0.1 Grams of Sub-Cutaneous Adipose Tissue (SCAT) as Source of Adult Multipotent Mesenchymal Stromal Cells (MSC) for Cell-Based Therapies.

The purification of multipotent mesenchymal stromal cell (MSC) from sub-cutaneous adipose tissue (SCAT) represents a promising approach for several clinical applications. The isolation procedures have been applied in humans only to fairly large amount (5–300 g) of harvested SCAT (Zuk PA et al., 2001; Van Harmelen V et al., 2004; Meyerrose TE et al., 2007). Thus, we tested whether it might be possible to isolate MSC from far smaller tissue volumes. SCAT specimens from healthy donors which underwent to plastic surgery operations (n=5; mean weight ± SD=116.3±91.9 mg) were processed with a protocol modified from Zuk et al. 2001. The released cells were filtered, centrifuged and seeded with expansion medium. Solely small, undifferentiated and adherent proliferating fibroblasts were soon evident in the cultures and cells reached the first confluence within 12.6±5.1 days. After 40 days of expansion, the SCAT-MSC displayed a robust proliferative potential, with a cell count of 1.32±0.53x10⁸. FACS analyses showed that SCAT-MSC were positive (>95%) for CD90, CD105, CD73 and negative (<1%) for CD45, CD14, HLA-DR, CD31 and 3G5. CD34 antigen was expressed (<50%) in 2 samples. In vitro differentiation assays successfully drove the cells towards osteogenic, adipogenic and chondrogenic phenotypes. Long-term cryopreservation (2 years) did not affect both phenotype and functional properties. Additionally, SCAT-MSC were gene-modified (57±12%) by retroviruses encoding for GFP and subcutaneously injected into NOD/SCID mice without evidence of uncontrolled proliferation. In 20% of these mice, the transplantation of GFP+ cells was followed by the finding of human derived SCAT three months after the trasplants. We here report a novel and safe approach to isolate multipotent cells from small SCAT specimens to be used as platform for cell based therapies with minimal donors’ discomfort.