Probing mechanisms of cryopreservation damage to natural killer cells.

Background Aims: Natural killer (NK) cells show significant potential in targeting hard to treat cancers, but these cells need effective preservation methods to maintain viability and efficacy after cryopreservation. Traditional methods of preserving NK cells result in low post-thaw recovery and function. Dimethyl sulfoxide (DMSO) is a very common cryoprotectant for preserving NK cells, but its infusion into patients post-thaw can cause dose-dependent adverse effects, including nausea, discomfort and cardiac arrest.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival.

There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives.