Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

Abstract

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not.

Figure 1. Purification of hADSCs from adipose tissue using the culture, membrane filtration, and membrane migration methods.

(a) Procedure for the purification of hADSCs. The primary adipose tissue cell solution (SVF) was prepared from fat tissue by collagenase digestion. The SVF solution was cultured on polystyrene (PS) or tissue culture polystyrene (TCPS) dishes to isolate hADSCs by the conventional culture method. In the membrane filtration method, SVF solution was permeated through the membranes and the permeation solution was collected. Subsequently, the membrane holder was inverted and the culture medium was permeated through the membranes where the recovery solution was collected. hADSCs were isolated in the permeation and/or recovery solution in the membrane filtration method. In the membrane migration method, the membranes were cultured on PS dishes in the culture medium after SVF solution and subsequently the culture medium was permeated through the membranes as described above. hADSCs migrated from the membranes on the PS dishes were collected in the migration method. (b) Morphology of nylon mesh of NY-11 mesh filter (i), NY-20 mesh filter (ii), NY-41 mesh filter (iii), NY-60 mesh filter (iv), and NY-80 mesh filter (v) used as membranes in membrane filtration and migration methods. The scale bars indicate 100 μm.

Figure 2. Effect of the pore sizes of the NY mesh filters on permeation, recovery, and residual rate of SVF solution in membrane filtration method.

SVF solution was permeated through NY mesh filters with different pore sizes (NY-11, NY-20, NY-41, NY-60, and NY-80 mesh filters). (i) The permeation rate of the cells where SVF solution was permeated through NY mesh filters. (ii) The recovery rate of the cells where SVF solution was permeated and subsequently the culture medium was permeated through NY mesh filters. (iii) The residual rates of the cells, which were calculated from eq. (3).

Figure 3. Cultivation of hADSCs isolated from the conventional culture and membrane migration methods.

(a) The morphology of the cells isolated by the culture method from SVF solution on PS dishes (i) and TCPS dishes (ii) and the morphology of the cells migrated from NY-11 (iii), NY-20 (iv), NY-41 (v), and NY-60 (vi) filters using the membrane migration method. (b) Growth curve of the cells isolated by the culture method from SVF solution on TCPS dishes (blue open circle) and the cells migrated from NY-11 (blue closed circle), NY-21 (green open square), NY-41 (green closed square), and NY-60 (red closed triangle) mesh filters using the membrane migration method. (c) Doubling time of the cells isolated from conventional culture method where SVF solution was cultured for 11 days on TCPS dishes (SVF on TCPS) and doubling time of the cells migrated from NY-11 (Cells from NY-11), NY-21 (Cells from NY-21), NY-41 (Cells from NY-41), and NY-60 (Cells from NY-60) mesh filters using the membrane migration method. * indicates statistical significance (p < 0.05).

Figure 4. CD34 and MSC surface marker expression of hADSCs isolated using the conventional culture, membrane filtration, and membrane migration methods.

(A) Flow cytometry analysis of the expression of CD34 (i) and MSC surface markers (CD44 [ii], CD73 [iii], and CD90 [iv]) on cells in SVF solution (SVF, the primary adipose tissue cells) (a), cells isolated by the culture method on TCPS dishes at first passage (SVF after culture on TCPS) (b), cells in permeation solution by the membrane filtration method through NY-20 mesh filters (Permeation solution through NY-20) (c), cells in recovery solution by the membrane filtration method through NY-20 mesh filters (Recovery solution through NY-20) (d), and cells that migrated out from NY-20 mesh filters that were subsequently cultured on PS dishes for 18 days after SVF solution and subsequently culture medium was permeated through the filters (Migrated cells through NY-20) (e). The dotted lines represent the cells stained with the isotype antibody as negative controls. (B) As analyzed by flow cytometry, the expression of CD34 (i), CD44 (ii), CD73 (iii) and CD90 (iv) on cells in SVF solution (SVF), cells isolated by the culture method on TCPS dishes at first passage, (SVF on TCPS), cells in permeation solution by the membrane filtration method through NY-11 (P via NY-11), NY-20 (P via NY-20), NY-41 (P via NY-41), NY-60 (P via NY-60), and NY-80 (P via NY-80) mesh filters, cells in recovery solution by the membrane filtration method through NY-11 (R via NY-11), NY-20 (R via NY-20), NY-41 (R via NY-41), NY-60 (R via NY-60), and NY-80 (R via NY-80) mesh filters, and cells that migrated out from NY-11 (M via NY-11), NY-20 (M via NY-20), NY-41 (M via NY-41), NY-60 (M via NY-60) mesh filters that were subsequently cultured on PS dishes.

Figure 5. Pluripotency of hADSCs isolated using the conventional culture, membrane filtration, and membrane migration methods.

(a–c) Relative gene expression levels of Oct3/4 (a), Sox2 (b), and Nanog (c) as analyzed by qRT-PCR in (i) cells in SVF solution (SVF), cells isolated by the culture method on TCPS dishes at first passage (SVF on TCPS), (ii) cells isolated by the culture method on Matrigel-coated dishes at first passage (SVF on Matrigel), (iii) cells in permeation solution by the membrane filtration method through NY-11 (P via NY-11), NY-20 (P via NY-20), and NY-41 (P via NY-41) mesh filters, and (iv) cells that migrated out from NY-11 (M via NY-11) and NY-20 (M via NY-20) mesh filters and were subsequently cultured on PS dishes as well as those of human ES cells (H9) and human iPS cells (HS0077) as positive controls. (d) The dependence of averaged pluripotent gene expression (Nanog, Sox2, and Oct3/4) of the cells on the cell culturing time where the cells were isolated by the conventional culture, membrane filtration, or membrane migration method. Cells in SVF solution were included for comparison.

Figure 6. Osteogenic differentiation of hADSCs isolated using the conventional culture, membrane filtration, and membrane migration methods.

(a) The ALP activity of the cells after osteogenic induction of cells in SVF solution (SVF), cells isolated by the culture method on TCPS dishes at first passage (SVF on TCPS), cells in permeation solution by the membrane filtration method through NY-20 mesh filters (P via NY-20), cells in recovery solution by the membrane filtration method through NY-21 mesh filters (R via NY-21), and cells that migrated out from NY-21 mesh filters and were subsequently cultured on PS dishes (M via NY-21) were cultured for 18 days. (b) Micrograph images of cells analyzed by Alizarin Red S staining (i–iv) and von Kossa staining (v–viii) after osteogenic differentiation of the cells that were isolated by the different methods. The cells were cultured for 28 days in osteogenic differentiation media. The scale bar represents 100 μm. (c) The level of osteogenic differentiation of cells analyzed by Alizarin Red S staining (calcium deposition) using Image J software after osteogenic differentiation for 28 days of the cells that were isolated by several methods. (d) The level of osteogenic differentiation of the cells analyzed by von Kossa staining (calcium phosphate deposition) using Image J software after culturing the cells that were isolated by several methods for 28 days in osteogenic differentiation media.

Figure 7. The relationship between osteogenic differentiation ability and MSC surface marker expression or pluripotent gene expression of hADSCs, which were purified from different isolation methods from SVF solution.

(a) The relationship between averaged MSC marker expression and averaged osteogenic differentiation rate of the cells, which were differentiated from (i) cells in SVF solution (SVF), (ii) cells isolated by membrane filtration method in permeation solution through NY-20 mesh filters (Cells in permeation), (iii) cells isolated by membrane filtration method in recovery solution through NY-20 mesh filters (Cells in recovery), (iv) migrated cells isolated by membrane migration from NY-20 mesh filters that were subsequently cultured on PS dishes for 18 days (Migrated cells), and (v) cells by culture method, which were cultured on TCPS dishes for 7 days (SVF on TCPS). (b) The relationship between averaged MSC marker expression and averaged pluripotent gene expression ratio of the cells, which were differentiated from (i) cells in SVF solution (SVF), (ii) cells isolated by membrane filtration method in permeation solution through NY-20 mesh filters (Cells in permeation), (iii) Cells by membrane migration from NY-20 mesh filters that were subsequently cultured on PS dishes for 18 days (Migrated cells), and (iv) cells by culturing on TCPS dishes for 7 days (SVF on TCPS).