Successful expansion and cryopreservation of human natural killer cell line NK-92 for clinical manufacturing.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.

Screening of Hydrophilic Polymers Reveals Broad Activity in Protecting Phages during Cryopreservation.

Bacteriophages have many biotechnological and therapeutic applications, but as with other biologics, cryopreservation is essential for storage and distribution. Macromolecular cryoprotectants are emerging for a range of biologics, but the chemical space for polymer-mediated phage cryopreservation has not been explored. Here we screen the cryoprotective effect of a panel of polymers against five distinct phages, showing that nearly all the tested polymers provide a benefit. Exceptions were poly(methacrylic acid) and poly(acrylic acid), which can inhibit phage-infection with bacteria, making post-thaw recovery challenging to assess. A particular benefit of a polymeric cryopreservation formulation is that the polymers do not function as carbon sources for the phage hosts (bacteria) and hence do not interfere with post-thaw measurements. This work shows that phages are amenable to protection with hydrophilic polymers and opens up new opportunities for advanced formulations for future phage therapies and to take advantage of the additional functionality brought by the polymers.

Engineered Test Tissues: A Model for Quantifying the Effects of Cryopreservation Parameters.

Engineered tissues are showing promise as implants to repair or replace damaged tissues in vivo or as in vitro tools to discover new therapies. A major challenge of the tissue engineering field is the sample preservation and storage until their transport and desired use. To successfully cryopreserve tissue, its viability, structure, and function must be retained post-thaw. The outcome of cryopreservation is impacted by several parameters, including the cryopreserving agent (CPA) utilized, the cooling rate, and the storage temperature. Although a number of CPAs are commercially available for cell cryopreservation, there are few CPAs designed specifically for tissue cryostorage and recovery. In this study, we present a flexible, relatively high-throughput method that utilizes engineered tissue rings as test tissues for screening the commercially available CPAs and cryopreservation parameters. Engineered test tissues can be fabricated with low batch-to-batch variability and characteristic morphology due to their endogenous extracellular matrix, and they have mechanical properties and a ring format suitable for testing with standard methods. The tissues were grown for 7 days in standard 48-well plates and cryopreserved in standard cryovials. The method allowed for the quantification of metabolic recovery, tissue apoptosis/necrosis, morphology, and mechanical properties. In addition to establishing the method, we tested different CPA formulations, freezing rates, and freezing points. Our proposed method enables timely preliminary screening of CPA formulations and cryopreservation parameters that may improve the storage of engineered tissues.

Proline pre-conditioning of Jurkat cells improves recovery after cryopreservation.

Cell therapies such as allogenic CAR T-cell therapy, natural killer cell therapy and stem cell transplants must be cryopreserved for transport and storage. This is typically achieved by addition of dimethyl sulfoxide (DMSO) but the cryoprotectant does not result in 100% cell recovery. New additives or technologies to improve their cryopreservation could have major impact for these emerging therapies. l-Proline is an amino acid osmolyte produced as a cryoprotectant by several organisms such as the codling moth Cydia pomonella and the larvae of the fly Chymomyza costata, and has been found to modulate post-thaw outcomes for several cell lines but has not been studied with Jurkat cells, a T lymphocyte cell line. Here we investigate the effectiveness of l-proline compared to d-proline and l-alanine for the cryopreservation of Jurkat cells. It is shown that 24-hour pre-freezing incubation of Jurkat cells with 200 mM l-proline resulted in a modest increase in cell recovery post-thaw at high cell density, but a larger increase in recovery was observed at the lower cell densities. l-Alanine was as effective as l-proline at lower cell densities, and addition of l-proline to the cryopreservation media (without incubation) had no benefit. The pre-freeze incubation with l-proline led to significant reductions in cell proliferation supporting an intracellular, biochemical, mechanism of action which was shown to be cell-density dependent. Controls with d-proline were found to reduce post-thaw recovery attributed to osmotic stress as d-proline cannot enter the cells. Preliminary analysis of apoptosis/necrosis profiles by flow cytometry indicated that inhibition of apoptosis is not the primary mode of action. Overall, this supports the use of l-proline pre-conditioning to improve T-cell post-thaw recovery without needing any changes to cryopreservation solutions nor methods and hence is simple to implement.

Cryopreservation of mouse thymus depletes thymocytes but supports immune reconstitution on transplantation.

Cryopreservation of mouse thymus depletes donor thymocytes but preserves thymus function when transplanted after thawing into athymic mice. No differences in immune reconstitution were observed between fresh and frozen/thawed transplants suggesting that donor thymocyte depletion does not affect outcome. Thus, cryopreservation of thymus may improve outcomes in thymus transplant patients.

Anionic Synthetic Polymers Prevent Bacteriophage Infection.

Bioprocessing and biotechnology exploit microorganisms (such as bacteria) for the production of chemicals, biologics, therapies, and food. A major unmet challenge is that bacteriophage (phage) contamination compromises products and necessitates shut-downs and extensive decontamination using nonspecific disinfectants. Here we demonstrate that poly(acrylic acid) prevents phage-induced killing of bacterial hosts, prevents phage replication, and that induction of recombinant protein expression is not affected by the presence of the polymer. Poly(acrylic acid) was more active than poly(methacrylic acid), and poly(styrenesulfonate) had no activity showing the importance of the carboxylic acids. Initial evidence supported a virustatic, not virucidal, mechanism of action. This simple, low-cost, mass-produced additive offers a practical, scalable, and easy to implement solution to reduce phage contamination.

A highly active mineral-based ice nucleating agent supports in situ cell cryopreservation in a high throughput format.

Cryopreservation of biological matter in microlitre scale volumes of liquid would be useful for a range of applications. At present, it is challenging because small volumes of water tend to supercool, and deep supercooling is known to lead to poor post-thaw cell viability. Here, we show that a mineral ice nucleator can almost eliminate supercooling in 100 µl liquid volumes during cryopreservation. This strategy of eliminating supercooling greatly enhances cell viability relative to cryopreservation protocols with uncontrolled ice nucleation. Using infrared thermography, we demonstrate a direct relationship between the extent of supercooling and post-thaw cell viability. Using a mineral nucleator delivery system, we open the door to the routine cryopreservation of mammalian cells in multiwell plates for applications such as high throughput toxicology testing of pharmaceutical products and regenerative medicine.

Red Blood Cell Cryopreservation with Minimal Post-Thaw Lysis Enabled by a Synergistic Combination of a Cryoprotecting Polyampholyte with DMSO/Trehalose.

From trauma wards to chemotherapy, red blood cells are essential in modern medicine. Current methods to bank red blood cells typically use glycerol (40 wt %) as a cryoprotective agent. Although highly effective, the deglycerolization process, post-thaw, is time-consuming and results in some loss of red blood cells during the washing procedures. Here, we demonstrate that a polyampholyte, a macromolecular cryoprotectant, synergistically enhances ovine red blood cell cryopreservation in a mixed cryoprotectant system. Screening of DMSO and trehalose mixtures identified optimized conditions, where cytotoxicity was minimized but cryoprotective benefit maximized. Supplementation with polyampholyte allowed 97% post-thaw recovery (3% hemolysis), even under extremely challenging slow-freezing and -thawing conditions. Post-thaw washing of the cryoprotectants was tolerated by the cells, which is crucial for any application, and the optimized mixture could be applied directly to cells, causing no hemolysis after 1 h of exposure. The procedure was also scaled to use blood bags, showing utility on a scale relevant for application. Flow cytometry and adenosine triphosphate assays confirmed the integrity of the blood cells post-thaw. Microscopy confirmed intact red blood cells were recovered but with some shrinkage, suggesting that optimization of post-thaw washing could further improve this method. These results show that macromolecular cryoprotectants can provide synergistic benefit, alongside small molecule cryoprotectants, for the storage of essential cell types, as well as potential practical benefits in terms of processing/handling.

Polymer-Mediated Cryopreservation of Bacteriophages.

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL-1 (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both -20 and -80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at -20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG's low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy.

The transfer temperature from slow cooling to cryogenic storage is critical for optimal recovery of cryopreserved mammalian cells.

Cryopreservation is a key step for the effective delivery of many cell therapies and for the maintenance of biological materials for research. The preservation process must be carefully controlled to ensure maximum, post-thaw recovery using cooling rates slow enough to allow time for cells to cryodehydrate sufficiently to avoid lethal intracellular ice. This study focuses on determining the temperature necessary at the end of controlled slow cooling before transfer to cryogenic storage which ensures optimal recovery of the processed cell samples. Using nucleated, mammalian cell lines derived from liver (HepG2), ovary (CHO) and bone tissue (MG63) this study has shown that cooling must be controlled to -40°C before transfer to long term storage to ensure optimal cell recovery. No further advantage was seen by controlling cooling to lower temperatures. These results are consistent with collected differential scanning calorimetry data, that indicated the cells underwent an intracellular, colloidal glass transition between -49 and -59°C (Tg'i) in the presence of the cryoprotective agent dimethyl sulfoxide (DMSO). The glass forms at the point of maximum cryodehydration and no further cellular dehydration is possible. At this point the risk of lethal intracellular ice forming on transfer to ultra-low temperature storage is eliminated. In practice it may not be necessary to continue slow cooling to below this temperature as optimal recovery at -40°C indicates that the cells have become sufficiently dehydrated to avoid further, significant damage when transferred into ultra-low temperature storage.

Freezing Technology: Control of Freezing, Thawing, and Ice Nucleation.

From early dry-ice-based freezers and passive coolers, cryopreservation devices have come a long way. With increasing interest in the field of cryobiology from new scientific applications, the importance of reliable, traceable, and reproducible cold chain devices is sure to increase, ensuring more precise cryopreservation and enabling better post-thaw outcomes, both for the user and for biological samples. As with any cryopreservation process, it is important to optimize each part of the cold chain for each lab's biological samples, cryocontainers used, and logistical restraints. In this chapter we describe how freezing technology can be used for cryopreservation of cells.

Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies-A Review.

Cryopreservation is a key enabling technology in regenerative medicine that provides stable and secure extended cell storage for primary tissue isolates and constructs and prepared cell preparations. The essential detail of the process as it can be applied to cell-based therapies is set out in this review, covering tissue and cell isolation, cryoprotection, cooling and freezing, frozen storage and transport, thawing, and recovery. The aim is to provide clinical scientists with an overview of the benefits and difficulties associated with cryopreservation to assist them with problem resolution in their routine work, or to enable them to consider future involvement in cryopreservative procedures. It is also intended to facilitate networking between clinicians and cryo-researchers to review difficulties and problems to advance protocol optimization and innovative design.

Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation.

Human pluripotent stem cells can be differentiated into midbrain dopaminergic (mDA) neurons by directing cells through a floor plate progenitor stage. The developmental identity of mDA neurons produced using floor plate protocols is similar to substantia nigra neurons, and this has improved the ability to model Parkinson's disease (PD) in a dish. Combined with the unlimited growth potential of pluripotent stem cells, mDA neural progenitor cell production can provide a scalable source of human dopaminergic (DA) neurons for diverse applications. However, due to the complexity and length of the protocols and inherent differences between cell lines, considerable variability of the final population of neurons is often observed. One solution to this problem is to cryopreserve committed mDA neural progenitor cells in a ready-to-use format. Creating a bank of cryopreserved mDA neural progenitor cells poised for neuronal differentiation could significantly improve reproducibility and facilitate collaborations. Here we have compared six (6) different commercial cryopreservation media and different freezing conditions for mDA neural progenitor cells differentiated from human embryonic stem cell (hESC) lines. Significant differences in cell recovery were observed at 24 h post-thawing, but no differences were observed immediately upon thawing. The presence of ROCK inhibitors improved cell recovery at 24 h for all cryopreservation media tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Importantly, cryopreservation of mDA neural progenitor cells did not alter their potential to resume differentiation into mDA neurons. Banks of cryopreserved committed mDA neural progenitor cells provide a method to generate human DA neurons with reduced batch-to-batch variability, and establish a mechanism to share lineage-primed cells for collaborative research.

Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender.

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues.

Cryopreservation of primary cultures of mammalian somatic cells in 96-well plates benefits from control of ice nucleation.

Cryopreservation of mammalian cells has to date typically been conducted in cryovials, but there are applications where cryopreservation of primary cells in multiwell plates would be advantageous. However excessive supercooling in the small volumes of liquid in each well of the multiwell plates is inevitable without intervention and tends to result in high and variable cell mortality. Here, we describe a technique for cryopreservation of adhered primary bovine granulosa cells in 96-well plates by controlled rate freezing using controlled ice nucleation. Inducing ice nucleation at warm supercooled temperatures (less than 5 °C below the melting point) during cryopreservation using a manual seeding technique significantly improved post-thaw recovery from 29.6% (SD = 8.3%) where nucleation was left uncontrolled to 57.7% (9.3%) when averaged over 8 replicate cultures (p < 0.001). Detachment of thawed cells was qualitatively observed to be more prevalent in wells which did not have ice nucleation control which suggests cryopreserved cell monolayer detachment may be a consequence of deep supercooling. Using an infra-red thermography technique we showed that many aliquots of cryoprotectant solution in 96-well plates can supercool to temperatures below -20 °C when nucleation is not controlled, and also that the freezing temperatures observed are highly variable despite stringent attempts to remove contaminants acting as nucleation sites. We conclude that successful cryopreservation of cells in 96-well plates, or any small volume format, requires control of ice nucleation.

Stepped vitrification technique for human ovarian tissue cryopreservation.

The advantage of stepped vitrification (SV) is avoiding ice crystal nucleation, while decreasing the toxic effects of high cryoprotectant concentrations. We aimed to test this method for human ovarian tissue cryopreservation. Ovarian cortex was taken from 7 fertile adult women. Samples were subjected to an SV protocol performed in an automatic freezer, which allowed sample transfer to ever higher concentrations of dimethyl sulfoxide (DMSO) as the temperature was reduced. Histological evaluation of the vitrified-warmed tissue showed large numbers of degenerated follicles after 24 hours of in vitro culture. We therefore evaluated DMSO perfusion rates by X-ray computed tomography, ice crystal formation by freeze-substitution, and cell toxicity by transmission electron microscopy, seeking possible reasons why follicles degenerated. Although cryoprotectant perfusion was considered normal and no ice crystals were formed in the tissue, ultrastructural analysis detected typical signs of DMSO toxicity, such as mitochondria degeneration, alterations in chromatin condensation, cell vacuolization and extracellular matrix swelling in both stromal and follicular cells. The findings indicated that the method failed to preserve follicles due to the high concentrations of DMSO used. However, adaptations can be made to avoid toxicity to follicles caused by elevated levels of cryoprotectants.

Recovery and Post-Thaw Assessment of Human Umbilical Cord Blood Cryopreserved as Quality Control Segments and Bulk Samples.

Quality control (QC) segments conjoined to a bulk sample container are used to evaluate the viability and quality of cryopreserved umbilical cord blood (UCB). Such QC segments are typically attached lengths of sealed tubing that are cooled concurrently with the bulk sample, both containing material from the same donor. QC segments are thawed independently of the bulk sample to assess the quality of the cryopreserved product. In current practice, there is typically post-thaw variation between the QC segment and the bulk sample which if suggestive of inadequate performance, could lead to material being needlessly discarded. In this study, these performance differences were quantified. Two cooling protocols in common use, 1 with and 1 without a "plunge" step to induce ice nucleation, gave equivalent results that maintained the QC segment versus bulk sample differences. Ice nucleated at significantly lower temperatures in the QC segments compared with the bulk samples, a consequence of their lower volume, thereby enhancing damaging osmotic stress. A reduction in total viable cells of approximately 10% was recorded in the QC segments compared with comparable bulk samples. It has been shown that CD45+ cells are more adversely impacted by this lower ice nucleation temperature than CD34+ cells, which can result in altered composition of the post-thaw cell population.

Physical events occurring during the cryopreservation of immortalized human T cells.

Cryopreservation is key for delivery of cellular therapies, however the key physical and biological events during cryopreservation are poorly understood. This study explored the entire cooling range, from membrane phase transitions above 0°C to the extracellular glass transition at -123°C, including an endothermic event occurring at -47°C that we attributed to the glass transition of the intracellular compartment. An immortalised, human suspension cell line (Jurkat) was studied, using the cryoprotectant dimethyl sulfoxide. Fourier transform infrared spectroscopy was used to determine membrane phase transitions and differential scanning calorimetry to analyse glass transition events. Jurkat cells were exposed to controlled cooling followed by rapid, uncontrolled cooling to examine biological implications of the events, with post-thaw viable cell number and functionality assessed up to 72 h post-thaw. The intracellular glass transition observed at -47°C corresponded to a sharp discontinuity in biological recovery following rapid cooling. No other physical events were seen which could be related to post-thaw viability or performance significantly. Controlled cooling to at least -47°C during the cryopreservation of Jurkat cells, in the presence of dimethyl sulfoxide, will ensure an optimal post-thaw viability. Below -47°C, rapid cooling can be used. This provides an enhanced physical and biological understanding of the key events during cryopreservation and should accelerate the development of optimised cryobiological cooling protocols.

The Impact of Varying Cooling and Thawing Rates on the Quality of Cryopreserved Human Peripheral Blood T Cells.

For the clinical delivery of immunotherapies it is anticipated that cells will be cryopreserved and shipped to the patient where they will be thawed and administered. An established view in cellular cryopreservation is that following freezing, cells must be warmed rapidly (≤5 minutes) in order to maintain high viability. In this study we examine the interaction between the rate of cooling and rate of warming on the viability, and function of T cells formulated in a conventional DMSO based cryoprotectant and processed in conventional cryovials. The data obtained show that provided the cooling rate is -1 °C min-1 or slower, there is effectively no impact of warming rate on viable cell number within the range of warming rates examined (1.6 °C min-1 to 113 °C min-1). It is only following a rapid rate of cooling (-10 °C min-1) that a reduction in viable cell number is observed following slow rates of warming (1.6 °C min-1 and 6.2 °C min-1), but not rapid rates of warming (113 °C min-1 and 45 °C min-1). Cryomicroscopy studies revealed that this loss of viability is correlated with changes in the ice crystal structure during warming. At high cooling rates (-10 °C min-1) the ice structure appeared highly amorphous, and when subsequently thawed at slow rates (6.2 °C min-1 and below) ice recrystallization was observed during thaw suggesting mechanical disruption of the frozen cells. This data provides a fascinating insight into the crystal structure dependent behaviour during phase change of frozen cell therapies and its effect on live cell suspensions. Furthermore, it provides an operating envelope for the cryopreservation of T cells as an emerging industry defines formulation volumes and cryocontainers for immunotherapy products.