Strategies in developing dimethyl sulfoxide (DMSO)-free cryopreservation protocols for biotherapeutics

Cryopreservation is a technology employed in long-term storage of biologics achieved by cooling to cryogenic temperatures (1,2). This preservation technique has become increasingly popular and relevant especially in the development and commercialization of cellular therapeutic products (3,4). Conventional cryopreservation protocols involve the application of the permeating cryoprotectant; dimethyl sulfoxide (DMSO) due its ability to restrict ice nucleation and promote post-thaw viability (4). Although DMSO application effectively preserves biologics, can impair functional recovery (5)(6)(7) and induce a variety of mild to severe toxic effects in patients which must be avoided at all cost when administering immunotherapeutic products (8,9).The situation is considerably more critical during vitrification; a freezing method that has attracted heightened recognition as a faster and economic substitute to slow freezing as the unorganized liquid state of water is rapidly transformed to a glassy solid state without ice crystallization (10). In vitrification, high cooling rates and high concentrations (4-8 M) of cryoprotectants are usually required (11); enforcing on the exigency of using non-toxic cryoprotectants because increasing DMSO concentration is not advisable.Efficacious cryopreservation and biobanking requires the development of safe and consistent storage protocols (12,13). Favorably, such procedures should be devoid of xenogeneic or toxic components and DMSO-Free Cryopreservation of Biotherapeutics 2 to this effect, many scientists have put forward for the replacement of DMSO. Several groups have discovered/ developed safer alternative cryoprotectants with a range of potential cryoprotection mechanisms like ice recrystallization inhibition, osmolality control, cell membrane stabilization and vitrification (14). Numerous methods have since emerged to lessen the quantity of DMSO used (mostly by supplementation with other cryoprotectants) (15)(16)(17)(18)(19) or to completely eliminate DMSO application and these methods may also require special adjunct treatments, reagents or freezing protocols as would be discussed in specifics in this article.But for the review by Weng et al. (20), studies on replacing DMSO are yet to be critically analyzed; a process that would track research accomplishments, expose novel supplementary techniques applied and encourage further research aimed at improving DMSO-free cryopreservation protocols for different biologicals. Through this article, we wish to draw the attention of researchers to possibility of a DMSOfree preservation era which is achievable in the nearest future. We provide prove of total exclusion of DMSO from cryopreservation solutions and summarize some of the supplementary techniques that have been applied to improve post-thaw viability and function. Our survey has also revealed the commercial availability of DMSO-free cryoprotectant solutions especially those used for cellular therapeutics, but there are limited studies to scrutinize or validate the potency of these products; which might be why most researchers are yet to largely patronizing products without DMSO as there is little to no evidence to back up these product claims. We therefore urge researchers to extend the application of these products to a wider range of biotherapeutics so as to speed up the availability of clinically approved products especially immunotherapeutics which is the answer to many complicated diseases like cancers.Cryopreservation is an important determinant of the stability and activity of biopharmaceutical formulations. Several studies have proven the hypothesis that the application of DMSO can induce temperature-, time-, and concentration-dependent toxicities (7,21). DMSO causes mitochondrial damage to astrocytes (22), and impacts negatively on cellular membrane/cytoskeleton structure and integrity by interacting with proteins and dehydrating lipids (23) as evident in the increased membrane permeability of erythrocytes (24) and altered chromatin conformation in fibroblasts (21). Also, the presence of DMSO in culture medium can induce unwanted stem cell differentiation (25).DMSO-Free Cryopreservation of Biotherapeutics 3 Furthermore, repeated DMSO use even at sub-toxic levels can affect cellular epigenetic profile resulting in undesirable phenotypic disturbances (26). For instance, DMSO interferes with DNA methyltransferases and histone modification enzymes of human pluripotent stem cells causing epigenetic variations and reduction in their pluripotency (27,28). Similarly, murine embryonic stem cells display disrupted mRNA expression levels of endodermal markers (Foxa2 and Sox17), mesodermal marker (Hand1) and ectodermal marker (β-tubulin3) following DMSO treatment (29).Adverse reactions from cardiac, neurological, and gastrointestinal systems have been reported in patients receiving DMSO-containing cellular products (30, 31). These discoveries have led to the design of several washing procedures as it has become pertinent to ensure complete DMSO removal. However, the washing protocol usually involves agitation and osmotic/mechanical stresses which are to be avoided due to the fragile and sensitive nature of biologics post-thaw (31). The washing step can also be time consuming, expensive and resource wasting as a significant number of cells are loss in the process.Potent and safe alternative cryoprotectants to DMSO are highly desirable in order to meet the demands in the development and manufacturing of cellular and genetic therapies. In numerous instances, the observed cryoprotective effect is derived from a combination of two or more strategies as discussed below. These strategies and their outcome are also summarized in Table 1.Replacing DMSO with other cryoprotectants is the typical and go-to approach in eradicating the use of DMSO in cryopreservation. Using a mixture of 40% v/v ethylene glycol (EG) and 0.6 M sucrose, Kuleshova et al. vitrified neural stem cells. The vitrification solution was removed at room temperature and post-storage evaluations revealed no substantial differences between fresh and vitrified cells in cell markers expression, proliferation or multipotent differentiation (32).Osmolyte-based freezing solutions containing varying blends of sucrose, glycerol, creatine, isoleucine and mannitol have supported the recovery and survival of mesenchymal stromal cells when compared to conventional preservation with DMSO. These solutions conferred cryoprotection, retained cell DMSO-Free Cryopreservation of Biotherapeutics 4 differentiation capacity and modulated the cytosine-phosphate-guanine epigenome (33). StemCell Keep™ has been proven effective for the cryopreservation of human induced pluripotent stem cells (hiPSCs) (34), human embryonic stem cells (hESCs) (35) and mesenchymal stem cells (MSCs) (36). Further investigation on the mechanism of cryoprotection showed that the polyampholyte is adsorbed on to the cell membrane suggesting that it can confer protection on cell surface and eliminate the use of proteins and DMSO (36).Other polyampholytes have also shown great potential as DMSO substitutes in storage of murine L929 cells and rat MSCs (37), natural killer cells (38) and other mammalian cell types (39).A cocktail of non-toxic and Food and Drug Administration (US-FDA)-approved infusible substances including sucrose, glycerol, isoleucine, human serum albumin, and poloxamer 188, have been applied for preservation of hiPSCs (40). In a recent study by Park et al., a block copolymer; PEG−PA (5000−500) has been presented as an excellent cryoprotectant where the recovered stem cells exhibited acceptable survival, proliferation and multilineage differentiation post-thaw (41).These studies portray the synergistic activity of several biocompatible cryoprotectants and suggests that this approach may be an innovative paradigm for safe preservation of biopharmaceuticals. Presently, there are some commercially available DMSO-free cryosolutions like Pentaisomaltose™, CryoScarless™, CryoNovo P24™, CryoProtectPureSTEM™, StemCell Keep™, CryoSOfree™ and XT-Thrive™, OptiFRZ™, PRIME-XV Stem FreezIS™, Bambanker DMSO Free™, FREEZEstem™, ReproCryo DMSO free™ and pZerve™, but their quality and capacity to protect a wide range of therapeutics is underinvestigated. Further research would involve testing the compatibility of these cryoprotectants with other biotherapeutics. In addition to substituting cryoprotectants, supporting techniques used to improve on solvent-free cryopreservation are discussed below.The pretreatment of biotherapeutics with cryoprotective and stabilizing agents prior to cryopreservation is largely becoming a viable approach to ensuring safe storage. Sugar pretreatment (during 24 h of culture), supplementation of expansion medium with 10% platelet lysate and slow freezing is reportedly an effective protocol in DMSO-less cryopreservation of adipose-derived stromal cells (42). Similarly, sugar pretreatment increased survival, metabolic activity, attachment, proliferation and multilineage differentiation after recultivation of dermal MSCs (43). Improved results are also obtained when the sugars are positioned intracellularly as performed by Mutsenko and coworkers who explored electroporation-DMSO-Free Cryopreservation of Biotherapeutics 5 aided delivery of cryoprotective sugars in human umbilical cord MSCs (44). Also, pre-incubation of MSCs with osmolyte-based freezing solutions could foster effective cryopreservation (33). These results corroborate the potential advantages of pre-cryopreservation treatment(s).Programmed freezing offers improved control over ice nucleation parameters. A technique involving a magnetic field driven freezer termed "Cells Alive System" has been used to prevent formation of intracellular ice for up to three months. The magnetic field vibration function prohibits water molecules from creating clusters during freezing. Although the optimal conditions needed for survival and viability of isolated human periodontal ligament cells (PDL), pulp tissue and tooth using CAS freezers were determined previously (45) ( 46) with DMSO, the technique proved equally effective without DMSO, promoting greater survival rates over that obtained with conventional freezers (47) Due to the low thermal conductivity of biological samples, the conventional approach of rewarming largevolume cryopreserved samples in a water bath heated at 37 °C is associated with non-uniform distribution of temperature, which can induce thermal stress (50). Preferably, a high heating rate is desirable during thawing, because devitrification and recrystallization may occur if the temperature cannot be elevated rapidly above the sample's melting point (51). Therefore, both the heating rate and uniformity of heating during rewarming are important to cryopreservation especially vitrification. Nevertheless, attaining the optimal rewarming rate remains a major factor complicating effective vitrification. Another nanotechnology assisted thawing approach involves the utilization of soft liquid metal nanoparticles possessing reproducible photothermal stability, high photothermal conversion efficiency, low cytotoxicity and the ability to suppress ice formation. This technique promotes less ice nucleation during freezing and ultrarapid rewarming while thawing. Human bone marrow stromal cells have been successfully rewarmed with this technique (53).These studies reveal that the advantages of nanotechnology can be capitalized on to promote safe rewarming post-cryopreservation after verifying the biocompatibility of the nanoparticles.A critical step prior to the clinical application of biotherapeutics is the optimization of cryopreservation protocols that minimize post-thaw alterations in the stability and potency of preserved materials. Aqueous solutions containing 10% of DMSO is a widely used cryopreservative but there is an increasing amount of evidence showing inconsistent results on its impact on post-cryopreservation performance of biologicals. This drawback forms the basis for the development of safer preservation protocols. DMSOfree strategies have the potential to alleviate the aforementioned obstacles as demonstrated by studies discussed in this article. The application of other cryoprotectants, combined with other techniques like programmed freezing, pretreatments and modified thawing protocols have shown good prospects.In conclusion, the development of effective DMSO-free cryopreservation techniques that will provide high post-thaw viability and preserve original morphology and functioning remains key because this is essential to hastening the industrialization and clinical application of biotherapeutics. We are of the opinion that more research efforts should be put into the development and performance validation of trademarked DMSO-free products. In cases where DMSO elimination is unavoidable, only confirmed safe DMSO-Free Cryopreservation of Biotherapeutics 7 concentrations should be applied preferably in combination with non-toxic cryoprotectants and other potent strategies. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.