Review of Rewarming Methods for Cryopreservation.

Cryopreservation is the most effective technology for the long-term preservation of biological materials, including cells, tissues, and even organs in the future. The process of cooling and rewarming is essential to the successful preservation of biological materials. One of the critical problems in the development of cryopreservation is the optimization of effective rewarming technologies. This article reviewed rewarming methods, including traditional boundary rewarming commonly used for small-volume biological materials and other advanced techniques that could be potentially feasible for organ preservation in the future. The review focused on various rewarming technique principles, typical applications, and their possible limitations for cryopreservation of biological materials. This article introduced nanowarming methods in the progressing optimization and the possible difficulties. The trends of novel rewarming methods were discussed, and suggestions were given for future development.

Single-Mode Electromagnetic Resonance Rewarming for the Cryopreservation of Samples with Large Volumes: A Numerical and Experimental Study.

Rapid and uniform rewarming has been proved to be beneficial, and sometimes indispensable for the survival of cryopreserved biomaterials, inhibiting ice-recrystallization-devitrification and thermal stress-induced fracture (especially in large samples). To date, the convective water bath remains the gold standard rewarming method for small samples in the clinical settings, but it failed in the large samples (e.g., cryopreserved tissues and organs) due to damage caused by the slow and nonuniform heating. A single-mode electromagnetic resonance (SMER) system was developed to achieve ultrafast and uniform rewarming for large samples. In this study, we investigated the heating effects of the SMER system and compared the heating performance with water bath and air warming. A numerical model was established to further analyze the temperature change and distribution at different time points during the rewarming process. Overall, the SMER system achieved rapid heating at 331.63 ± 8.59°C min-1 while limiting the maximum thermal gradient to <9°C min-1, significantly better than the other two warming methods. The experimental results were highly consistent, indicating SMER is a promising rewarming technology for the successful cryopreservation of large biosamples.

Simultaneous multiparameter whole blood hemostasis assessment using a carbon nanotube-paper composite capacitance sensor.

Rapid and accurate clinical assessment of hemostasis is essential for managing patients who undergo invasive procedures, experience hemorrhages, or receive antithrombotic therapies. Hemostasis encompasses an ensemble of interactions between the cellular and non-cellular blood components, but current devices assess only partial aspects of this complex process. In this work, we describe the development of a new approach to simultaneously evaluate coagulation function, platelet count or function, and hematocrit using a carbon nanotube-paper composite (CPC) capacitance sensor. CPC capacitance response to blood clotting at 1.3 MHz provided three sensing parameters with distinctive sensitivities towards multiple clotting elements. Whole blood-based hemostasis assessments were conducted to demonstrate the potential utility of the developed sensor for various hemostatic conditions, including pathological conditions, such as hemophilia and thrombocytopenia. Results showed good agreements when compared to a conventional thromboelastography. Overall, the presented CPC capacitance sensor is a promising new biomedical device for convenient non-contact whole-blood based comprehensive hemostasis evaluation.

Modelling Analysis of COVID-19 Transmission and the State of Emergency in Japan.

To assess the effectiveness of the containment strategies proposed in Japan, an SEIAQR (susceptible-exposed-infected-asymptomatic-quarantined-recovered) model was established to simulate the transmission of COVID-19. We divided the spread of COVID-19 in Japan into different stages based on policies. The effective reproduction number Re and the transmission parameters were determined to evaluate the measures conducted by the Japanese Government during these periods. On 7 April 2020, the Japanese authority declared a state of emergency to control the rapid development of the pandemic. Based on the simulation results, the spread of COVID-19 in Japan can be inhibited by containment actions during the state of emergency. The effective reproduction number Re reduced from 1.99 (before the state of emergency) to 0.92 (after the state of emergency). The transmission parameters were fitted and characterized with quantifiable variables including the ratio of untracked cases, the PCR test index and the proportion of COCOA app users (official contact confirming application). The impact of these variables on the control of COVID-19 was investigated in the modelling analysis. On 8 January 2021, the Japanese Government declared another state of emergency. The simulated results demonstrated that the spread could be controlled in May by keeping the same strategies. A higher intensity of PCR testing was suggested, and a larger proportion of COCOA app users should reduce the final number of infections and the time needed to control the spread of COVID-19.

Management and Data Sharing of COVID-19 Pandemic Information.

The coronavirus disease 2019 (COVID-19) is an ongoing global pandemic caused by severe acute respiratory syndrome coronavirus 2. During the past 10 months, COVID-19 has killed over 1 million people worldwide. Under this global crisis, data sharing and management of the COVID-19 information are urgently needed and critical for researchers, epidemiologists, physicians, bioengineers, funding agencies, and governments to work together in developing new vaccines, drugs, methods, therapeutics, and strategies for the prevention and treatment of this deadly and rapidly spreading disease. The COVID-19 pandemic information includes the database of COVID-19-patient biospecimen resources in hospitals or biorepositories, electronic patient health records, ongoing clinical trials and research results on this disease, policies, guidelines, and regulations related to COVID-19, and the COVID-19 outbreak tracking records, and so on. A study of the current management and data-sharing approaches, tools, software, network, and internet systems developed in the United States is conducted in this article. Based on this study, it is revealed that the existing data-sharing and management systems are facing many big challenges and problems associated with data decentralization, inconsistencies, security and legal issues, limited financial support, international communications, standardization, and globalization. To overcome and solve these problems, several integrated platform models for national and international data-sharing and management are developed and proposed in this article to meet the unprecedented need and demand for COVID-19 pandemic information sharing and research worldwide.

A Study of Policies and Guidelines for Collecting, Processing, and Storing Coronavirus Disease 2019 Patient Biospecimens for Biobanking and Research.

Biobanking has been playing a crucial role in the development of new vaccines, drugs, biotechnology, and therapeutics for the prevention and treatment of a wide range of human diseases. This puts biobanks at the forefront of responding to the ongoing worldwide outbreak of the severe pandemic, coronavirus disease 2019 (COVID-19). The leading public health institutions around the world have developed and established interim policies and guidelines for researchers and biobank staff to handle the infectious biospecimens safely and adequately from COVID-19 patients. A study of these important and complementary policies and guidelines is conducted in this study. It should be emphasized that the COVID-19 biospecimens must be collected, processed, and preserved by trained personnel equipped with right personal protective equipment to prevent the transmission of the coronavirus and ensure the specimen quality for testing and research. Six of the leading global public health organizations or institutions included in this study are the World Health Organization, the Pan American Health Organization, the U.S. Centers for Disease Control and Prevention, the Public Health England, the U.S. Food and Drug Administration, and the Office of Research at the University of California, San Francisco. In conclusion, following the recommended guidance and policies with extreme precautions is essential to ensure the quality of the collected COVID-19 biospecimens and accuracy of the conducted research or treatment, and prevent any possible transmission. Efforts from cryobiologist and biobanking engineers to optimize the protocol of COVID-19 biospecimen cryopreservation and develop the user-friendly and cost-effective devices are urgently required to meet the urgent and increased needs in the specimen biobanking and transportation.

H4S1ph, an alternative epigenetic marker for sperm maturity.

Histone phosphorylation, an epigenetic post-translational modification, plays essential roles in male gamete chromatin compaction during spermatogenesis and sperm maturity. Previously, we studied the epigenetic marker of phosphorylated serine 1 of histone H2A and H4 (HS1ph) during spermatogenesis in mice and crabs, which was shown to be closely related to the sperm maturity. To further investigate the correlation between phosphorylated serine 1 of histone H4 (H4S1ph) and sperm maturation, a comparison study was conducted in this work between the healthy and the precocious crabs. It was discovered that the distribution of H4S1ph was similar for the two groups of crabs during spermatogenesis before maturity, but totally different in the sperm nuclei. H4S1ph vanished in the nuclei of healthy crab spermatozoa mostly, while retained in the precocious crabs just like what it was in elongated spermatid of both kinds of crabs. The results showed that a high level of H4S1ph conservation was closely associated with immaturity and might indicate inefficient fertility of male precocious crabs. Thus, H4S1ph was suggested to be an epigenetic marker of sperm maturity.

Determination of the Membrane Transport Properties of Jurkat Cells with a Microfluidic Device.

The Jurkat cell is an immortalized line of human acute lymphocyte leukemia cells that is widely used in the study of adoptive cell therapy, a novel treatment of several advanced forms of cancer. The ability to transport water and solutes across the cell membrane under different temperatures is an important factor for deciding the specific protocol for cryopreservation of the Jurkat cell. In this study we propose a comprehensive process for determination of membrane transport properties of Jurkat cell. using a novel microfluidic controlled single cell-trapping system. The osmotic behavior of an individual Jurkat cell to water and dimethyl sulfoxide (DMSO), a commonly used cryoprotective agent (CPA), under constant temperature, was recorded under a microscope utilizing the modified microfluidic system. The images of the Jurkat cell under osmotic change were processed to obtain a relationship between cell volume change and time. The experimental results were fitted using a two-parameter transport numeric model to calculate the Jurkat cell membrane permeability to water and DMSO at room temperature (22 °C). This model and the calculated parameters can help scientists optimize the cryopreservation protocol for any cell type with optimal cryoprotective agents and cooling rate for future experiments.

Investigation of Electromagnetic Resonance Rewarming Enhanced by Magnetic Nanoparticles for Cryopreservation.

The lack of an effective rewarming technique restricted the successful cryopreservation of organ or large tissues by vitrification. The conversion of electromagnetic (EM) energy into heat provides a possible solution for the rewarming process for the cryopreservation. In this work, an EM resonance rewarming system was set up with dynamic feedback control and power feeding optimization. In addition, we take advantage of magnetic nanoparticles (MNPs) to absorb magnetic field energy to further enhance the energy conversion efficiency. We achieved a >200 °C min-1 rewarming rate for tens of milliliters of cryopreserved samples. Besides, we also investigated the effect of nanoparticle size and concentration based on thermal properties by analyzing the contribution of nanoparticles and the utilization of field energy. The closed system reduced the possible concomitant side effects when increasing the number of nanoparticles or increasing the EM source power. With the remarkably low dosage of nanoparticles (0.1 mg mL-1 Fe) compared to that for other MNP-based rewarming applications, this study opens the door to new approaches for exploring novel techniques for tissue and organ preservation.

Cryopreservation of human mucosal tissues.

BACKGROUND: Cryopreservation of leukocytes isolated from the cervicovaginal and colorectal mucosa is useful for the study of cellular immunity (see Hughes SM et al. PLOS ONE 2016). However, some questions about mucosal biology and sexually transmitted infections are better addressed with intact mucosal tissue, for which there is no standard cryopreservation protocol. METHODS AND FINDINGS: To find an optimal preservation protocol for mucosal tissues, we tested slow cooling (1°C/min) with 10% dimethylsulfoxide (designated "cryopreservation") and fast cooling (plunge in liquid nitrogen) with 20% dimethylsulfoxide and 20% ethylene glycol ("vitrification"). We compared fresh and preserved human cervicovaginal and colorectal tissues in a range of assays, including metabolic activity, human immunodeficiency virus infection, cell phenotype, tissue structure by hematoxylin-and-eosin staining, cell number and viability, production of cytokines, and microbicide drug concentrations. Metabolic activity, HIV infectability, and tissue structure were similar in cryopreserved and vitrified vaginal tissues. However, vitrification led to poor cell recovery from the colorectal mucosa, with 90% fewer cells recovered after isolation from vitrified colorectal tissues than from cryopreserved. HIV infection rates were similar for fresh and cryopreserved ectocervical tissues, whereas cryopreserved colorectal tissues were less easily infected than fresh tissues (hazard ratio 0.7 [95% confidence interval 0.4, 1.2]). Finally, we compared isolation of cells before and after cryopreservation. Cell recoveries were higher when cells were isolated after freezing and thawing (71% [59-84%]) than before (50% [38-62%]). Cellular function was similar to fresh tissue in both cases. Microbicide drug concentrations were lower in cryopreserved explants compared to fresh ones. CONCLUSIONS: Cryopreservation of intact cervicovaginal and colorectal tissues with dimethylsulfoxide works well in a range of assays, while the utility of vitrification is more limited. Cell yields are higher from cryopreserved intact tissue pieces than from thawed cryopreserved single cell suspensions isolated before freezing, but T cell functions are similar.

Determination of Dielectric Properties of Cryoprotective Agent Solutions with a Resonant Cavity for the Electromagnetic Rewarming in Cryopreservation.

In the rewarming process during cryopreservation, preventing ice recrystallization and thermal stress is important, especially for large tissues and organs. Uniform and rapid heating is essential in ameliorating the problem and maintaining the viability of cryopreserved biological samples. Currently, the most promising method is heating by application of electromagnetic (EM) waves, the effectiveness of which is dependent on the dielectric properties (DP) of the cryopreserved materials. In this work, the cavity perturbation method was adopted to measure the DP of cryoprotectant solutions. Based on the values of DP, the cryoprotectant solutions most amenable to EM heating can be identified. A system composed of a rectangular resonant cavity, a network analyzer, and a fiber optic temperature meter was implemented for the measurement. The DP of three cryoprotectant solutions during cooling to -80°C were measured and presented. The data can be used to optimize the rewarming process with the numerical method. The results show that a cryoprotectant solution consisting of 41% (w/v) dimethyl sulfoxide and 6% (w/v) polyvinylpyrrolidone has the highest dielectric loss for EM rewarming among the tested solutions. In addition, the developed DP measurement system could not only improve the EM heating in cryopreservation but also benefit hyperthermia or other therapies associated with EM waves.

Determination of the temperature-dependent cell membrane permeabilities using microfluidics with integrated flow and temperature control.

We developed an integrated microfluidic platform for instantaneous flow and localized temperature control. The platform consisted of a flow-focusing region for sample delivery and a cross-junction region embedded with a microheater for cell trapping and localized temperature control by using an active feedback control system. We further used it to measure the membrane transport properties of Jurkat cells, including the osmotically inactive cell volume (Vb) and cell membrane permeabilities to water (Lp) and to cryoprotective agent (CPA) solutions (dimethyl sulfoxide (DMSO) in this study) (PS) at various temperatures (room temperature, 30 °C, and 37 °C). Such characteristics of cells are of great importance in many applications, especially in optimal cryopreservation. With the results, the corresponding activation energy for water and CPA transport was calculated. The comparison of the results from the current study with reference data indicates that the developed platform is a reliable tool for temperature-dependent cell behavior study, which provides valuable tools for general cell manipulation applications with precise temperature control.

Quantification of Intracellular Ice Formation and Recrystallization During Freeze-Thaw Cycles and Their Relationship with the Viability of Pig Iliac Endothelium Cells.

Quantitative evaluation of the inherent correlation between cell cryoinjuries and intracellular ice formation (IIF) together with recrystallization (IIR) is of primary importance for both optimization of biopreservation and cryotherapy. The objective of this study is to thoroughly explore the roles of IIF on cell viability by using pig iliac endothelium cells (PIECs) as model cells during freezing and thawing. The experimental results indicated that both the probabilities of IIF (PIF) and IIR (PIR) increased along with the increase of cooling rates (p < 0.05) during the freeze-thaw cycles at cooling rates of 40, 60, 80, 100, and 150°C/min and the same warming rates of 100°C/min in phosphate-buffered saline-based solutions with or without 1 M DMSO. Viability evaluation with Hoechst 33342/propidium iodide double staining showed that most of the cells were killed (viability <20%) by the abovementioned freeze-thaw cycles, which indicated that the cooling rates investigated were all too rapid since large amounts of IIF and IIR were introduced. Another interesting phenomenon is that the presence of a low concentration of DMSO (1 M) tends to improve cell viability while increasing the PIF and PIR during freezing/thawing, contrary to the common belief that larger PIF corresponds to greater cryoinjury. This may be attributed to the intrinsic protection effect of DMSO by reduction of solution injury or other potential injuries. These findings may be of potential application value for both cryopreservation and cryosurgery by providing helpful additions to the existing studies on investigation of cryoinjuries of PIECs.

Comparative Study on Two Different Methods for Determination of Hydraulic Conductivity of HeLa Cells During Freezing.

BACKGROUND: The measurement of hydraulic conductivity of the cell membrane is very important for optimizing the protocol of cryopreservation and cryosurgery. There are two different methods using differential scanning calorimetry (DSC) to measure the freezing response of cells and tissues. Devireddy et al. presented the slow-fast-slow (SFS) cooling method, in which the difference of the heat release during the freezing process between the osmotically active and inactive cells is used to obtain the cell membrane hydraulic conductivity and activation energy. Luo et al. simplified the procedure and introduced the single-slow (SS) cooling protocol, which requires only one cooling process although different cytocrits are required for the determination of the membrane transport properties. To the best of our knowledge, there is still a lack of comparison of experimental processes and requirements for experimental conditions between these two methods. This study made a systematic comparison between these two methods from the aforementioned aspects in detail. METHODS: The SFS and SS cooling methods mentioned earlier were utilized to obtain the reference hydraulic conductivity (Lpg) and activation energy (ELp) of HeLa cells by fitting the model to DSC data. RESULTS: With the SFS method, it was determined that Lpg = 0.10 µm/(min·atm) and ELp = 22.9 kcal/mol; whereas the results obtained by the SS cooling method showed that Lpg = 0.10 µm/(min·atm) and ELp = 23.6 kcal/mol. CONCLUSIONS: The results indicated that the values of the water transport parameters measured by two methods were comparable. In other words, the two parameters can be obtained by comparing the heat releases between two slow cooling processes of the same sample according to the SFS method. However, the SS method required analyzing heat releases of samples with different cytocrits. Thus, more experimental time was required.

Cryopreservation of Human Mucosal Leukocytes.

BACKGROUND: Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. METHODS AND FINDINGS: To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. CONCLUSIONS: Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Determination of the Membrane Permeability to Water of Human Vaginal Mucosal Immune Cells at Subzero Temperatures Using Differential Scanning Calorimetry.

To study mucosal immunity and conduct HIV vaccine trials, it is important to be able to cryopreserve mucosal specimens and recover them in functional viable form. Obtaining a good recovery depends, in part, on cooling the cells at the appropriate rate, which is determined by the rate of water transport across the cell membrane during the cooling process. In this study, the cell membrane permeabilities to water at subzero temperatures of human vaginal mucosal T cells and macrophages were measured using the differential scanning calorimetry method proposed by Devireddy et al. in 1998. Thermal histograms were measured before and after cell lysis using a Slow-Fast-Fast-Slow cooling program. The difference between the thermal histograms of the live intact cells and the dead lysed cells was used to calculate the temperature-dependent cell membrane permeability at subzero temperatures, which was assumed to follow the Arrhenius relationship, [Formula: see text], where Lpg is the permeability to water at the reference temperature (273.15 K). The results showed that Lpg = 0.0209 ± 0.0108 µm/atm/min and Ea = 41.5 ± 11.4 kcal/mol for T cells and Lpg = 0.0198 ± 0.0102 µm/atm/min and Ea = 38.2 ± 10.4 kcal/mol for macrophages, respectively, in the range 0°C to -40°C (mean ± standard deviation). Theoretical simulations predicted that the optimal cooling rate for both T cells and macrophages was about -3°C/min, which was proven by preliminary immune cell cryopreservation experiments.

A study of the osmotic characteristics, water permeability, and cryoprotectant permeability of human vaginal immune cells.

Cryopreservation of specimens taken from the genital tract of women is important for studying mucosal immunity during HIV prevention trials. However, it is unclear whether the current, empirically developed cryopreservation procedures for peripheral blood cells are also ideal for genital specimens. The optimal cryopreservation protocol depends on the cryobiological features of the cells. Thus, we obtained tissue specimens from vaginal repair surgeries, isolated and flow cytometry-purified immune cells, and determined fundamental cryobiological characteristics of vaginal CD3(+) T cells and CD14(+) macrophages using a microfluidic device. The osmotically inactive volumes of the two cell types (Vb) were determined relative to the initial cell volume (V0) by exposing the cells to hypotonic and hypertonic saline solutions, evaluating the equilibrium volume, and applying the Boyle van't Hoff relationship. The cell membrane permeability to water (Lp) and to four different cryoprotective agent (CPA) solutions (Ps) at room temperature were also measured. Results indicated Vb values of 0.516 V0 and 0.457 V0 for mucosal T cells and macrophages, respectively. Lp values at room temperature were 0.196 and 0.295 µm/min/atm for T cells and macrophages, respectively. Both cell types had high Ps values for the three CPAs, dimethyl sulfoxide (DMSO), propylene glycol (PG) and ethylene glycol (EG) (minimum of 0.418 × 10(-3) cm/min), but transport of the fourth CPA, glycerol, occurred 50-150 times more slowly. Thus, DMSO, PG, and EG are better options than glycerol in avoiding severe cell volume excursion and osmotic injury during CPA addition and removal for cryopreservation of human vaginal immune cells.

Sensing and Sensibility: Single-Islet-based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules.

Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.