Supplementary MaterialsSupplementary Information srep34393-s1. and viabilities for HUVEC cryopreservation studies reported in the literature. Furthermore, HUVECs AZD8055 inhibitor cryopreserved using our improved procedure showed high tube forming capability in a post-thaw angiogenesis assay, a standard indicator of endothelial cell function. As well as presenting superior cryopreservation procedures for HUVECs, the methods developed here can serve as a model to optimize the cryopreservation of other cells. Human AZD8055 inhibitor umbilical vein endothelial cells (HUVECs) have become a model system for vascular biology research since their successful culture in 19731. HUVECs are used to study physiology and pathophysiology of vascular disorders2, biomaterials in tissue engineering3,4 and drug delivery systems5,6. Investigations and applications include: vasoregulation7, coagulation8, fibrinolysis9, atherosclerosis10, vasculogenesis and angiogenesis11 and as a healthy counterpart to dysfunctional endothelial cells12. Their availability has been facilitated through routine cryopreservation procedures13,14,15 that were originally designed for corneal cells16,17. Despite substantial research on HUVECs, the key variables in their cryopreservation have not been optimized. Cell response to freeze-thaw stress is an important first step to investigate cryopreservation of cells, and the plasma membrane is of particular interest18. Ice excludes solutes to the unfrozen fraction19, thus increasing solute concentration and creating osmotic imbalance. The cells restore equilibrium either by undergoing intracellular ice formation or by becoming sufficiently dehydrated20. The mechanism by which intracellular ice formation occurs has been linked directly Rabbit Polyclonal to NMDAR2B (phospho-Tyr1336) to membrane damage, with the proposition that intracellular ice is a result rather than a cause of damage21. On the other hand, cells can AZD8055 inhibitor only lose water to a certain extent before it becomes lethal22. Mazur developed the two-factor hypothesis of freezing injury to explain observations of optimal cooling rates23. Cooling cells slower than the optimal rate in the presence of ice results in cell death by excessive dehydration and solute toxicity24,25 while cooling cells faster than the optimal rate results in cell death by intracellular ice formation21. Many types of cells which are rapidly cooled can be saved from freezing injury by rapid thawing26. Cryoprotectants also mitigate slow cooling damage and enable survival of cells at lower cooling rates. Cryoprotectants can be classified based on their ability to permeate cell membranes27. Permeating cryoprotectants pass through cell membranes, protecting cells by increasing intracellular and extracellular osmolality28,29, depressing the freezing temperature thereby reducing the amount of ice formed29,30,31, and reducing the extent of cell shrinkage28. Dimethyl sulfoxide (DMSO) is a water-soluble permeating cryoprotectant and was first demonstrated for human and bovine red blood cells and bull spermatozoa32,33,34. Non-permeating cryoprotectants, which are incapable of diffusing through intact cell membranes, protect cells by increasing extracellular osmolality, causing cells to dehydrate and reducing the likelihood of intracellular ice formation and the amount of ice formed35,36,37. Hydroxyethyl starch (HES) was first demonstrated as a non-permeating cryoprotectant for erythrocytes38, and a low molecular weight HES (Pentastarch) has been used as a plasma volume expander39. The use of HES in clinical settings makes it an ideal cryoprotectant for human health therapeutics. A combination of DMSO and HES has been used to cryopreserve many cells, including: the multiple steps that take place during angiogenesis. These include: disruption of the basement membrane, migration of AZD8055 inhibitor endothelial cells, and the proliferation and differentiation into capillaries, via adhesion molecule signaling and extracellular matrix remodeling, which can be observed as three-dimensional capillary-like tubular structures by microscopy72,73. The primary objective of this work was to study cryoinjury to HUVECs by applying interrupted cooling protocols which can identify key variables for optimizing HUVEC cryopreservation. Figure 2 is a schematic diagram of the experimental design to systematically investigate the effects of: two-step freezing in the absence or presence of 10% DMSO. Next, the effect of two cooling rates (0.2?C/min or 1?C/min) on graded freezing was examined. Then, graded freezing using a 1?C/min cooling.