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

Abstract

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.

High glucose exposure promotes proliferation and in vivo network formation of adipose-tissue-derived microvascular fragments

“…The isolated ad-MVF were cultivated for 24 h under humidified conditions in 1 % agarose-coated 24-well plates containing 4 °C University of Wisconsin (UW) solution (Belzer UW Cold Storage Solution, Bridge to Life Ltd., Columbia, SC, USA). These culture conditions have previously been proven to be suitable for the xeno-free cultivation of ad-MVF, as it would be necessary in future clinical practice (Laschke et al, 2019b). The UW solution was supplemented with 30 mM glucose (high glucose; Carl Roth, Karlsruhe, Germany) or vehicle (control; PBS).…”

“…1c). A detailed description of the scaffold preparation and seeding procedure is given in Laschke et al (2019b). MW Laschke et al ad-MVF and high glucose exposure Morphology of ad-MVF The morphology of vehicle-and high-glucose-exposed ad-MVF seeded on to collagen-glycosaminoglycan scaffolds was analysed by means of a FEI XL-30 ESEM FEG scanning electron microscopy device (FEI, Hillsboro, OR, USA).…”

“…Vehicle-and high-glucose-exposed ad-MVF from GFP + donor mice were seeded on to collagenglycosaminoglycan scaffolds, which were implanted into dorsal skinfold chambers of GFP − recipient mice ( Fig. 1d,e) and analysed by means of intravital fluorescence microscopy, as described previously in detail (Laschke et al, 2019a). For contrast enhancement by intravascular staining of plasma, 100 µL 5 % FITC-labelled dextran 150,000 (Sigma-Aldrich) were injected before each microscopy analysis into the retrobulbary plexus of the anaesthetised mice.…”

“…Moreover, www.ecmjournal.org they contain mesenchymal stem cells within their physiological niche, which exhibit a high capacity for angiogenic, neurogenic, adipogenic and osteogenic differentiation (McDaniel et al, 2014). In addition, ad-MVFs can be pre-cultivated for up to 24 h prior to their in vivo application without losing their physiological vessel morphology (Laschke et al, 2019a). This offers the possibility of further improving their angiogenic activity by exposing them to different physical and biochemical stimuli, such as subnormothermic temperatures and pro-angiogenic factors Laschke et al, 2019a;Laschke et al, 2019b).…”

“…In addition, ad-MVFs can be pre-cultivated for up to 24 h prior to their in vivo application without losing their physiological vessel morphology (Laschke et al, 2019a). This offers the possibility of further improving their angiogenic activity by exposing them to different physical and biochemical stimuli, such as subnormothermic temperatures and pro-angiogenic factors Laschke et al, 2019a;Laschke et al, 2019b).…”