Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells

Abstract

Background A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at –196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required. Method As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at –196 °C for 7 days. Then, cell recovery was assayed. Results We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants. Conclusions PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system. Graphical Abstract

Injectable smart stimuli-responsive hydrogels: pioneering advancements in biomedical applications

“…5,169 As a result of its non-toxicity, low immunogenicity, good biocompatibility and biodegradation, PEG is one of the most widely used synthetic materials in the preparation of injectable hydrogels. 5,170 PEG is a hydrophilic polymer which has been approved by the Food and Drug Administration (FDA), and PEG-based injectable hydrogels demonstrate good performance as a drug/ bioactive molecule carrier. Meng et al prepared an injectable PEG hydrogel which was loaded with an anthracycline anticancer drug (epirubicin).…”

“…5,169 As a result of its non-toxicity, low immunogenicity, good biocompatibility and biodegradation, PEG is one of the most widely used synthetic materials in the preparation of injectable hydrogels. 5,170…”

Poly(l-alanine-co-l-threonine succinate) as a Biomimetic Cryoprotectant

“…Additives have extensively been developed as synthetic cryoprotectants that are expected to eliminate the concerns above related to the long-term low temperature storage of biologics including proteins and cells. − Cell recovery from cryopreservation is significantly enhanced by using cryoprotectants, such as carboxylated ε-poly­( l -Lys), graphene oxide, block copolymer/poly­(vinyl alcohol) (PVA) composite systems, oligo­( l -Pro), amphiphilic poly­( l -Ala) block copolymers, amphiphilic poly­( N , N -dimethyl acrylamide- co -alkyl acrylamide), and low molecular weight poly­(ethylene glycol)­s (PEGs). − The enhanced cell recovery with those cryoprotectants mainly results from ice recrystallization inhibition (IRI) and relief of the osmotic stress inside or outside of cells. Coacervates were prepared by liquid–liquid phase separation for the dodecyl trimethylammonium bromide/5-methyl salicylate, sodium dodecyl sulfate/1,2-diphenylethylenediamine, oleic acid/spermine, and polyethylene glycol/sodium citrate .…”

Thermosensitive Triblock Copolymer for Slow-Release Lubricants under Ocular Conditions

“…For example, aqueous polymer polyethylene glycol (PEG), a common chemical composition of an ocular lubricant additive with mild effect, is usually used to reduce shear stress and allow uniform spread of lubricants . Recently, PEGs with small molecular weight (300–400 kDa) have been widely applied in eye drop compositions, and they exhibit excellent intracellular transport properties . In addition, a polysorbate surfactant (Tween) also exhibits good dispersibility and emulsification, which effectively reduces the viscosity of lubrication system and enhances flowability.…”