Autologous fat grafting is an increasingly popular option for breast reconstruction following mastectomy as it has the ability to create a natural cosmetic outcome with minimal associated surgical risks. However, retention of fat grafts is highly variable and only 30-70% of the grafted tissue volume will be retained. Recently, there has been considerable focus on enrichment of adipose derived stem cells (ADSCs) in the grafted tissue to promote retention. A caveat of this approach is that very little is known about how ADSCs interact with resident cells of the breast cavity, such as macrophages and fibroblasts. The aim of this study is to examine extracellular vesicle (EV) release from ADSCs and their functional effects on stromal cells in vitro.
Following written informed consent, subcutaneous fat tissue was obtained from women undergoing autologous fat grafting at Wellington Hospital. ADSCs were isolated by enzymatic digestion and cultured up to passage five. Cultured cells were confirmed to be ADSCs by positive expression of CD105, CD10, and CD166 and negative expression of CD14 and CD31 by flow cytometry. ADSCs were cultured at a density of 1x107 cells in T75 flasks with media containing EV-depleted serum replaced every 3 days. ADSC-EVs were isolated from culture media using size exclusion columns on an automated fraction collector (qEV, Izon) and measured by tunable resistive pulse sensing (NP200, qNano, Izon). At baseline, ADSCs released a mean concentration of 5.07x108 EVs/mL media ranging in diameter from 80-600 nm with a mean diameter of 166 nm (n=5). Expression of transmembrane (CD9) and cytosolic (Alix) EV markers was confirmed by Western blotting. Preliminary data on co-culture of ADSC-EVs with macrophages derived from healthy donors suggests that EVs increase mRNA expression of the pro-inflammatory markers TNFα (12 fold) and IL-1β (83 fold) and the anti-inflammatory marker IL-10 (7 fold) in cultured macrophages (n=3 healthy donors, n=1 ADSC donor).
This study has established the isolation and culture of human ADSCs in our laboratory and will continue to investigate the relationship between ADSC-EVs and stromal cells of the breast cavity. In the future, this research could lead to improvements in fat graft retention.