To address this challenge, we recently developed a novel method that can eliminate the primary mechanisms of ice crystallization and thus, achieve stable storage of large-volume water and red blood cell suspensions at deep subzero temperatures (< ?10 C) without freezing [22]. In this study, we applied the deep-supercooling (DSC) approach to preserve human adipose-derived stem cells (hADSCs). tissue/organ transplantation (including blood transfusion) [10; 12], cell therapeutics [45; 46; 59], and tissue regeneration and repairing [25; 48]. Conventional long-term preservation (cryopreservation) is achieved by cooling biospecimens to deep subzero temperatures (e.g. ?196 C), storing them in a state of suspended animation, and then warming them back to normothermic temperature (e.g. 37 C) on demand as necessary. There are two methods for cryopreservation, slow-freezing and vitrification [18]. The former is to cool biospecimens at a low cooling rate (e.g. 1 C/min) to gradually dehydrate cells and minimize intracellular ice formation, but it can cause osmotic shock and extensive dehydration and deformation [18; 27; 31]. The latter is to cool the biospecimens at a high cooling rate without ML221 ice formation, but it requires a high concentration of cryoprotectant (CPA) and/or limits the sample volume within the order of 100 l [18; 19]. Both of these methods require cell membrane-permeable CPAs (e.g. dimethyl sulfoxide) to minimize cryoinjuries. The presence of cytotoxic CPAs not only requires rigorous removal before further applications via tedious washing and centrifugation [8; 23], but also causes spontaneous differentiations [5], intravascular hemolysis[33], and cell loss[43]. Thus, these traditional cryopreservation approaches, while critical for theoretically infinite storage time, have shown a series of inadequacies and bottlenecks which currently hinder some of Mouse monoclonal to CD69 their promises. Mesenchymal stem cells (MSCs) recently have attracted great interest for scientific research and clinical applications [51]. They are adult stem cells that can be found in many organs and tissues, such as bone marrow, adipose tissue, and amniotic fluid [52]. Due to their self-renewal capacity, multilineage differentiation ability, and extraordinary potential of paracrine secretions, MSCs are widely used as cell therapeutic agents for immunoregulation, antimicrobial medicine, tissue regeneration and repair [32; 44]. Adipose-derived stem cells (ADSCs) are MSCs derived from adipose tissues, which are abundant, accessible, and reliable sources of stem cells [4]. Their easy isolation procedure and high isolation yield make them a perfect candidate for cell-based therapies [35]. Therefore, an effective and efficient biopreservation method of ADSCs would have a significant impact on their widespread dissemination for research and clinical applications [24]. Hypothermic storage below normothermic temperature (37 C) is an alternative approach for short-term biopreservation. In this method, biospecimens are usually stored above freezing temperatures so that phase transition will not occur, cytotoxic CPA will not be required, and thus, cryoinjuries (such as osmotic shock, intracellular ML221 and extracellular ice formation, and freezing concentration) associated with cryopreservation can be avoided. It has been used to preserve various mammalian cell (e.g. primary human hepatocytes [13; 37], cardiomyocytes [47], multipotent stromal cells [41], and blood cells [26; 38; 54]) and cell-biomaterial constructs (e.g. two-dimensional (2D) cell monolayers [7], three-dimensional (3D) cell aggregates [7], and cell/tissue/organ-on-a-chip [14; 57]). Since there is ML221 no ice formation, hypothermic storage is preferred for preserving large-volume tissues and organs with complex and delicate structures (e.g. microcapillaries) that are highly susceptible to ice crystal formation [12]. Therefore, it was utilized to preserve livers [1; 15], kidneys [1; 56], and other organs [16] for transportation and transplantation. However, due to relatively high storage temperatures (usually above 0 C), ML221 biospecimens in hypothermic storage still undergo significant metabolic activities, and thus, they gradually decay and deteriorate as storage proceeds. Depending ML221 on physicochemical properties and characteristics of biospecimens, the storage time is usually short, varying from several hours (e.g. 4C6 hours for hearts and.