Intracellular Delivery of Trehalose for Cell Banking

Abstract

Advances in stem cell technology and regenerative medicine have underscored the need for effective banking of living cells. Cryopreservation, using very low temperatures to achieve suspended animation, is widely used to store or bank cells for later use. This process requires the use of cryoprotective agents (CPAs) to protect cells against damage caused by the cooling and warming process. However, current popular CPAs like DMSO can be toxic to cells and must be thoroughly removed from cells before they can be used for research or clinical applications. Trehalose, a nontoxic sugar found in organisms capable of withstanding extreme cold or desiccation, has been explored as an alternative CPA. The disaccharide must be present on both sides of the cellular membrane to provide cryo-protection. However, trehalose is not synthesized by mammalian cells nor has the capability to diffuse through their plasma membranes. Therefore, it is crucial to achieve intracellular delivery of trehalose for utilizing the full potential of the sugar for cell banking. In this review, various methods that have been explored to deliver trehalose into mammalian cells for their banking at both cryogenic and ambient temperatures are surveyed. Among them, the nanoparticle-mediated approach is particularly exciting. Collectively, studies in the literature demonstrate the great potential of using trehalose as the sole CPA for cell banking, to facilitate the widespread use of living cells in modern medicine.

Nanoparticle‐Mediated Intracellular Protection of Natural Killer Cells Avoids Cryoinjury and Retains Potent Antitumor Functions

“…Images indicate that the internalized FITC nanoparticle could be localized to the cytoplasm of the cells, but not the nucleus. For its cryoprotective activity, trehalose does not have to be confined to a specific subcellular location, [ 53 ] confirming that the observed uptake results of FITC‐nano by NK cells are indicative of potential biological relevance. Nuclear staining of NK cells revealed that the nanoparticles were localized to the cytoplasm of the cell (Figure S3, Supporting Information).…”

“…Encapsulating trehalose inside CS:TPP nanoparticles enables it to be shuttled across the NK cell membrane, allowing for its intracellular presence where it can induce cryoprotection. [ 53 ] To identify the maximum amount of trehalose that can be encapsulated inside the CS:TPP nanoparticles, various concentrations of trehalose ranging from 1 to 20 mg mL −1 were directly dissolved in the TPP solution. Using a CS:TPP ratio of 3:1 and corresponding pH of 5.0 for chitosan and 9.5 for TPP, different concentrations of trehalose/TPP solution were added into the chitosan solution drop wise to assemble the encapsulated nanoparticle (trehalose‐loaded nanoparticle, nTre).…”

“…[ 66 ] Through a meticulous physicochemical optimization process, we identified conditions which enabled the assembly of nanocarriers that can efficiently deliver CPAs inside NK cells. [ 40,53 ] Using this platform, we were able to reduce the need for cryoprotectants to only trehalose, though using other CPAs such as those described in our previous studies is an obvious opportunity. This represents the first example, to our knowledge, of the use of nanoparticles to achieve any kind of intracellular delivery to NK cells and modulate their cellular responses.…”

Advanced Biotechnology for Cell Cryopreservation

“…In recent years, several studies have reported efficient delivery of trehalose into cells using nanomaterials. The nanoparticles generally utilize the natural process of endocytosis to specifically deliver trehalose into cells without any harmful effects [29]. The use of nanoparticles for trehalose delivery and cryopreservation of cells with trehalose as sole CPA has been reported to maintain cell viability and functions.…”

“…In order to avoid the toxicity of organic solvents, several advanced biotechnology tools have been used to deliver trehalose into cells to provide both intracellular and extracellular cryopreservation. These techniques help to achieve organic solvent-free cell cryopreservation and high post-thaw cell survival efficiency [29]. These biotechnology tools, including both physical and chemical methods, can increase cellular membrane permeability and thus transport non-permeable trehalose into cells (Table 1).…”