There was evidence of interactions between sucrose and isoleucine (p?=?0.012) and sucrose and glycerol (p?=?0.014). formation was observed more often in the presence of single osmolytes Sulfo-NHS-Biotin as well as non-optimized multi-component solution compositions. Introduction Over the past several years, immunotherapy has emerged and been called the fourth pillar of cancer treatment. Chimeric antigen receptor (CAR) T-cell therapy is usually a rapidly growing therapy for the treatment of cancer1. The U.S. Food and Drug Administration (FDA) approved two CAR T-cell therapies in 2017, Kymriah developed by Novartis for the DPP4 treatment of children with acute lymphoblastic leukemia and Yescarta developed by Kite for adults with advanced lymphomas. Further progress with the use of immunotherapies for the treatment of cancer as well as other diseases is also anticipated. Dimethyl sulfoxide (DMSO) has been the standard cryopreservation agent for freezing cells since the 1960?s2. However, DMSO is usually toxic upon infusion to patients and can lead to side effects from moderate (such as nausea and vomiting) to severe (such as cardiovascular) or even cause death3. When exposed to DMSO, cells drop viability and function with time of exposure4. For hematopoietic cells, exposure to DMSO is typically limited to 30 min5. Sulfo-NHS-Biotin This practice adds to the complexity of the workflow associated with preservation of cells using DMSO. There is a demand for DMSO-free cryoprotectants that maintain cell viability and function after thaw. Diverse biological systems (plants, insects, etc.) survive high salt environments, dehydration, drought, freezing temperatures and other Sulfo-NHS-Biotin stresses through the use of osmolytes6. In the human kidney, a mixture of five osmolytes are used to stabilize the cells7. Recently we developed a method of preserving cells with combinations of osmolytes8C10. These studies exhibited that a combination of three different osmolytes including sugar, sugar alcohol and amino acids/proteins could stabilize Jurkat cells and mesenchymal stromal cells (MSCs) during freezing. Each of the components plays a role in stabilization of the cell during freezing. Sugars are associated with stabilization of the cell membrane11 and conversation via hydrogen bonding with water12, thereby changing solidification patterns. Glycerol also interacts strongly with water13 via hydrogen bonds, penetrates the cell membrane14 and is associated with stabilization of proteins15. Amino acids help stabilize Sulfo-NHS-Biotin sugars during freezing so that they do not precipitate out of solution16. It is noteworthy that higher levels of osmolytes did not necessarily correspond to higher post-thaw viability17. The osmolytes appeared to act in concert to improve post-thaw recovery. The objective of this investigation is usually to understand in more detail the relationships amongst the osmolytes present in these solutions and Jurkat cell recovery. Raman spectroscopy has been widely used in characterizing subcellular structures such as mitochondrion, lysosome and nucleus because it is usually label-free and has?high spatial resolution18. Moreover, Raman spectroscopy can identify the phase of water (liquid or solid) and the location of cryoprotective brokers. For this study, low temperature Raman spectroscopy was used to interrogate freezing responses of cells cryopreserved in different combinations of osmolytes. This tool enables us to quantify intracellular ice formation (IIF), distribution of cryoprotective brokers, damage to subcellular compartments and other cell behaviors during freezing17,19. In a previous study, we exhibited that osmolytes act in concert to improve cell viability17. A recent study exhibited that combinations of osmolytes had a strong effect on crystallization of water and form natural deep eutectic systems (NADES)20. The next phase of the investigation will involve characterizing the role of a given osmolyte and its interactions with other osmolytes on post-thaw recovery using a statistical model. This type of analysis will provide the foundation for a molecular model of protection and osmolyte interaction. This knowledge is critical for the development of improved cryopreservation protocols, in particular, for high value cells such as cell therapies. Materials and Methods Cell culture Jurkat cells (ATCC TIB-152), a T-cell line, whose identity was confirmed by Short Tandem Repeat (STR) profiling were used in this investigation. Jurkat cells are a model cell line for T-cells and have also been used the production of IL-2 and studies of T-cell receptor signaling18. The cells were cultured in high-glucose RPMI 1640 (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; Qualified, Life Technologies, Carlsbad, CA, USA). Cultures were maintained at densities ranging between 1??105 and 3??106 cells/mL. Cells for Raman spectroscopy were prepared by washing and centrifuging cells twice in Dulbeccos Phosphate Buffered Saline at 125??g for 10?min. Cells were then.