Effects of cryopreservation on adipose tissue-derived microvascular fragments

Adipose tissue-derived microvascular fragments (ad-MVF) are effective vascularization units for tissue engineering. They rapidly reassemble into new microvascular networks after seeding on scaffolds and subsequent in vivo implantation. Herein, we analyzed whether the vascularization capacity of ad-MVF is affected by cryopreservation. Ad-MVF were isolated from the epididymal fat pads of C57BL/6 mice and cryopreserved for 7 days to compare their morphology, viability, cellular composition, and protein expression with freshly isolated control ad-MVF. Moreover, cryopreserved and control ad-MVF from green fluorescent protein (GFP)(+) donor mice were seeded on collagen-glycosaminoglycan matrices (Integra((R))), which were implanted into dorsal skinfold chambers of GFP(-) recipient animals to study their vascularization and incorporation using intravital fluorescence microscopy, histology, and immunohistochemistry. Cryopreservation of ad-MVF did not affect vessel morphology and cellular composition. However, cryopreservation was associated with an increased rate of necrotic cells and a significantly reduced number of transplantable ad-MVF. This was compensated by a higher angiogenic activity of the remaining ad-MVF, as indicated by significantly elevated expression levels of pro-angiogenic factors when compared to controls. Accordingly, cryopreserved and control ad-MVF induced a comparable vascularization and incorporation of implanted Integra((R)) without differences in microvascular network formation, maturation, and remodeling. Enhanced angiogenic sprouting even resulted in a higher fraction of GFP(+) microvessels within the implants of the cryopreservation group. These findings indicate that cryopreservation of ad-MVF is feasible and, thus, offers the exciting opportunity to build up stocks of readily available vascularization units for future tissue engineering applications.