Characterizing cellular membrane partitioning of DMSO using low-temperature Raman spectroscopy.

Additives that help cells survive the stresses of freezing and thawing are known as cryoprotective agents (CPAs). Two different types of CPAs have been identified: penetrating and non-penetrating. Common penetrating CPAs include dimethylsulfoxide (DMSO) and glycerol. The location of a CPA (intracelluar or extracellular) is important for understanding the molecular mechanisms of action for the agent. Low-temperature Raman spectroscopy is a label-free method of detecting the location of CPAs at low temperature with high spatial resolution and chemical specificity. To this end, cells cryopreserved in DMSO using a variety of cooling rates and DMSO concentrations and imaged using Raman spectroscopy were analyzed using automated image analysis to determine the partitioning ratio (concentration of DMSO outside/concentration of DMSO inside the cell). The partitioning ratio was roughly 1 for Jurkat cells frozen at 1°C/min in varying concentrations of DMSO with the exception of 1% DMSO which had a partitioning ratio of 0.2. The partitioning ratio increased from 1 to 1.3 as the cooling rate increased from 1°C to 5°C/min. Different cell types, specifically sensory neurons cells and human induced pluripotent stem cells, exhibited differences in partitioning ratio when frozen in 10% DMSO and 1°C/min suggesting that differences in freezing response may result from differences in solute partitioning. The presence of intracellular ice changed the distribution of DMSO inside the cell and also the partitioning ratio.

Characterization of eutectic mixtures of sugars and sugar-alcohols for cryopreservation.

Natural Deep Eutectic Systems (NADES) composed of sugar and sugar alcohols have been studied and applied in a variety of biological applications. Understanding their interaction with water across dilution and temperature is inherently important for maximizing the utility of NADES. Herein a wide range of sugar:sugar-alcohol molar ratios were synthesized and characterized by viscosity, molar excess volume, differential scanning calorimetry, water activity, and confocal Raman cryomicroscopy. NADES were found to have greater viscosity, reduced heat of fusion, greater absolute molar excess volume, lower water activity, and stronger hydrogen bonding of water than non-NADES mixtures. This is hypothesized to be due to cumulatively stronger hydrogen bonding interactions between components in pure and diluted NADES with the strongest interactions in the water-rich region. This work provides useful data and further understanding of hydrogen bonding interaction strength for a wide range of molar ratios in pure to well-diluted forms.

Long-term storage of gametes and gonadal tissues at room temperatures: the end of the ice age?

Long-term preservation of viable spermatozoa, eggs, embryos, and gonadal tissues of good quality is essential in human reproductive medicine and for the population management of livestock, laboratory, and wild species. Instead of using freezing temperatures, encouraging findings indicate that structures and functions of gametes or gonadal tissues can be suspended in trehalose glass after dehydration and then preserved at supra-zero temperatures. As a new era in fertility preservation and biobanking is about to start, the advantages, needs, and implications of germplasm storage at room temperatures must be carefully examined. Although very promising, the development of alternate biobanking strategies does not necessarily mean that the end of the "ice age" (cryopreservation) is near.

Differentiation of Human iPS Cells Into Sensory Neurons Exhibits Developmental Stage-Specific Cryopreservation Challenges.

Differentiation of human induced pluripotent stem cells (hiPSCs) generates cell phenotypes valuable for cell therapy and personalized medicine. Successful translation of these hiPSC-derived therapeutic products will rely upon effective cryopreservation at multiple stages of the manufacturing cycle. From the perspective of cryobiology, we attempted to understand how the challenge of cryopreservation evolves between cell phenotypes along an hiPSC-to-sensory neuron differentiation trajectory. Cells were cultivated at three different stages to represent intermediate, differentiated, and matured cell products. All cell stages remained ≥90% viable in a dimethyl sulfoxide (DMSO)-free formulation but suffered ≥50% loss in DMSO before freezing. Raman spectroscopy revealed higher sensitivity to undercooling in hiPSC-derived neuronal cells with lower membrane fluidity and higher sensitivity to suboptimal cooling rates in stem cell developmental stages with larger cell bodies. Highly viable and functional sensory neurons were obtained following DMSO-free cryopreservation. Our study also demonstrated that dissociating adherent cultures plays an important role in the ability of cells to survive and function after cryopreservation.

Natural deep eutectic systems for nature-inspired cryopreservation of cells.

Natural deep eutectic systems (NADES) are emerging as potential cryoprotective agents (CPA) for cell preservation. In this investigation, we develop an optimized CPA formulation using trehalose-glycerol NADES (T:G) diluted in Normosol-R and supplemented with isoleucine. Differential scanning calorimetry (DSC) is used to define the thermophysical properties of NADES-based solutions, and Raman spectroscopy is used to characterize the effect of NADES on ice formation and hydrogen bonding. Jurkat cells are cryopreserved in each solution, and post-thaw cell recovery, apoptosis, and growth are quantified. Raman spectra and heat maps show that NADES suppresses both ice formation and dehydration of the nonfrozen region. Supplementing NADES with isoleucine does not affect the solution's thermophysical properties but significantly improves the cells' survival and proliferation post-thaw. The study indicates that thermophysical properties of CPA solutions alone cannot predict optimal cell survival, suggesting that stabilization of biological structures by CPAs may play a role in successful cryopreservation.

Comparative analysis of cell therapy infusion workflows at clinical sites.

BACKGROUND AIMS: Cell therapies are an emerging treatment option for a variety of diseases, especially with the success of chimeric antigen receptor T-cell therapies. With 18 FDA-approved cell therapy products as of December 2020 and a growing number in clinical trials, standards for most aspects of the cell therapy lifecycle are well-established by professional organizations like AABB and FACT; however, there are limited standardized protocols regarding the day-of infusion. METHODS: Infusions were observed at three academic medical centers in the United States, and the workflows were analyzed and compared based on factors including facility layout, product verification processes, cryobag design, timing restrictions, and use of electronic medical records. RESULTS: Variations between the facilities were identified with product thawing location and cell therapy lab location being the most important factors in time from thaw to infusion. CONCLUSIONS: Based on this analysis, opportunities were identified for standardization and streamlining the infusion workflow which may help facilitate adoption of new and existing cell therapies at a wider range of hospitals.

Raman Cryomicroscopic Imaging and Sample Holder for Spectroscopic Subzero Temperature Measurements.

Raman spectroscopy has been gaining in popularity for noninvasive analysis of single cells. Raman spectra and images deliver meaningful information regarding the biochemical, biophysical, and structural properties of cells in various states. Low-temperature Raman spectroscopy has been applied to verify the presence of ice inside a frozen cell and to illustrate the distribution of both penetrating and non-penetrating cryoprotectants. This chapter delineates Raman cryomicroscopic imaging of single cells as well as sample handling for spectroscopic measurements at subzero temperature. The experimental setup is depicted with a special emphasis on a custom-built temperature-controlled cooling stage. The use of Raman cryomicroscopic imaging is demonstrated using Jurkat cells cryopreserved in a sucrose solution. Moreover, strategies for determining intracellular ice formation (IIF) and analysis of sucrose partitioning across the cell membrane are presented.

Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants.

BACKGROUND: This study examined the freezing responses of peripheral blood mononuclear cells (PBMCs) and specific white blood cell subsets contained therein when cryopreserved in three combinations of osmolytes composed of sugars, sugar alcohols and amino acids. METHODS: A differential evolution algorithm with multiple objectives was used to optimize cryoprotectant composition and thus the post-thaw recoveries for both helper and cytotoxicity T cells simultaneously. RESULTS: The screening of various formulations using a differential evolution algorithm showed post-thaw recoveries greater than 80% for the two subsets of T cells. The phenotypes and viabilities of PBMC subsets were characterized using flow cytometry. Significant differences between the post-thaw recovery for helper T cells and cytotoxic T cells were observed. Statistical models were used to analyze the importance of individual osmolytes and interactions between post-thaw recoveries of three subsets of T cell including helper T cells, cytotoxic T cells and natural killer T cells. The statistical model indicated that the preferred concentration levels of osmolytes and interaction modes were distinct between the three subsets studied. PBMCs were cultured for 72 h post-thaw to determine the stability of the cells. Because post-thaw apoptosis is a significant concern for lymphocytes, apoptosis of helper T cell and cytotoxic T cells frozen in a DMSO-free cryoprotectant was analyzed immediately post-thaw and 24 h post-thaw. Both cell types showed a decrease in cell viability 24 h post-thaw compared with immediately post-thaw. Helper T cell viability dropped 17%, and cytotoxic T cells had a 10% drop in viability. Immediately post-thaw, both cell types had >30% of cells in early apoptosis, but after 24 h the number of cells in early apoptosis decreased to below 20%. CONCLUSION: This study helped us identify the freezing responses of different human PBMC subsets using combinations of osmolytes.

Cryopreservation of Human iPS Cell Aggregates in a DMSO-Free Solution-An Optimization and Comparative Study.

Human induced pluripotent stem cells (hiPSCs) are an important cell source for regenerative medicine products. Effective methods of preservation are critical to their clinical and commercial applications. The use of a dimethyl sulfoxide (DMSO)-free solution containing all non-toxic molecules offers an effective alternative to the conventional DMSO and alleviates pain points associated with the use of DMSO in the cryopreservation of hiPSCs. Both hiPSCs and cells differentiated from them are commonly multicellular systems, which are more sensitive to stresses of freezing and thawing than single cells. In this investigation, low-temperature Raman spectroscopy visualized freezing behaviors of hiPSC aggregates in different solutions. These aggregates exhibited sensitivity to undercooling in DMSO-containing solutions. We demonstrated the ability to replace DMSO with non-toxic molecules, improve post-thaw cell survival, and reduce sensitivity to undercooling. An accelerated optimization process capitalized on the positive synergy among multiple DMSO-free molecules, which acted in concert to influence ice formation and protect cells during freezing and thawing. A differential evolution algorithm was used to optimize the multi-variable, DMSO-free preservation protocol in 8 experiments. hiPSC aggregates frozen in the optimized solution did not exhibit the same sensitivity to undercooling as those frozen in non-optimized solutions or DMSO, indicating superior adaptability of the optimized solution to different freezing modalities and unplanned deviations. This investigation shows the importance of optimization, explains the mechanisms and advantages of a DMSO-free solution, and enables not only improved cryopreservation of hiPSCs but potentially other cell types for translational regenerative medicine.

Differential Evolution for the Optimization of DMSO-Free Cryoprotectants: Influence of Control Parameters.

This study presents the influence of control parameters including population (NP) size, mutation factor (F), crossover (Cr), and four types of differential evolution (DE) algorithms including random, best, local-to-best, and local-to-best with self-adaptive (SA) modification for the purpose of optimizing the compositions of dimethylsufloxide (DMSO)-free cryoprotectants. Post-thaw recovery of Jurkat cells cryopreserved with two DMSO-free cryoprotectants at a cooling rate of 1 °C/min displayed a nonlinear, four-dimensional structure with multiple saddle nodes, which was a suitable training model to tune the control parameters and select the most appropriate type of differential evolution algorithm. Self-adaptive modification presented better performance in terms of optimization accuracy and sensitivity of mutation factor and crossover among the four different types of algorithms tested. Specifically, the classical type of differential evolution algorithm exhibited a wide acceptance to mutation factor and crossover. The optimization performance is more sensitive to mutation than crossover and the optimization accuracy is proportional to the population size. Increasing population size also reduces the sensitivity of the algorithm to the value of the mutation factor and crossover. The analysis of optimization accuracy and convergence speed suggests larger population size with F > 0.7 and Cr > 0.3 are well suited for use with cryopreservation optimization purposes. The tuned differential evolution algorithm is validated through finding global maximums of other two DMSO-free cryoprotectant formulation datasets. The results of these studies can be used to help more efficiently determine the optimal composition of multicomponent DMSO-free cryoprotectants in the future.

Preservation of cell-based immunotherapies for clinical trials.

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5-10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.

Cryopreservation of Hematopoietic Stem Cells: Emerging Assays, Cryoprotectant Agents, and Technology to Improve Outcomes.

Hematopoietic stem cell (HSC) therapy is widely used to treat a growing number of hematological and non-hematological diseases. Cryopreservation of HSCs allows for cells to be transported from the site of processing to the site of clinical use, creates a larger window of time in which cells can be administered to patients, and allows sufficient time for quality control and regulatory testing. Currently, HSCs and other cell therapies conform to the same cryopreservation techniques as cells used for research purposes: cells are cryopreserved in dimethyl sulfoxide (DMSO) at a slow cooling rate. As a result, HSC therapy can result in numerous adverse symptoms in patients due to the infusion of DMSO. Efforts are being made to improve the cryopreservation of HSCs for clinical use. This review discusses advances in the cryopreservation of HSCs from 2007 to the present. The preclinical development of new cryoprotectants and new technology to eliminate cryoprotectants after thawing are discussed in detail. Additional cryopreservation considerations are included, such as cooling rate, storage temperature, and cell concentration. Preclinical cell assessment and quality control are discussed, as well as clinical studies from the past decade that focus on new cryopreservation protocols to improve patient outcomes.

The role of preservation in the variability of regenerative medicine products.

Regenerative medicine (RM) has the potential to restore or establish normal function of cells, tissues and organs that have been lost due to age, disease or injury. It is common for the site of raw material collection, site of manufacture and site of clinical use to be different for RM products, and at the same time cells must remain viable and functional during transportation among different sites. Freezing products down to cryogenic temperatures along with cold chain transportation has become an effective method of preserving RM products. The quality of RM products along this supply chain represents the cumulative effects of all of the processing steps and all of the reagents used in the process. A variety of sources of variability in the preservation of RM products can result in both cell losses and greater variability in the quality of RM products. The purpose of this article is to review the sources of variability in the preservation process as well as the methods by which variability can be controlled or avoided.

Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells.

This study examined the post-thaw recovery of Jurkat cells cryopreserved in three combinations of five osmolytes including trehalose, sucrose, glycerol, mannitol, and creatine. Cellular response was characterized using low-temperature Raman spectroscopy, and variation of post-thaw recovery was analyzed using statistical modeling. Combinations of osmolytes displayed distinct trends of post-thaw recovery, and a nonlinear relationship between compositions and post-thaw recovery was observed, suggesting interactions not only between different solutes but also between solutes and cells. The post-thaw recovery for optimized cryoprotectants in different combinations of osmolytes at a cooling rate of 1°C/min was comparable to that measured with 10% dimethyl sulfoxide. Statistical modeling was used to understand the importance of individual osmolytes as well as interactions between osmolytes on post-thaw recovery. Both higher concentrations of glycerol and certain interactions between sugars and glycerol were found to typically increase the post-thaw recovery. Raman images showed the influence of osmolytes and combinations of osmolytes on ice crystal shape, which reflected the interactions between osmolytes and water. Differences in the composition also influenced the presence or absence of intracellular ice formation, which could also be detected by Raman. These studies help us understand the modes of action for cryoprotective agents in these osmolyte solutions.

Interfacial Interactions of Sucrose during Cryopreservation Detected by Raman Spectroscopy.

There is considerable interest in the use of sugars to preserve cells. In this study, low temperature Raman spectroscopy was used to characterize the behaviors of sucrose during freezing. The hydrogen bond network between sucrose and water was investigated at -10 °C and -50 °C, and the Raman spectra showed strengthened sucrose-water and sucrose-sucrose hydrogen bonds in more concentrated sucrose solution at -50 °C. The concentration of sucrose at the ice interface increased as the ice density decreased, and it plateaued across a narrow channel of nonfrozen sucrose solution before it decreased toward the next ice interface. The biophysical environment at interfaces between the cell and nonfrozen sucrose solution and between the cell and extracellular ice was also studied. A thin layer of nonfrozen sucrose solution was observed at the interface between the cell and extracellular ice. The extracellular concentration of sucrose at this interface was generally lower than that of bulk nonfrozen sucrose solution. The variation of sucrose concentration outside different regions of the cell membrane suggests that the chemical environment around the cell during freezing may be more heterogeneous than previously thought. Raman spectra and images also showed colocalization of nonfrozen sucrose solution and the cell, which implied that direct interaction between sucrose and cell membrane might be responsible for protective properties of sucrose. Sucrose was predominantly distributed outside the cell, and the observation of strong partitioning of sucrose across the cell membrane is consistent with substantial cell dehydration detected by the Raman spectra. This work enhances our understanding of the behaviors of sucrose solution and its interactions with cells at low temperature and can improve cryopreservation protocols of cells frozen in a sucrose-based media.

Characterizing the "sweet spot" for the preservation of a T-cell line using osmolytes.

This study examined the post-thaw recovery of Jurkat cells cryopreserved in single osmolyte solutions containing sucrose, glycerol or isoleucine, as well as in a combination of the three osmolytes. Cell response was determined using low temperature Raman Spectroscopy and variation in post-thaw recovery with composition was analyzed using statistical modeling. Post-thaw recovery of Jurkat cells in single osmolyte was low. A combination of the osmolytes displayed a non-linear relationship between composition and post-thaw recovery, suggesting that interactions exist between the different solutes. The post-thaw recovery for an optimized multicomponent solution was comparable to that observed using 10% dimethyl sulfoxide and a cooling rate of 1 °C/min. Statistical modeling was used to characterize the importance of each osmolyte in the combination and test for interactions between osmolytes. Higher concentrations of glycerol increase post-thaw recovery and interactions between sucrose and glycerol, as well as sucrose and isoleucine improve post-thaw recovery. Raman images clearly demonstrated that damaging intracellular ice formation was observed more often in the presence of single osmolytes as well as non-optimized multi-component solution compositions.

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Characterizing Intracellular Ice Formation of Lymphoblasts Using Low-Temperature Raman Spectroscopy.

Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols.

High-Throughput Processing to Preserve Viable Cells: A Precision Medicine Initiative Cohort Program Workshop.

Conventionally, biobanks supporting clinical research studies have preserved serum, plasma, urine, saliva, a variety of tissue types, and stool. With the emergence of increasingly sophisticated technologies for analyzing single cells, there is growing interest in preserving viable blood cells for future functional studies. The new All of Us Research Program (formerly the Precision Medicine Initiative Cohort Program) biobank plans to house samples from a million or more individuals as part of a cohort with rich phenotypic data and longitudinal follow-up ( www.nih.gov/research-training/allofus-research-program ). Storage of viable cells for future single-cell analysis offers the promise of new biology, discovery of novel biomarkers, and advances toward the goal of precision medicine. A workshop was held in the summer of 2016 to evaluate the case for preservation of viable mononuclear blood cells and its feasibility within the collection plan for the biobank.