopreservation with Raman spectroscopy.

The cryopreservation method of microdroplets has steadily become widely employed in the cryopreservation of microscale biological samples such as various types of cells due to its fast cooling rate, significant reduction of the concentration of cryoprotectants, and practical liquid handling method. However, it is still necessary to consider the corresponding relationship between droplet size and concentration and the impact of crystallization during the cooling process on cell viability. The key may be a misunderstanding of the influencing factors of crystallization and vitrification behavior with concentration during cooling on the ultimate cell viability, which may be attributable to the inability to analyze the freezing state inside the microdroplets. Therefore, in this work, an in situ Raman observation system for droplet quenching was assembled to obtain Raman spectra in the frozen state, and the spectral characteristics of the crystallization and vitrification processes of microdroplets with varied concentrations and volumes were investigated. Furthermore, the degree of crystallization inside the droplets was quantitatively analyzed, and it was found that the ratio of the crystalline peak to hydrogen bond shoulder could clearly distinguish the degree of crystallization and the vitrified state, and the Raman crystallization characteristic parameters gradually increased with the decrease of concentrations. By obtaining the cooling curve and the overall cooling rate of quenching droplets, the vitrification state of the microdroplets was confirmed by theoretical analysis of the cooling characteristics of a DMSO solution system. In addition, the effect of cell cryopreservation was investigated using the microdroplet quenching device, and it was found that the key to cell survival during the quenching process of low-concentration microdroplets was dominated by the cooling rate and the internal crystallization degree, while the main influencing factor on high concentration was the toxic effect of a protective agent. In general, this work introduces a new nondestructive evaluation and analysis method for the cryopreservation of quenching microdroplets.

Droplet Generation, Vitrification, and Warming for Cell Cryopreservation: A Review.

Cryopreservation is currently a key step in translational medicine that could provide new ideas for clinical applications in reproductive medicine, regenerative medicine, and cell therapy. With the advantages of a low concentration of cryoprotectant, fast cooling rate, and easy operation, droplet-based printing for vitrification has received wide attention in the field of cryopreservation. This review summarizes the droplet generation, vitrification, and warming method. Droplet generation techniques such as inkjet printing, microvalve printing, and acoustic printing have been applied in the field of cryopreservation. Droplet vitrification includes direct contact with liquid nitrogen vitrification and droplet solid surface vitrification. The limitations of droplet vitrification (liquid nitrogen contamination, droplet evaporation, gas film inhibition of heat transfer, frosting) and solutions are discussed. Furthermore, a comparison of the external physical field warming method with the conventional water bath method revealed that better applications can be achieved in automated rapid warming of microdroplets. The combination of droplet vitrification technology and external physical field warming technology is expected to enable high-throughput and automated cryopreservation, which has a promising future in biomedicine and regenerative medicine.

Inhibition of ice recrystallization by tamarind (Tamarindus indica L.) seed polysaccharide and molecular weight effects.

This study aimed to investigate the inhibition effects of tamarind seed polysaccharide (TSP) on ice recrystallization and to figure out its possible molecular weight-dependent effects. TSP fractions (2412.38-20.75 kDa) were prepared while preserving the natural structure. Ice recrystallization was effectively inhibited by TSP. Decreasing the molecular weight to a certain range, such as 224.04 kDa and 90.41 kDa, could enhance the activity of TSP due to the reduction of self- and intermolecular aggregation. Adding TSP into water decreased the melting temperature of bulk ice. Raman spectra showed that partial group vibrations or deformations of TSP molecules were restricted upon solution freezing and also revealed a destructuring effect of TSP on the H-bond network of water. These findings suggested the potential of TSP as a novel food cryoprotectant and help produce TSP fractions with enhanced activity, and shed new light on understanding the antifreeze mechanism of natural polysaccharides.

New Cryoprotectant Loading Method for Cell Droplet Vitrification with Continuous Evaporation.

Droplet-based vitrification is considered to be a promising cryopreservation method, which achieves high cell viability through high cooling rates and low concentrations of cryoprotective agents (CPAs). However, the droplet vitrification cryopreservation process needs in-depth research, such as the balance of the CPA concentration and the cooling rate, the CPA loading process, and the droplet encapsulation method. Here, we developed a chip with a high cooling rate for vitrification droplet encapsulation and provided a new method for continuous loading of low-concentration CPA droplets by evaporation. The results showed that the CPA droplet volume decreased exponentially with the evaporation time, and the larger the initial droplet size, the longer the evaporation time to achieve the critical vitrification concentration. There was no significant difference in the viability of MSCs, NHEK, and A549 cells between the evaporation loading vitrification method and the traditional slow freezing method, but the former was easier to operate and can balance the cooling rate and concentration by controlling the evaporation time. Moreover, a theoretical model was proposed to predict the CPA concentration inside the microdroplets dependent on the evaporation time. The current work provides a potential method to load low-concentration CPAs for cell vitrification preservation, which is more beneficial for cell therapy and other regenerative medicine applications.

Quantitative assessment of intracellular/extracellular dimethyl sulfoxide concentrations during freezing with low-temperature confocal Raman micro-spectroscopy.

Although cryopreservation plays an indispensable role in the clinical application of cell therapy, the research on the osmotic behavior of cells during freezing is still at the level of theoretical models, and quantitative experimental data are still lacking. Therefore, the Raman spectra of dimethyl sulfoxide (DMSO) solutions with different standard concentrations (5%-80% v/v) were recorded experimentally to establish a quantitative evaluation method with the intensity ratio of different labeled peaks to the hydrogen bonding peak (as the internal standard) of water molecules in relation to different DMSO concentrations. By using this method, the characteristics of quantitative changes in intra- and extracellular concentrations under three different freezing methods were explored, including direct freezing, ice seeding freezing and vitrification. It was found that the intracellular concentration (@ -50 °C) after the ice seeding (@ -7 °C) freezing (1 °C min-1) method could reach 41.6%-49.2%, significantly higher than that of the direct freezing method (1 °C min-1 to -50 °C) of 32.4%-39.1%. Moreover, it is worth noting that the quantitative values of concentrations (@ -50 °C) of the ice seeding freezing are more consistent with the primary saturation curve of the DMSO solution. Thus, for the first time, it was revealed from the experimental data that the fundamental reason for the improvement of cell survival after ice seeding operation was pre-dehydration, higher concentration and smaller osmotic pressure difference between the inside and outside of the cell. These results also confirmed the validity of the famous two-factor hypothesis and more work will be carried out in depth.