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 20.9 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.
- Citation: Lai SY, Jiang Y, Yuan R, Wang Z, Wang YC, Zhao ZT, Cai CY, Ruan MY, Zeng YX, Ren B, Wang XF. Density gradient centrifugation vs adherence for rat intestinal mesenchymal stem cell isolation. World J Stem Cells 2026; 18(9): 124197
- URL: https://www.wjgnet.com/1948-0210/full/v18/i9/124197.htm
- DOI: https://dx.doi.org/10.4252/wjsc.124197
The ability of mesenchymal stem cells (MSCs) to differentiate into three cell lineages has been validated in various tissue sources and has become a major focus of global research[1,2]. As a source of MSCs, the intestine is unique because its lamina propria is rich in mesenchymal components, and the local hypoxic environment shapes a distinctive immu
The adherent culture method relies on the preferential adhesion properties of MSCs; preliminary target cell enrichment can be achieved by directly seeding the enzymatic digestion suspension of intestinal cells[5]. This strategy is derived from experience with bone marrow MSC isolation. The cell composition resulting from the enzymatic digestion of intestinal tissue is complex, including mesenchymal, epithelial, endothelial, and hematopoietic cells, among others; some fibroblast-like cells also possess the ability to adhere. In actual culture, passages 0-2 (P0-2) often contain a mixture of spindle-shaped and non-spindle-shaped cells; flow cytometry analysis shows that the proportion of CD34+ or CD45+positive populations fluctuates significantly[6,7], and there are marked inter-batch variations when the same protocol is repeated.
Percoll density gradient centrifugation relies on differences in cell sedimentation coefficients to achieve stratification and has previously been widely used for the isolation of peripheral blood mononuclear cells and bone marrow MSCs[8,9]. When the medium density is set to 1.073 g/cm3, most red blood cell fragments and density gradient centrifugation hematopoietic cells can be removed, facilitating the extraction of mesenchymal components[10]. The parameters for this method are relatively well-established for the isolation of bone marrow MSCs; the density distribution characteristics of enzymatic digestion products from intestinal tissue remain unclear, and factors such as intestinal epithelial cell clumps and mucus components may interfere with the stratification process. In the existing literature, the CD90+positive rate of MSCs derived from porcine ileum can reach over 85% after Percoll purification[11,12], while reports on rat intestinal tissue have focused on collagenase-mediated differential adhesion or immunomagnetic bead sorting; a systematic evaluation of the Percoll protocol is still lacking[13,14].
This study used a controlled approach to compare the efficacy of adherent culture and Percoll density gradient centrifugation in isolating rat jejunal MSCs[15,16]. Cells were expanded to P1-3, and then compared across multiple dimen
Within the existing literature, there have been sporadic reports of both Percoll density gradient centrifugation and adherent culture being used for the isolation of intestinal MSCs; nevertheless, there is a lack of comparisons between the two methods, particularly in rats - a commonly used model for intestinal regeneration research. On a technical level, the Percoll gradient is not a novel approach; the focus of this study is to systematically apply this method to rat jejunal tissue to establish optimal enzymatic digestion conditions, centrifugation parameters, along with criteria for interface recovery. The established protocol enables P0-generation cells to achieve high purity, reducing the risk of phenotypic drift during subsequent passaging and, in part, mitigating the batch-to-batch variability commonly observed with the adherent culture method. The reagent costs for this strategy are comparable to those of conventional adherent culture, and it requires no specialized equipment, rendering it feasible for most laboratories. Currently, despite using the same tissue source, different research groups report surface marker positivity rates ranging up to 30% or higher due to variations in isolation procedures[17-19].
The animal experiment protocol was approved by the Animal Care and Use Committee of Guang’anmen Hospital, China Academy of Chinese Medical Sciences (Approval No. IACUC-GAMH-2025-060), and animal housing and procedures were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. Twelve male Sprague-Dawley rats, 2 weeks old and weighing 35-45 g, supplied by Shanghai SLAC Laboratory Animal Co., Ltd., SCXK Hu] 2022-0004, were used in the experiment. The animals were housed in a specific pathogen-free (SPF) environment at a temperature of 22-24 °C and a humidity of 50%-60%, with a 12-hour light/dark cycle, and had free access to food and water. The animals were anesthetized via intraperitoneal injection of 40 mg/kg sodium pentobarbital; upon completion of cell isolation, the experimental animals were euthanized by an overdose of the same drug at 120 mg/kg. Within the experimental design, the depth of anesthesia and postoperative temperature maintenance were key considerations for animal welfare.
In aseptic conditions, a segment of intestine was excised from a rat. The intestine was opened longitudinally and rinsed thoroughly with D-Hanks balanced salt solution (Beijing Solabio Co., Ltd., Beijing, China) to remove the intestinal contents. The mucosal layer was then carefully dissected using ophthalmic scissors to obtain tissue from the submucosal and muscular layers. Tissues were minced into 1-2 mm3 fragments and subjected to enzymatic digestion with 0.1% Type I collagenase (Biosharp, Hefei, Anhui Province, China) in DMEM medium (Gibco, Grand Island, NY, United States) at
Conventional adherence culture (group A). The cell pellet was resuspended in complete medium and seeded into T25 culture flasks at a density of 1 × 106 cells/mL. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Replace the cell culture medium after 48 hours of culture to remove non-adherent cell clusters; thereafter, exchange the medium every 2-3 days. As the cell confluence reaches 80%, digest the cells with 0.25% trypsin (Solarbio, Beijing, China) at 37 °C for 2 minutes to detach them, and add an equal volume of complete medium to terminate the digestion reaction. When cell confluence reaches 80%-90%, passage the cells at a 1:3 or 1:4 ratio, limiting trypsin digestion to no more than 2 minutes. The cells are continuously expanded to P9, using P4 cells for subsequent surface marker analysis and differentiation experiments.
Density gradient centrifugation (group B). Following collagenase digestion, the cell pellet was resuspended in D-Hanks solution and carefully layered on top of anequal-volume Percoll gradient (1.077 g/mL; GE Healthcare, Chicago, IL, United States). Centrifugation was carried out at 800 × g for 20 minutes without braking, using a Beckman Coulter Allegra X-30R centrifuge with an SX4750A rotor (brake setting: 0, acceleration setting: 5). Aspirate the white membrane layer using a Pasteur pipette, wash twice with D-Hanks, and transfer to culture (400 × g, 10 minutes each). Washed cells were resuspended in complete medium, then cultured under the same conditions as group A and passaged up to P9. Cells at the P4 were harvested for subsequent experiments.
At the time points P0, P3, P6, and P9, cell morphology was visually examined and photographed using an upright differential interference contrast microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany).
To identify the physical characteristics of the isolated cells, P4 cells first underwent collection, digestion with 0.25% trypsin, washing with D-Hanks balanced salt solution, and subsequent adjustment to a concentration of 1 × 106 cells/mL. According to product instructions, the cells were labeled with the following hematological markers: CD73, CD90, and CD44 (positive markers) and CD34, CD45, and CD11b/c (negative markers) (products supplied by Shanghai Saigen Biotechnology Co., Ltd.). Corresponding isotype controls were prepared. Following 30 minutes of incubation at 4 °C in the dark, the cells were washed twice with D-Hanks balanced salt solution and subsequently analyzed using a BD Accuri C6 flow cytometer (Becton, Dickinson & Company, Franklin Lake, NJ, United States) equipped with a 488 nm laser. Data were collected from more than 104 cells per sample, fitted with a 488 nm laser. Event data from more than 104 cells per sample were collected and analyzed using FlowJo software. All experiments were repeated three times. The flow cytometry screening strategy was as follows: First, dead cells were excluded by screening the forward scatter area (FSC-A) and side scatter area. First, debris was excluded based on FSC-A and SSC. Doublets were then excluded using FSC-H vs FSC-A gating to select single cells. Finally, live cells were identified as 7-AAD-negative cells. A minimum of 10000 viable, single-cell events were acquired for each sample. All gates were established using appropriate isotype control staining.
Cell viability was assessed using the CCK-8 assay. P4 cells were seeded at a density of 2 × 103 cells per well in a 96-well plate (200 μL per well) in DMEM medium containing 10% foetal bovine serum (FBS). Cell Counting Kit-8 (10 μL/well, Beyotime Biotechnology, Shanghai, China) was added at 0, 24, 48, 72, 96, and 120 hours, respectively, and incubated at
The multipotent differentiation potential of P4 cells was evaluated using the Cyagen Three-Lineage Differentiation Induction Kit, employing a non-induced control group for comparison (Cyagen, Guangzhou, Guangdong Province, China).
Osteogenic differentiation: The cells were seeded at 2 × 104 cells/cm2 in 6-well plates coated with 0.1% gelatin. When confluence reached 70%, the medium was replaced with osteogenic induction medium: High-glucose DMEM, 10% FBS, 100 nM dexamethasone, 10 mmol/L sodium β-glycerophosphate, and 50 μM ascorbic acid. The medium was refreshed every 3 days. After 3 weeks, calcium nodule formation was detected by Alizarin Red S staining (Solarbio, Beijing, China).
Adipogenic differentiation: Upon reaching 100% confluence, cells were cultured sequentially in Induction Medium A (DMEM/10% FBS/1 μM dexamethasone/0.5 mmol/L IBMX/10 μg/mL insulin/1 μM rosiglitazone) for 3 days, followed by Maintenance Medium B (DMEM/10% FBS/10 μg/mL insulin) for 1 day. This cycle was repeated three times, after which the cells were kept in Medium B until lipid droplets appeared. Lipid accumulation was visualized by Oil Red O staining (Solarbio, Beijing, China).
Chondrogenic differentiation: A total of 2.5 × 105 cells were pelleted by centrifugation at 250 × g for 4 minutes in 15 mL conical tubes to form micromasses. Culture medium: High-glucose DMEM/1% FBS/100 nM dexamethasone/50 μM ascorbic acid/1% insulin-transferrin-selenium/10 ng/mL transforming growth factor (TGF)-β3. Replace half the medium every 2 days. After 3 weeks, the micromasses were fixed and cartilage matrix production was evaluated by Alcian blue staining (Solarbio, Beijing, China).
Images were acquired using an inverted microscope (Leica DMi8, Leica Microsystems, Wetzlar, Germany) and an upright microscope (Olympus BX43, Tokyo, Japan). Staining-positive areas were quantified using ImageJ software version 1.53t (National Institutes of Health, Bethesda, MD, United States). For quantification, images were converted to 8-bit grayscale, and the threshold was set using the default Otsu algorithm to minimize inter-observer variability. Background subtraction was performed using the rolling ball method with a radius of 50 pixels. For each sample, five randomly selected fields of view were analyzed, and the percentage of positive staining area was calculated as (positive area/total field area) × 100%. All quantification was performed by two independent investigators blinded to the group assignment.
Flow cytometry and trilineage differentiation experiments were independently repeated 3 times, while cell proliferation assays (CCK-8) were repeated 5 times. Data are expressed as mean ± SEM. Comparisons between two groups were performed using independent samples t-test. For the CCK-8 time-course data, two-way analysis of variance (ANOVA) with repeated measures was performed to compare proliferation curves between groups, followed by Tukey’s post hoc test for multiple comparisons at each time point. A P-value < 0.05 was considered statistically significant. Statistical analyses were conducted using GraphPad Prism 9.0 software.
To obtain rat intestinal-derived MSCs, experimental animals were first subjected to anatomical dissection. Figure 1A shows a whole-body view of a representative SD rat, with glossy fur consistent with experimental requirements. Figure 1B illustrates the abdominal cavity exposed in the supine position; the skin and abdominal muscles were incised along the ventral midline to fully expose the abdominal cavity. The jejunum and ileum were located in the middle-lower abdomen, exhibiting a ruddy color and good blood supply. Figure 1C shows a close-up view of the jejunum. The intestinal wall appeared thin and the mucosal folds were clearly visible, providing useful anatomical landmarks for tissue collection.
After seeding of P0 primary cells, distinct growth patterns were observed between the two groups. As shown in Figure 2, at 48 hours post-culture, due to the presence of a few incompletely digested tissue fragments, cells in group A (conventional adherence) migrated radially from the edges of tissue fragments (a1 in Figure 2A), whereas cells in group B (density gradient centrifugation) rapidly adhered, forming typical “halos” with relatively homogeneous morphology (b1 in Figure 2B). At day 7, group A formed island-like colonies contaminated with heterogeneous cells (a2 in Figure 2A), while group B developed uniform colonies without obvious contamination (b2 in Figure 2B). At day 10, group A exhibited coexistence of target cells and contaminants, with partial detachment in some regions (a3 in Figure 2A), whereas group B showed highly uniform fibroblast-like morphology with orderly arrangement (b3 in Figure 2B). Overall, group B indicated rapid adhesion, high purity, and homogeneous morphology, whereas group A showed contamination resulting from cell migration out of tissue fragments.
To evaluate the efficiency of the density gradient separation procedure, cell recovery and viability were assessed immediately after Percoll centrifugation. The mean cell recovery rate was 86.4% ± 4.7% (n = 3) relative to the pre-centrifugation cell count, and Trypan blue exclusion assays revealed a cell viability of 96.2% ± 1.8% (n = 3). Taken together, these results suggest that density gradient centrifugation maintains high cell viability and yields a purer target cell population.
As observed under an inverted phase-contrast microscope, morphological differences were observed between cell groups A and B during passaging (Figure 3). At P3, group A consisted primarily of spindle-shaped cells, while also containing polygonal and flat, epithelium-like cells; group B consisted of uniform spindle-shaped cells arranged in a swirl pattern. At P6, group A exhibited a prolonged doubling time, increased cytoplasmic granules, and vacuoles in some cells; group B had well-defined boundaries, high refractive index, and maintained orderly monolayer growth even upon confluence. By P9, cells in group A had significantly enlarged, becoming flat and epithelium-like, with a “cicada wing”-like cytoplasm and a decreased nucleus-to-cytoplasm ratio; although occasional large cells were observed in group B, the majority remained slender and spindle-shaped, rapidly spreading upon attachment to the surface and exhibiting a stable passage cycle.
To characterize the phenotypic profiles of isolated cells, surface marker expression in P4 cells was analyzed by flow cytometry. Representative histograms of positive markers for group A (conventional adherence) and group B (density gradient centrifugation) are shown in Figure 4, and those of negative markers in Figure 5. For positive markers, group B cells showed significantly higher expression of CD90 (93.70% ± 0.20% vs 70.63% ± 1.15%, P < 0.001), CD73 (91.67% ± 0.21% vs 81.93% ± 0.06%, P < 0.001), and CD44 (90.70% ± 0.36% vs 60.50% ± 5.85%, P < 0.01) than group A. These results indicate that cells isolated using density gradient centrifugation exhibited higher proportions of CD90, CD73, and CD44positive cells. For negative markers, expression of CD34, CD45, and CD11b/c was below 5% in both groups. Notably, CD34 expression was significantly lower in group B (0.52% ± 0.35%) than in group A (3.24% ± 0.28%) (P < 0.01), whereas no differences were observed for CD45 or CD11b/c between the two groups (P > 0.05). Detailed data are presented in Table 1.
| Marker | Group A | Group B | P values |
| CD90+ | 70.63% ± 1.15% | 93.70% ± 0.20% | < 0.001 |
| CD73+ | 81.93% ± 0.06% | 91.67% ± 0.21% | < 0.001 |
| CD44+ | 60.50% ± 5.85% | 90.7% ± 0.36% | < 0.01 |
| CD34+ | 3.24% ± 0.28% | 0.52% ± 0.35% | < 0.01 |
| CD45+ | 3.54% ± 3.84% | 4.59% ± 3.40% | > 0.05 |
| CD11b/c+ | 1.02% ± 1.05% | 1.44% ± 1.17% | > 0.05 |
Cell proliferation capacity was assessed by CCK-8 assay in P4 cells from both groups over 6 days of culture (Figure 6). No difference in absorbance was observed between the two groups at days 1-2 (P > 0.05). From day 3 onward, group B (density gradient centrifugation) showed significantly higher absorbance values than group A (conventional adherence) (P < 0.05), indicating higher proliferative activity upon entry into the logarithmic growth phase. At day 6, the absorbance in group B was roughly 1.5 times that of group A, indicating better proliferative activity for cells obtained by density gradient centrifugation (Table 2).
| Culture time | Group A | Group B | P values |
| Day 0 | 0.105 ± 0.006 | 0.102 ± 0.005 | > 0.05 |
| Day 1 | 0.152 ± 0.008 | 0.148 ± 0.007 | > 0.05 |
| Day 2 | 0.213 ± 0.012 | 0.221 ± 0.010 | > 0.05 |
| Day 3 | 0.345 ± 0.018 | 0.412 ± 0.015 | < 0.05 |
| Day 4 | 0.521 ± 0.025 | 0.678 ± 0.022 | < 0.01 |
| Day 5 | 0.734 ± 0.032 | 0.998 ± 0.028 | < 0.01 |
| Day 6 | 0.892 ± 0.035 | 1.356 ± 0.031 | < 0.001 |
After 3 weeks of osteogenic induction, no obvious calcium nodule deposition was observed in the control group (Figure 7A). Group A (conventional adherence) showed positive Alizarin Red S staining with visible red calcium nodules (Figure 7B). Group B (density gradient centrifugation) exhibited a marked increase in both the number and area of calcium nodules (Figure 7C). Quantitative analysis revealed that the Alizarin Red S-positive area in group B (72.0% ± 5.0%) was significantly higher than that in group A (33.5% ± 3.2%) (P < 0.001), representing approximately 2.15-fold that of group A (Figure 7D).
Following adipogenic induction, no obvious lipid droplet deposition was observed in the control group (Figure 8A). Group A (conventional adherence) showed positive Oil Red O staining with visible red lipid droplets (Figure 8B). Group B (density gradient centrifugation) exhibited a modest increase in lipid droplet area compared with group A (Figure 8C). Quantitative analysis revealed that the Oil Red O-positive area in group B (46.2% ± 2.1%) was significantly higher than that in group A (36.6% ± 4.5%) (P < 0.05) (Figure 8D).
After 3 weeks of chondrogenic induction, no obvious cartilage matrix deposition was observed in the control group (Figure 9A). Group A (conventional adherence) showed positive Alcian blue staining with visible blue cartilage matrix (Figure 9B). Group B (density gradient centrifugation) exhibited cartilage matrix staining similar to that of group A (Figure 9C). Quantitative analysis revealed no difference in the Alcian blue-positive area between the two groups (74.6% ± 4.5% vs 73.5% ± 5.1%, P > 0.05) (Figure 9D).
Intestinal-derived MSCs hold great potential in China for intestinal regeneration and the treatment of inflammatory bowel disease (IBD). Previous studies across multiple species have explored efficient isolation methods for these cells[15,16]. While the traditional adherent culture method remains the most commonly used approach, the application of density gradient centrifugation as an alternative method in rat intestinal-derived MSCs has not yet been systematically evaluated[20]. The study indicated that both methods can isolate MSCs from the rat intestine, and that the morphology and proliferation rates of these two cell populations are similar during the early stages of expansion. The study found that cells isolated using the high-density separation method exhibited extremely high expression levels of positive markers (CD73, CD90, and CD44) and extremely low expression levels of negative markers (CD34, CD45, and CD11b/c). Both methods were able to induce cell differentiation into three cell lineages: Osteoblasts, adipocytes, and chondrocytes; but cells isolated by density gradient centrifugation exhibited stronger osteogenic and adipogenic differentiation capabilities, while the chondrogenic differentiation potential of the two cell populations was comparable.
To the best of our knowledge, this study represents the first systematic comparison of conventional adherent culture and density gradient centrifugation for isolating MSCs from the rat intestine. A comprehensive characterization of cell surface marker expression, cell surface marker expression and multipotent differentiation potential, and multipotent differentiation potential provides strong evidence that density-based sorting methods offer advantages in obtaining highly pure cell populations. Density gradient centrifugation improved the purity of intestinal MSCs, and the results were comparable to those obtained using this method with MSCs derived from bone marrow and adipose tissue[21,22]. The CD73, CD90, and CD44 phenotypes expressed by Percoll-treated cells met the International Society for Cell & Gene Therapy (ISCT) immunophenotypic criteria[13,23]. The levels of hematopoietic markers (CD34, CD45, CD11b/c) fell below the thresholds that would typically hinder functional analysis. Difference became apparent in the proliferation curve as early as day 2, followed by accelerated proliferation in group B during this delayed phase - a phenomenon similar to that observed in MSC preparations from other sources[24].
Although the specific molecular mechanisms underlying why MSCs isolated via density gradient centrifugation exhibit higher purity and greater differentiation potential have not yet been fully explained, our team has proposed several testable hypotheses. First, the Percoll density gradient centrifugation method may, through a physical separation process, enrich the CD73+/CD90+/CD44+ cell subpopulation while simultaneously removing hematopoietic cells (CD34+/CD45+) and cellular debris commonly co-captured during adherent culture; second, the removal of non-target cells may create a microenvironment conducive to the activation of the Wnt/β-catenin or TGF-β/Smad signaling pathways - both of which have been shown to regulate MSC self-renewal and osteogenic differentiation. Finally, reduced exposure to oxidative stress and inflammatory cytokines during the early stages of culture may be one of the reasons why cells isolated by density gradient separation exhibit delayed senescence. Future plans include identifying differentially expressed genes and signaling pathways that regulate such functional differences by comparing transcriptomic (RNA sequencing) and phosphoproteomic analyses of the two cell populations during early and late passages.
The purity of co-cultured MSCs isolated by density gradient centrifugation is improved because monocytes are preferentially enriched in different density layers, thereby reducing the failure to remove red blood cells, granulocytes, and cellular debris associated with adherent cell culture techniques. This preliminary purification process facilitates the subsequent isolation of a MSC population with high purity and minimal hematopoietic cell contamination. It can be hypothesized that the increased differentiation toward osteoblasts and adipocytes observed in cells isolated by density gradient centrifugation may be due to their higher mesenchymal potential and reduced exposure to immunosuppressive factors or growth factors present in the mixed cell populations generated under adherent culture conditions. The team initially hypothesized that increased cellular purity would improve differentiation simply by reducing contaminant interference. Homogeneous MSC populations may establish more efficient juxtacrine signaling, accelerating reciprocal differentiation cues.
Correctly applying this testing protocol to the rat intestine is no easy task. Young rats were chosen because they produce large numbers of mesenchymal cells; their intestinal walls, however, no longer reflect the complex structure of the basal layer of the adult colon mucosa. In preliminary experiments, we attempted to use tissue from adult rats (12 months old) and found that the digestion time had to be extended to 90 minutes. Based on our previous experience with human colon tissue preparation, the digestion time must be extended to 90 minutes or longer, the concentration of chemical reagents must be increased to 0.15%-0.2%, and mechanical mincing must be performed to increase the tissue’s surface area. Based on the literature on bone marrow MSCs, a density of 1.077 g/mL was used; however, the intestinal cell aggregates obtained after enzymatic digestion were not uniform. In two preliminary experiments, discontinuous density gradients (1.055, 1.065, 1.077, and 1.090 g/mL) were tested, the 1.065 g/mL density gradient contained a population of CD45+ cells, whereas in the 1.077 g/mL density gradient, the majority of CD73+/CD90+ cells were retained. To address this issue, we are currently negotiating to obtain surgically discarded tissue with segment anno
Setting up Percoll gradient centrifugation requires minimal equipment - a standard refrigerated centrifuge is
Unlike systemic immunosuppressants, gut-derived MSCs exert a regulatory effect in the treatment of IBD by simultaneously suppressing T cells, inducing regulatory T cells, and inducing M1/M2 macrophage polarization[10,26,27]. CD34+ residual cells were lower in the density gradient group, whereas CD45 and CD11b/c levels were comparable between groups, suggesting a reduction in hematopoietic cell contamination. This reduction in hematopoietic cell contamination may be associated with changes in inflammatory cytokines such as tumor necrosis factor-α and interleukin-6, which antagonize the immunosuppressive effects of MSCs[28,29]. These effects are cell-to-cell contact-dependent and paracrine-mediated, suggesting their potential to achieve local rather than systemic immunosuppression. However, the current evidence base is insufficient; studies using bone marrow- and adipose-derived MSCs dominate the literature, while fewer than ten published studies have focused on intestinal MSCs, despite their close association with the mucosa. This has prompted us to focus on isolation methods as a means of validating their function[30-35].
Studies have shown that using Percoll can achieve purity levels that meet ISCT standards while preserving differentiation potential; however, two issues fall outside the scope of our current data. First, it is unclear whether the ~45-minute Percoll separation step is scalable. Our experience is limited to conducting two to three runs simultaneously; at higher th
Despite these encouraging findings, it is important to acknowledge that the study has several limitations. Primary among these is that the molecular mechanisms underlying the enhanced cell purity and differentiation potential remain to be clarified; further investigation using transcriptomic or proteomic analyses will help identify key signaling pathways and molecular determinants. The team has proposed several testable mechanistic hypotheses in the aforementioned section and plans to conduct RNA sequencing and phosphoproteomics analyses in subsequent studies. Second, the in vivo regenerative capacity of these density-gradient-separated cells still needs to be validated in vivo using animal models of intestinal injury.
Though this study primarily focuses on in vitro analyses - a standard and necessary first step in establishing a novel isolation method - in vivo efficacy data are indispensable for translational applications. With limited time for manuscript revision, this study was unable to conduct in vivo experiments. However, our in vitro results (improved cell purity and enhanced proliferation and trilineage differentiation capabilities) clearly show that intestinal MSCs isolated by density gradient centrifugation warrant further in vivo investigation. Plans are underway to establish rat models of colitis and intestinal anastomosis to evaluate their mucosal repair efficacy.
Additionally, although this study used rats as the experimental subjects, the translation of this method to human intestinal MSCs requires further exploration; species differences and the direction of experimental adjustments for human tissue applications have been discussed earlier in this paper. The long-term effects of the isolation method on cellular senescence and genetic stability have not yet been evaluated using specific markers (such as SA-β-gal staining or p16INK4a expression); quantitative senescence assays will be added at the P3, P6, and P9 to validate the conclusion that density gradient centrifugation delays senescence. Immunomodulatory properties of intestinal MSCs obtained via different isolation methods warrant further evaluation through immunosuppression assays using co-culture. In addition, the differentiation capacity of cells beyond P4 was not examined in this study[36-38].
A key limitation of this study is that functional validation was focused on P4 cells; whether higher passage numbers maintain the ability for tripartite differentiation requires further verification. For example, a dextran sulfate sodium-induced colitis model would directly test the homing efficiency and local immunomodulatory effects of Percoll-purified MSCs, which represents a direction for future research. Based on the current data, Percoll gradient centrifugation enables rat intestinal MSCs to meet reproducible phenotypic criteria as early as P1-P4, reducing the early senescence and batch-to-batch variability commonly associated with the adherent culture method; its low barriers to entry in terms of equipment and reagents make it suitable as a benchmark protocol for initial screening in this field.
Percoll gradient centrifugation was used to isolate intestinal MSCs from rats. The resulting P4 cells met the ISCT phenotypic criteria, with CD90, CD73, CD44 positivity rates > 90% and CD34, CD45, and CD11b/c positivity rates were all < 5%. When compared to the concurrent adherent culture group, the proliferative capacity of group B cells was superior to that of group A from P4 cells; the area of calcium nodules increased following osteogenic induction; and Oil Red O staining was enhanced following adipogenic differentiation. Cell morphological stability was maintained through P9, with no signs of cell flattening or growth arrest. This may be due to the removal of hematopoietic and fibroblast-like cells during the initial screening step, rather than the culture conditions themselves. The reagent costs for this method are comparable to those of traditional adherent culture, and it does not require magnetic beads or flow cytometry equipment, while yielding cells of high purity. That said, further in vivo studies are still needed to confirm the therapeutic potential and clinical translatability of MSCs isolated by this method.
We thank the staff of the Animal Experimental Center of Guang’anmen Hospital, China Academy of Chinese Medical Sciences for their technical support. We are also grateful to Wu Jing for her assistance in flow cytometry analysis and cell culture.
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