The Optimal Processing Time of Adipose-Derived Mesenchymal Stem Cell after Lipoaspiration.

Background: Our aims were to characterize the phenotype of these cells, to investigate their immunoregulatory properties in vitro, and to determine the optimal processing time of AD-MSC after lipoaspiration.

Methods: ASCs were isolated from lipoaspirated adipose tissues by treatment of collagenase A and cultured in a Dulbecco’s Modified Eagle’s Medium (DMEM). To evaluate the optimal processing time of ASCs, the nucleated cells were processed by collagenase treatment at 3, 6, 9, 12, 15, 24, 30 and 36 hours after lipoaspiration respectively. To know the characteristics of ASCs, the expression of cell surface antigens was analyzed by flow cytometry, and the proliferation potentials were estimated by colony forming abilities or capacities of population doubling. The differentiation potentials into adipocytes or osteoblasts were confirmed by accumulation of neutral lipid vacuoles stained with Oil-red O and expression of alkaline phosphatases.

Results: When the nucleated cells were isolated by collagenase treatment after lipoaspiration, the mean cell yield was about 3.1 × 10⁶ or 1.2 × 10⁶cells per gram of lipoaspirate (n=8) processed. Nucleated cell counts were increased with time but plateau between 15h and 24h after initial processing and decreased after 24h. Flowcytometric analysis showed that Adipose tissue-derived stem cells (ASCs) have a marker expression that is similar to that of bone marrow stromal cells (BMSCs). ASCs expressed CD44, CD73, CD90, and CD105 and were absent for CD14, CD31 and CD45 expression. When primary cells were plated at 50 or 1000 cells/cm² on 6-well plates, cumulative population doublings were about 50 times until passage 7 or 13 (approximately 130 days), respectively, and ASCs expanded to 10¹⁸ cells. ASCs were multipotent, differentiating along the adipocyte and osteoblast lineages. ASCs did not provoke in vitro alloreactivity of incompatable lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogen.

Conclusion: The optimal processing time for AD-MSCs from lipoaspirates should before 24h after removal RBC and AD-MSCs can be a alternate source of multilineage MSCs for clinical use such as tissue repair and transplantation.