Lipoaspirate Storage Time and Temperature: Effects on Stromal Vascular Fraction Quality and Cell Composition.

The adipose tissue-derived stromal vascular fraction (SVF) is a promising candidate for use in cell therapy and tissue engineering due to its regenerative and immunomodulatory properties. Some therapies are based on using the complete SVF product, whereas others depend on the expansion of adipose-derived stromal cells (ASCs) in culture. The latter application often involves a time delay between adipose tissue harvest and SVF isolation. This study investigated how storage time and temperature affected cell quality and composition. Aliquots of lipoaspirate were stored cold (4°C), at room temperature (18-20°C), or at 37°C. SVF was isolated on sequential time points over a period of 48 h, and the following were assessed: cell viability, vitality, composition, and the proliferative potential of the ASCs. When the lipoaspirate was stored cold, the viability of the SVF remained stable for up to 48 h; however, the vitality of the SVF decreased significantly after 24 h. When stored at higher temperatures (room temperature or 37°C), the vitality of the SVF decreased after 8 h. The ASC fraction in the SVF decreased rapidly after 8 h when stored at higher temperatures, whereas this change was delayed significantly when the lipoaspirate was stored cold. Tendencies towards increases in the lag phase, population doubling time (PDt), and time to reach confluency were observed when the lipoaspirate was stored at higher temperatures. The vitality of the SVF was correlated significantly with the time of the lag phase and the time required to reach confluence, whereas no correlation was observed with the PDt. Both prolonged storage time and increased temperature during lipoaspirate storage negatively affected the quality of the obtained SVF. Our results suggest that lipoaspirate should be stored for no longer than 24 h at 4°C to maintain the optimal quality for the isolation of SVF and the expansion of ASCs.

Cryopreservation of adipose-derived stromal/stem cells using 1-2% Me2SO (DMSO) in combination with pentaisomaltose: An effective and less toxic alternative to comparable freezing media.

Mesenchymal stromal/stem cells (MSCs) derived from bone marrow, umbilical cord and especially adipose tissue are increasingly being explored for their therapeutic potential to treat a wide variety of diseases. A prerequisite for most allogeneic off-the-shelf and some autologous MSC therapies is the ability to safely and efficiently cryopreserve cells during production or for storage prior to treatment. Dimethyl sulfoxide (Me2SO) is still the commonly used gold standard cryoprotectant (CPA). However, undesirable cellular impacts and side effects of Me2SO have led to an increasing demand for the development of safe and effective alternatives. This study investigated the effect of pentaisomaltose as a CPA for cryopreservation of adipose-derived stromal/stem cells (ASCs). We compared pentaisomaltose-based freezing media containing 1% Me2SO (PIM1) or 2% Me2SO (PIM2) to our in-house freezing media formulation containing 10% Me2SO (STD10) and to CryoStor freezing media containing 2% or 10% Me2SO (CS2 and CS10). We assessed the recovery of viable ASCs, their phenotype, differentiation potential, proliferation potential, and migratory potential. Further, their immunomodulatory potential was assessed by measuring their ability to suppress T cell proliferation and express immunomodulatory markers. The results showed that the post-thaw viability of ASCs cryopreserved with STD10, CS10 and PIM2 was improved compared to that of CS2. The recovery of ASCs with PIM1 and PIM2 was also improved compared to that of CS2. Proliferation and migration were comparable among the tested freezing media. The results showed no difference in the induction of PDL1, PDL2 or IDO1 expression. Nevertheless, the potential of cryopreserved ASCs to suppress T cell proliferation was reduced when the Me2SO concentration was reduced (CS10>STD10>CS2 and PIM2>PIM1). Altogether, the migratory and immunomodulatory potential combined with improved recovery indicate that the addition of pentaisomaltose in the freezing media may allow for the reduction of the Me2SO concentration to 2% while retaining a more potent cell product that what is recovered using comparable freezing media. With the desire to reduce the amount of Me2SO, these results suggest that 2% and potentially even 1% Me2SO in combination with 10% pentaisomaltose could be an effective and less toxic alternative to comparable freezing media.

MAIT cell heterogeneity across paired human tissues reveals specialization of distinct regulatory and enhanced effector profiles.

Mucosal-associated invariant T (MAIT) cells are unconventional T cells that recognize microbial riboflavin pathway metabolites presented by evolutionarily conserved MR1 molecules. We explored the human MAIT cell compartment across organ donor-matched blood, barrier, and lymphoid tissues. MAIT cell population size was donor dependent with distinct tissue compartmentalization patterns and adaptations: Intestinal CD103+ resident MAIT cells presented an immunoregulatory CD39highCD27low profile, whereas MAIT cells expressing NCAM1/CD56 dominated in the liver and exhibited enhanced effector capacity with elevated response magnitude and polyfunctionality. Both intestinal CD39high and hepatic CD56+ adaptations accumulated with donor age. CD56+ MAIT cells displayed limited T cell receptor-repertoire breadth, elevated MR1 binding, and a transcriptional profile skewed toward innate activation pathways. Furthermore, CD56 was dynamically up-regulated to a persistent steady-state equilibrium after exposure to antigen or IL-7. In summary, we demonstrate functional heterogeneity and tissue site adaptation in resident MAIT cells across human barrier tissues with distinct regulatory and effector signatures.