Subnormothermic short-term cultivation improves the vascularization capacity of adipose tissue-derived microvascular fragments

Adipose tissue-derived microvascular fragments (ad-MVFs) are promising vascularization units for tissue engineering. In this study, we analysed the effects of normothermic (37°C) and subnormothermic (20°C) short-term cultivation on their viability and network forming capacity. Ad-MVFs from green fluorescent protein (GFP)+ and GFP- C57BL/6 mice were cultivated for 24 hr at 37°C or 20°C. Freshly isolated, noncultivated ad-MVFs served as controls. Number, length, viability, proliferation, and angiogenic activity of the ad-MVFs were assessed by microscopic analysis and proteome profiling. GFP+ ad-MVFs were seeded onto collagen-glycosaminoglycan matrices, which were implanted into dorsal skinfold chambers of GFP- mice to analyse their vascularization by means of intravital fluorescence microscopy, histology, and immunohistochemistry. Depending on the temperature, short-term cultivation of ad-MVFs markedly changed their expression of multiple proangiogenic and antiangiogenic factors. Moreover, cultivation at 37°C significantly increased the number of apoptotic cells within ad-MVFs, whereas 20°C preserved the viability of ad-MVFs and even promoted the proliferation of endothelial and perivascular cells. Accordingly, ad-MVFs cultivated at 20°C also exhibited an enhanced in vivo vascularization capacity when compared with normothermically cultivated ad-MVFs and noncultivated controls. This was indicated by an accelerated network formation, an increased microvascular remodelling, and a higher density of GFP+ microvessels within implanted matrices. Thus, if ad-MVFs require short-term storage before in vivo application, subnormothermic cultivation should be preferred to normothermic cultivation.