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Wet milling of large quantities of human excision adipose tissue for the isolation of stromal vascular fraction cells.

ABSTRACT: The isolation of stromal vascular fraction (SVF) cells from excised human adipose tissue, for clinical or research purposes, implies the tedious and time consuming process of manual mincing prior to enzymatic digestion. Since no efficient alternative technique to this current standard procedure has been proposed so far, the aim of this study was to test a milling procedure, using two simple, inexpensive and commercially available manual meat grinders, to process large amounts of adipose tissue. The procedure was assessed on adipose tissue resections from seven human donors and compared to manual mincing with scalpels. The processed adipose tissues were digested and the resulting SVF cells compared in terms of number, clonogenicity and differentiation capacity. After 10 min of processing, either device tested yielded on average sixfold more processed material for subsequent cell isolation than manual mincing. The isolation yield of SVF cells (isolated cells per ml of adipose tissue), their viability, phenotype, clonogenicity and osteogenic/adipogenic differentiation capacity, tested by production of mineralized matrix and lipid vacuoles, respectively, were comparable. This new method is practical and inexpensive and represents an efficient alternative to the current standard for large scale adipose tissue resection processing. A device based on the milling principle could be embedded within a streamlined system for isolation and clinical use of SVF cells from adipose tissue excision.

SUBMITTER: Menzi N

PROVIDER: S-EPMC5851973 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0-150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.

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Wet milling of large quantities of human excision adipose tissue for the isolation of stromal vascular fraction cells.

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