Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

Abstract

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

Seeding density is a crucial determinant for the in vivo vascularisation capacity of adipose tissue-derived microvascular fragments

“…Ad-MVF were isolated from epididymal fat pads of GFP + donor mice, as previously described in detail (Frueh et al, 2017b). Briefly, the bilateral epididymal fat pads were transferred into 10 % Dulbecco's modified eagle medium (DMEM; 100 U/mL penicillin, 0.1 mg/mL streptomycin; Biochrom, Berlin, Germany) and washed thrice with phosphate-buffered saline (PBS).…”

“…1a,b). The total number of ad-MVF was subsequently extrapolated to the whole isolate (Frueh et al, 2017b). Furthermore, the average ad-MVF length was measured by means of microscopic analysis.…”

“…Of interest, ad-MVF can be isolated in large amounts from fat tissue within a short time and represent fully functional vessel segments (Frueh et al, 2017b). Hence, they bear the major advantage that their reassembly into new microvascular networks induces a rapid implant vascularisation and blood perfusion (Laschke et al, 2012;Laschke and Menger, 2015).…”

Enoxaparin does not affect network formation of adipose tissue–derived microvascular fragments

“…Emerging experimental approaches to further accelerate and improve the vascularization of these dermal substitutes involve their seeding with stem cells, keratinocytes, and adipose tissue–derived microvascular fragments (ad‐MVF) . Ad‐MVF represent fully functional vessel segments that are enzymatically isolated from fat samples . Hence, they rapidly reassemble into new microvascular networks within Integra ® and, thus, markedly improve the incorporation of the matrix at the defect site …”

The Crucial Role of Vascularization and Lymphangiogenesis in Skin Reconstruction

“…adipose tissue using a specific isolation protocol and can be immediately transplanted without any further in vitro processing [54]. After transfer into a tissue defect, ad-MVF only have to develop interconnections with each other and the surrounding recipient microvessels to develop into blood-perfused microvascular networks, which avoids complex and timeconsuming in vitro or in situ incubation steps [53,55].…”