Density gradient centrifugation vs adherence for rat intestinal mesenchymal stem cell isolation

Figure 1 Rat intestinal tissue dissection. A: Whole-body image of an SD rat. Scale bar: 5 cm; B: Abdominal cavity opened in the supine position, with the intestine and surrounding structures exposed. Scale bar: 5 cm; C: Close-up view of the jejunum. Scale bar: 2 cm.

Figure 2 Morphological comparison of primary cells. A: Conventional adherence (group A): 48 hours, cells migrated out (a1); 7 days, island-like colonies with contaminants (a2); 10 days, target cells and contaminants coexisted (a3); B: Density gradient centrifugation (group B): 48 hours, rapid adhesion (b1); 7 days, homogeneous colonies (b2); 10 days, uniform fibroblast-like morphology (b3). Scale bars: 500 μm (a1, a2, b1, b2) and 200 μm (a3, b3).

Figure 3 Morphological comparison of passaged cells. A: Group A: Passage 3 (P3) cells exhibited a mixture of fusiform and polygonal cells (a1); P6 cells showed an increase in cytoplasmic granules (a2); P9, senescent cells with a flattened, widened “cicada-wing-like” appearance (a3); B: Group B: P3 cells are uniformly spindle-shaped and arranged in a swirl pattern (b1); P6, vigorous proliferation (b2); P9, dense and uniform morphology with good adhesion (b3). Scale bar: 500 μm.

Figure 4 Expression of positive markers. Representative histograms showing the expression of positive markers (CD90, CD73, CD44) in group A and group B. Data are presented as overlays with isotype controls.

Figure 5 Expression of negative markers. Representative histograms showing the expression of negative markers (CD34, CD45, CD11b/c) in group A and group B. Data are presented as overlays with isotype controls.

Figure 6 Proliferation curves of passage 4 cells isolated by the two methods. Cell proliferation was assessed by CCK-8 assay at days 0-6. Data are expressed as mean ± SEM (n = 5). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs group A.

Figure 7 Osteogenic differentiation of passage 4 cells isolated by the two methods. A: Control (non-induced), scale bar: 500 μm; B: Group A (conventional adherence), scale bar: 500 μm; C: Group B (density gradient centrifugation), scale bar: 500 μm; D: Quantitative analysis of Alizarin Red S-positive area. Data are expressed as mean ± SD (n = 3). ^cP < 0.001 vs group A.

Figure 8 Adipogenic differentiation of passage 4 cells isolated by the two methods. A: Control (non-induced), scale bar: 200 μm; B: Group A (conventional adherence), scale bar: 200 μm; C: Group B (density gradient centrifugation), scale bar: 200 μm; D: Quantitative analysis of Oil Red O-positive area. Data are expressed as mean ± SD (n = 3). ^aP < 0.05 vs group A.

Figure 9 Chondrogenic differentiation of passage 4 cells isolated by the two methods. A: Control (non-induced), scale bar: 500 μm; B: Group A (conventional adherence), scale bar: 500 μm; C: Group B (density gradient centrifugation), scale bar: 500 μm; D: Quantitative analysis of Alcian blue-positive area. Data are expressed as mean ± SD (n = 3). NS: Not significant.