Isolation and Characterization of Mesenchymal Stem Cells from the Fat Layer on the Density Gradient Separated Bone Marrow

Abstract

The density gradient centrifugation method was originally designed for the isolation of mononuclear peripheral blood cells and rapidly adapted to fractionate bone marrow (BM) cells. This method involves the use of gradient density solutions with low viscosity and low osmotic pressure that allows erythrocytes and more mature cells gravitate to the bottom at a density fraction superior to 1.080 g/dL; mononuclear cells (MNCs) held in the plasma-solution to interphase at a density between 1.053 and 1.073 g/dL; plasma, dilution medium and anticoagulant to occupy a density less than 1.050 g/dL and the fat cells to float due to their very low density. BM-mesenchymal stem cells (MSCs) are usually obtained after the separation and cultures of BM-MNCs from the plasma-solution interphase, which is traditionally considered the only source of progenitor cells (hematopoietic and nonhematopoietic). In this study evidences that MSCs could be isolated from the very low-density cells of the fat layer are presented. In addition, we demonstrated that the MSCs obtained from these cells have similar immunophenotypic characteristics, and similar proliferative and differentiation potential to those obtained from the MNCs at plasma-solution interphase. The method represents a simple and cost effective way to increase the MSCs yield from each BM donor, without the need to look for other sources, additional manipulation of cells, and risks of contamination or disturbances of the potential of differentiation. These cells might serve as a complementary source of MSCs to facilitate preclinical and clinical application in tissue engineering and cell therapy.

Cytotoxicity and bioactivity of various pulpotomy materials on stem cells from human exfoliated primary teeth

“…The results suggest that the immunophenotypical and morphological profiles of SHEDs are the same as those of MSCs isolated from bone marrow (Insausti et al . , ).…”

The Impact of Cell Source, Culture Methodology, Culture Location, and Individual Donors on Gene Expression Profiles of Bone Marrow-Derived and Adipose-Derived Stromal Cells

“…Adding to the degree of heterogeneity is the anatomical location of harvest sites for these cells [21][22][23][24][25][26]. Since AT can be obtained with low-invasive procedures and donors tend to easily donate AT, this source of MSCs became popular in contrast to BM aspiration [27]. Notwithstanding the higher frequency of MSCs in AT tissue than in BM [28,29], the frequency of MSCs is still rather low in both tissues, and an expansion step is needed to obtain enough cells for experimentation, which further leads to increased heterogeneity [29][30][31].…”

Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

“…A density gradient of Ficoll ( Fig. S1D, Supplementary Information) allowed for the experimental determination of the density of the hMSCs, which is known to be lower than 1.073 g*ml -1 [53,54]. Both separation by gradient centrifugation and by buoyancy forces determined that the density of the population of hMSCs lied in between 1.034 and 1.052 g*ml -1 corresponding to 40 vol% and 60 vol% Ficoll in CM ( Fig.…”