Published online Sep 26, 2026. doi: 10.4252/wjsc.124197
Revised: July 24, 2026
Accepted: September 15, 2026
Published online: September 26, 2026
Processing time: 107 Days and 21.3 Hours
Intestinal mesenchymal stem cells (MSCs) hold potential for applications in intestinal regeneration; adherent culture remains the method of choice in majority of studies, but this approach suffers from insufficient purity and variability among samples, thereby limiting the reproducibility of experiments. Currently, there is no standardized protocol for isolating rat intestinal MSCs.
To compare the effectiveness of traditional adherent culture and density gradient centrifugation in isolating rat intestinal MSCs, and to determine which method yields superior cell purity, proliferative capacity, and differentiation potential.
Either the adherent culture method or the Percoll density gradient centrifugation method was used to isolate rat intestinal MSCs derived from the jejunum; the resulting cells were designated as group A and group B. These were subsequently compared in terms of changes in cell morphology during passaging, the expression of CD73, CD90, and CD44 (positive) and CD34, CD45, and CD11b/c (negative) as detected by flow cytometry, proliferation activities measured by the CCK-8 assay, and the outcomes of induced differentiation into osteoblasts, adipocytes, and chondrocytes.
The proportions of CD90+, CD73+, and CD44+positive cells in the MSCs obtained via density gradient centrifugation were 93.7%, 91.7%, and 90.7%, respectively, all of which were higher than those in the adherent culture group (70.6%, 81.9%, and 60.5%); the proportion of CD34-positive cells decreased from 3.2% to 0.5%. The proliferative activity of group B cells was superior to that of group A starting from day 3, and their osteogenic and adipogenic differentiation outcomes were also better; their morphological characteristics did not undergo changes after being passaged to the 9th generation. No statistical difference was observed between the two groups in terms of chondrogenic differentiation potential.
In the experimental conditions described here, the Percoll density gradient centrifugation method was used to isolate rat intestinal MSCs; a treatment took approximately 90 minutes, and the cost of reagents was comparable to that of conventional adherent culture. The obtained cell population maintained CD90 and CD73 positivity rates above 90% through passage 4, and both the in vitro expansion rate and the efficiency of osteogenic and adipogenic induction were better than in the corresponding adherent culture group. The limitations of this method include the need for a certain level of operator experience and the requirement to pre-optimize the Percoll concentration.
Core Tip: Intestinal mesenchymal stem cells (MSCs) hold therapeutic potential for intestinal diseases, but standardized isolation protocols for rat intestinal MSCs are lacking. This study systematically compares conventional adherence culture and Percoll density gradient centrifugation for isolating rat intestinal MSCs. Density gradient centrifugation yields cells with higher purity, enhanced proliferation, and superior osteogenic/adipogenic differentiation potential. This simple, cost-effective method addresses the limitations of conventional adherence culture and provides a standardized cell source for intestinal regenerative medicine research.