Lyophilized mesenchymal stromal cell-derived extracellular vesicles for the oral treatment of inflammatory bowel disease: a novel cell-free therapeutic strategy

Abstract

Background

Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. Current standard treatments, including aminosalicylates, corticosteroids, immunomodulators, and biologic therapies, can induce and maintain remission; however, a considerable proportion of patients show limited responsiveness or experience adverse effects. This highlights the urgent need for safer and more effective therapeutic alternatives. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) represent a promising cell-free therapeutic modality owing to their immunomodulatory and reparative capabilities. These vesicles replicate many of the therapeutic benefits of mesenchymal stromal cells (MSCs) while avoiding the risks associated with conventional cell-based treatments. Nevertheless, clinical translation of MSC-EVs for IBD management remains constrained by issues such as limited storage stability and invasive delivery routes.

Methods

Extracellular vesicles (EVs) were isolated from the culture supernatant of human umbilical cord-derived MSCs via tangential flow filtration (TFF) followed by ultracentrifugation, and were characterized using transmission electron microscopy, western blotting, and nanoflow cytometry. To enhance their stability, MSC-EVs were lyophilized with trehalose and mannitol serving as cryoprotectants. The lyophilized powder was subsequently encapsulated in enteric-coated capsules for oral administration. The therapeutic efficacy of this formulation was evaluated in a dextran sulfate sodium (DSS)-induced rat colitis model. Disease activity was recorded daily, and post-experimental analyses included histopathology, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and serum biochemistry to evaluate hepatorenal function.

Results

A robust lyophilization protocol was successfully established to enhance MSC-EV stability, along with an innovative oral delivery system for IBD therapy. In DSS-induced colitis rats, lyophilized MSC-EVs markedly alleviated disease severity, as indicated by a reduced disease activity index (DAI), preserved colon length, and improved histological architecture. MSC-EV administration restored intestinal barrier integrity, modulated cytokine expression profiles, mitigated systemic inflammation, and improved hepatic and renal function.

Conclusions

This study demonstrated the therapeutic efficacy of a lyophilized, orally administered MSC-EV formulation in experimental colitis. This approach not only enhances vesicle stability and simplifies storage but also provides a non-invasive, patient-friendly route of administration. These findings underscore the strong translational potential of lyophilized MSC-EVs as a practical and effective cell-free therapeutic strategy for IBD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-026-05033-1.

Introduction

Inflammatory bowel disease (IBD) represents a group of chronic inflammatory disorders of the gastrointestinal tract, primarily Crohn’s disease (CD) and ulcerative colitis (UC). Its primary pathological features include chronic recurrent intestinal inflammation, mucosal damage, and impaired barrier function. IBD is a complicated multifactorial disease, and its pathogenesis involves genetic susceptibility, abnormal immune responses, intestinal microbiota dysbiosis, and environmental factors [1]. As early as the 19th century, IBD began to emerge in highly industrialized regions of Western Europe, North America, and Oceania. By the early 21st century, it had appeared in newly industrialized areas such as Asia, South America, and Africa. As the incidence of IBD in emerging countries and the number of cases in developed nations continue to increase, IBD poses a growing threat to global public health [2]. Contemporary IBD management strategies encompass both medical therapy and surgical options, which are selected based on disease severity and complications. The pharmacological management of IBD includes corticosteroids, aminosalicylates, small-molecule drugs, immunomodulators, and biologics [3]. Moreover, 5-aminosalicylic acid (5-ASA) compounds serve as first-line treatments for inducing and maintaining remission of mild-to-moderate UC [4]. Glucocorticoids can effectively induce remission of moderate-to-severe UC and CD; however, they are not suitable for long-term maintenance therapy due to safety concerns [5]. Thiopurines (such as azathioprine) are used to maintain remission, although their adverse reactions (such as bone marrow suppression and heightened susceptibility to infection) require close monitoring [6]. Treatment with biologics, such as infliximab, is efficacious; however, the single-target mechanism of action of these agents limits their clinical application, with approximately 50% of patients experiencing primary or secondary failure [7]. The application of emerging therapeutic strategies such as fecal microbiota transplantation (FMT) and probiotics is limited, and these techniques are recommended only on a case-by-case basis when conventional treatments fail [5]. Pharmacological treatments face challenges such as limited drug options, high dosage requirements, prolonged maintenance periods, severe side effects, and suboptimal efficacy or high rates of drug dependency. Moreover, surgical interventions may lead to complications such as anastomotic leakage and poor wound healing [8, 9]. For refractory IBD patients, existing treatment strategies often fail to achieve satisfactory outcomes, emphasizing that innovative treatment methods need to be developed.

Mesenchymal stromal cells (MSCs) have garnered significant attention as core research subjects in both basic research and clinical applications. Over 1000 clinical trials have been conducted globally, encompassing various pathological fields, including autoimmune diseases and inflammatory disorders [10, 11]. Certain MSC-based products have been approved for clinical applications [12–14]. MSCs are historically classified as stem cells based on their capacity for multipotent differentiation in vitro. These cells demonstrate adipogenic, osteogenic, and chondrogenic potential under specific culture conditions. However, their therapeutic mechanism does not stem from differentiation, but rather from their role as sentinels and regulators of tissue homeostasis, which modulates tissue repair through paracrine signaling [15, 16]. Moreover, MSC therapy in IBD patients has yielded poor results due to inconsistent cell supply, high costs, potential infusion-related toxicity, and cell rejection [17]. To overcome these challenges, mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising alternatives. These double-membrane-enclosed nanovesicles (30–150 nm in diameter) carry complex proteins, nucleic acids, and lipids, enabling them to convey their contents to recipient cells and exert anti-inflammatory and tissue-repair effects [18].

Studies on IBD animal models have revealed that EVs exhibit therapeutic effects similar to or even better than those of MSCs while avoiding the potential risks associated with MSC infusion. This finding offers new ways of performing cell-free therapy for managing IBD [19]. Research findings on the functionality, safety, and potential applications of EVs highlight their industrial value. However, their poor storage stability is a key factor limiting their widespread adoption. EVs are generally stored at − 80 °C; however, this method is not only inconvenient for handling and transportation but also may lead to degradation of the morphology, structure, and function of EVs [20, 21]. Therefore, a more stable and user-friendly storage method needs to be developed for advancing the biomedical applications of EVs. Lyophilization (or freeze-drying) is a suitable preservation technique for various biological materials. It simplifies storage and transportation by eliminating the dependence on expensive cold chain systems [22–24]. Moreover, research on the lyophilization of EVs and long-term stability data is scarce. EVs are generally administered through weekly intravenous infusions. While this approach is recommended for treating various diseases, its efficacy is limited by its invasiveness, operational inconvenience, and poor targeting ability. In contrast, oral administration offers several significant advantages, including patient self-administration and high treatment compliance [25]. Therefore, establishing an oral delivery strategy that prevents drug degradation due to gastric acid is extremely important for managing IBD.

In this study, we isolated high-purity MSC-EVs and developed a lyophilization protocol to increase their stability during prolonged storage. To apply this technique for treating IBD, we established an oral delivery strategy for MSC-EVs and investigated its therapeutic effects and mechanism of action in experimental IBD animal models.

Materials and methods

Separation and phenotypic characterization of MSCs

Clinical-grade MSCs were aseptically harvested from human umbilical cord tissue donated by healthy, rigorously screened donors who were seronegative for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), and syphilis. The entire process, from tissue procurement to cell isolation, was performed under Good Manufacturing Practice (GMP) conditions, and the Medical Ethics Committee approved the protocol. The steps involved separating Wharton’s jelly from the umbilical cord, homogenization, and centrifugation to extract primary cells. The master cell bank (MCB) was subsequently established in the second generation, and the postproduction cell bank (PPCB) was subsequently established in the fourth generation. The characteristics, safety, and functional activity of MSCs were evaluated and validated while a database was constructed. When needed, cryopreserved MSCs from PPCB were thawed and seeded at a density of 10,000 cells/cm² in a cell factory system (UCF011005, Jetbiofil, Guangzhou, China). The cells were cultured in 500 mL of a basal medium specifically formulated for MSCs supplemented with 5% UltraGRoTM-Advanced (GMP Grade, Helios, HPCFDCGL50) and maintained at 37 °C with 5% CO₂. Flow cytometry analysis was performed to characterize the MSCs by detecting surface markers (CD44, CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR). To validate their multilineage differentiation potential, MSCs were cultured in specific induction media and then subjected to histological staining. Following adipogenesis, the cells were stained with Oil Red O. Following osteogenesis, the cells were stained with Alizarin Red, and after chondrogenesis, the cells were individually stained with Alcian Blue and Safranin O. More details can be found in the Supporting Methods section of the Supplementary Information on the characterization of MSCs. Supplementary Table 1 lists the antibodies used in the experimental procedures.

Enrichment and identification of MSC-EVs

For the actual production of EVs, supernatants were collected from MSCs at passages 5 to 7. When the cell confluence reached 80–90%, the conditioned medium was harvested and preserved at − 80 °C for subsequent experiments. To harvest the MSC-EVs, the medium was gently thawed overnight at 4 °C and then pooled to form a total volume of 10 L. After mixing, the mixture was filtered through a membrane with a 5 μm pore size to eliminate large vesicles and particles. The filtrate was subjected to tangential flow filtration (TFF) via a hollow fiber membrane with a molecular weight cutoff (MWCO) of 300 kDa to obtain a 20-fold concentrated solution. To remove the cell debris from the mixture, the above mixture was centrifuged for 30 min at 3000 ×g and for 30 min at 16,500 ×g. Next, the clarified mixture was ultracentrifuged at 100,000 ×g for 70 min to precipitate the EVs. The above processes were conducted at 4 °C to avoid the degradation of EVs. Finally, the EV precipitate was dissolved in phosphate-buffered saline (PBS, Gibco, Grand Island, USA), adjusted to the corresponding concentration, and frozen at − 80 °C. The EVs were identified via transmission electron microscopy (TEM), high-sensitivity flow cytometry (NanoFCM), and western blotting (WB) analysis. TEM was used to examine the morphological characteristics of EVs, WB was performed to assess the expression levels of exosomal markers within the EVs, and NanoFCM was performed to quantify the particle concentration and determine the distribution of particle sizes in the samples. More details can be found in the Supporting Methods section of the Supplementary Information on the Western blot analysis.

Lyophilization and characterization of MSC-EVs

Trehalose and mannitol were used as cryoprotectants, and MSC-EVs were lyophilized following the protocol outlined in Table 1. Trehalose and mannitol were prepared as mixed solutions at specific concentrations in PBS and mixed with purified MSC-EVs. The final concentration of trehalose and mannitol was 6% (w/v), while the final concentration of MSC-EVs was 2.5 × 10¹⁰ particles/mL. Subsequently, 1 mL of the resulting MSC-EV suspension was transferred into 2 mL cryogenic vials and freeze-dried. We used a differential scanning calorimeter (DSC) to measure the sample onset freezing temperature, whereas a freeze-drying microscope was used to determine its collapse temperature. The lyophilized MSC-EVs (Lyo MSC-EVs) were identified via NanoFCM, TEM, and WB techniques. FITC-conjugated CD9 antibody (2.5 µL) was added to the 25 µL EV suspension, and the CD9 ratio of the vesicles before and after freeze-drying was detected via NanoFCM. To assess the effect of storage time, three batches of Lyo MSC-EVs were stored at 4 °C for 12 months, and the changes in particle size, quantity, and moisture content were evaluated. The moisture content of the lyophilized MSC-EV powder was measured via an electronic hygrometer.

Table 1.

The lyophilization procedure

Temperature (°C)	Vacuum (Pa)	Time (h)
-50	/1	2
-20	/	2
-50	/	2
-40	4	5
-20	4	10
2	4	2
36	4	10

¹“/” means atmospheric pressure

Functional evaluation of Lyo MSC-EVs

To evaluate the potential influence of the freeze-drying process on the function of MSC-EVs, this study conducted in vitro scratch experiments and immunomodulation experiments. For the scratch assay, human umbilical vein endothelial cells (HUVECs), obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and cultured in complete medium supplemented with 10% fetal bovine serum (FBS, C2830-0500, XP BioMed, Shanghai, China). When the cells reached 90% confluency, the monolayer was scratched with a pipette tip. Following two washes with PBS, serum-free basal medium containing either MSC-EVs or lyophilized MSC-EVs (1 × 10⁹ particles) was added, and the mixture was cultured at 37 °C for 18 h. Scratch images were captured at 0 and 18 h of cultivation and then analyzed via ImageJ. PBS and FBS were used as the negative control and positive control, respectively, for the experiment.

For the immunomodulation experiment, peripheral blood mononuclear cells (PBMCs, SLB-HP010B, Xuanfeng Biotechnology, Shanghai, China) were seeded into 6-well plates at a density of 1 × 10⁶ cells per well. PBMCs cultured alone served as the negative control group, while the remaining groups were supplemented with PBS, MSC-EVs (1 × 10⁹ particles) or lyophilized MSC-EVs (1 × 10⁹ particles). Additionally, 10 µg/mL phytohemagglutinin (PHA, L2769, Sigma, Darmstadt, Germany), a non-specific T lymphocyte activator, was added to each of these three groups. After 48 h of cocultivation, 25 ng/mL phorbol 12-myristate 13-acetate (PMA, 50601ES03, Yeasen, Shanghai, China), 1 µg/mL ionomycin (19657, Sigma, Darmstadt, Germany), and 10 µg/mL brefeldin A (BFA, 50502ES03, Yeasen, Shanghai, China) were added to the coculture system to stimulate the cells for 5 h. The PBMCs were subsequently harvested for cell staining. For the detection of Th1 and Th17 cells, the cells were first coincubated with a FITC-conjugated anti-human CD3 antibody (300405, BioLegend, San Diego, California, United States) and a PE-conjugated anti-human CD8 antibody (344706, BioLegend, San Diego, California, United States) at room temperature in the dark for 15 min. After being fixed and permeabilized according to the manufacturer’s instructions for the true-nuclear transcription factor buffer set (562574, BD Pharmingen, Franklin Lakes, New Jersey, United States), the samples were incubated with an APC-conjugated anti-human IFN-γ antibody (502512, BioLegend, San Diego, California, United States) and an APC-conjugated anti-human IL-17 A monoclonal antibody (17-7179-42, Thermo Fisher, Waltham, Massachusetts, United States) at 4 °C in the dark for 50 min. To analyze the percentage of Treg cells, the cells were initially costained with a FITC-conjugated anti-human CD25 antibody (302604, BioLegend, San Diego, California, United States) and a PE-conjugated anti-human CD4 antibody (300508, BioLegend, San Diego, California, United States). Following membrane permeabilization, staining was performed with an Alexa Fluor 647-conjugated anti-human FOXP3 antibody (320114, BioLegend, San Diego, California, United States). After staining, flow cytometry was used to analyze the quantity and characteristics of these immune cell subsets.

Preparation of MSC-EV enteric capsules

To protect Lyo MSC-EVs from destruction by gastric acid, they were encapsulated in enteric-coated capsules (YG00-2, Yuyan Instruments, Shanghai, China) according to the scheme shown in Fig. 5 Subsequently, sodium bicarbonate and citric acid were used as simulated cargos for filling the capsules. The stability of the enteric capsules was tested in acidic (pH 2) and weakly alkaline (pH 7.4) environments [26].

Fig. 5.

Preparation process and pH responsiveness of enteric-coated capsules. A Preparation process: The capsule body is placed on the mold, sleeved into the drug-loading device, the contents are loaded, the capsules are pressed into the capsule body, and finally, the capsule cap is fastened. B Disintegration tests within 20 min of capsules containing sodium bicarbonate and citric acid in solutions with pH 2.0 and pH 7.4, respectively

Biodistribution study of Lyo MSC-EVs

The study was conducted and reported in accordance with the ARRIVE guidelines 2.0 (Animal Research: Reporting of In Vivo Experiments). The experimental protocols involving animals were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Xiamen University. Male Sprague–Dawley (SD) rats, aged five weeks and weighing between 170 and 190 g, were sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd. All the animals were housed under specific pathogen-free (SPF) conditions, quarantined for one week under standard laboratory conditions (24 °C, room temperature; 45%–55% air humidity; and a 12:12 h light/dark cycle), and provided food and water ad libitum. Health and welfare were monitored daily by trained staff. Following a period of acclimatization to standard environmental conditions, the in vivo biodistribution of Lyo MSC-EVs in rats was examined by employing 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DiD) fluorescent labeling and an in vivo imaging system (IVIS, IVIS Spectrum, PerkinElmer, Massachusetts, USA) for tracking purposes. The DiD-labeled MSC-EVs (DiD-EVs) were produced according to the manufacturer’s guidelines, subsequently lyophilized, and encapsulated within enteric-coated capsules via the aforementioned procedures. The labeling efficacy was determined through NanoFCM analysis. The experimental cohort, comprising six rats, was administered a single enteric-coated capsule via oral gavage. The dosage administered was about 2 × 10¹⁰ particles per rat. The rats were humanely euthanized at 6 h and 24 h post-administration, after which the stomach, small intestine, colon, and major organs, including the heart, liver, spleen, lungs, and kidneys, were excised. The control group consisted of three rats, which were gavaged with exosome-free, lyophilized, enteric-coated capsules. Immediate imaging was conducted via the IVIS (exposure time = 1 s, f/stop = 2, binning = 8, excitation = 745 nm, emission = 800 nm, lamp level = high, FOV = 22.8 cm) to monitor the accumulation of DiD.

Development of a rat model of colitis and study design

In this study, male SD rats were used to establish a DSS-induced colitis model. This choice is consistent with common practices in preclinical IBD research, which aim to minimize the potential confounding effects of hormonal fluctuations associated with the female estrous cycle, thereby enhancing experimental reproducibility and reducing baseline variability [27]. Male SD rats (aged five weeks; weighing 170–190 g) were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. After the rats were acclimated to standard feeding conditions, experimental colitis induction was performed through the oral administration of dextran sulfate sodium (DSS; molecular weight 36,000–50,000 g/mol; Yisheng Biotechnology), following methods described in other studies [28, 29]. Next, 18 SD rats were randomized into three groups (n = 6 rats/group): a healthy control (Control), a DSS model (DSS + PBS), and a treatment group (DSS + EVs). The rats in the control group received regular drinking water ad libitum, whereas the other rats were administered 5% (w/v) DSS in their drinking water for 10 consecutive days to induce colitis.

To evaluate the efficacy of Lyo MSC-EVs, each rat in the treatment group received one enteric capsule orally every day from days 5 to 10. The dosage was determined based on the particle concentration measured via nanoflow cytometry. Each rat received a daily dose of 5 × 10⁹ EV particles for seven consecutive days. This dose was not adjusted according to individual body weight, and the same dose was administered to all animals in the treatment group throughout the treatment period. The rats in the DSS group and control group were also administered enteric capsules, but these capsules were prepared with PBS instead of MSC-EVs during the lyophilization process. The capsules for these two groups contained all the components of the formulation except for the EVs, meaning that only the lyoprotectant-excipient matrix was included. By establishing a control group, it was possible to assess the potential effects of capsule materials, the lyophilization process, and other non-EV components. The rats were deeply anesthetized via intraperitoneal injection of tribromoethyl alcohol (300 mg/kg) and euthanized via cervical dislocation under anesthesia, in accordance with the guidelines established by the International Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Evaluation of the severity of colitis

Throughout the experimental period, all animals underwent daily assessments, including body weight measurement and evaluation of disease activity parameters (stool consistency, fecal occult blood, and weight loss). The disease activity index (DAI), determined daily for each animal, was calculated as the sum of the scores for stool consistency, presence of bloody stool, and weight loss, divided by 3 (Table 2) [30]. A scale with a score ranging from 0 (lowest score) to 4 (highest score) was applied to the relevant parameters. The colon tissues of the rats in each group were fixed with formalin and stained with hematoxylin and eosin (H&E). Histopathological damage was evaluated in the DSS-induced colitis model to assess the degree of inflammation (Table 3) [31]. Immunohistochemical staining was performed to analyze the levels and distribution of tight junction proteins in colon samples. For immunostaining, colon samples were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4-µm slices. The following primary antibodies were used: ZO-1 (zonula occludens-1) and occludin. Photomicrographs were analyzed using a light microscope (magnification: 400 ×) and visualized using appropriate detection methods.

Table 2.

Standard DAI scores

Score	Decline in body weight (%)	Stool	Hematochezia
0	None	Normal	Negative of occult blood
1	1–5	Mild soft stool	Occult blood weakly positive (+)
2	5–10	Severe soft stool	Occult blood positive (++)
3	10–15	Mild diarrhea	Occult blood strongly positive (+++)
4	> 15	Severe diarrhea	Bloody naked eye

Table 3.

Standard HS scores

Feature	Score	Description
Damage	0	None
	1	Loss of the basal 1/3 of the crypt
	2	Loss of the basal 2/3 of the crypt
	3	Loss of the entire crypt but intact surface epithelial cells
	4	Loss of entire crypt and surface epithelial cells (erosion)
Extension	0	None
	1	Focal
	2	Lesions affecting l/3 of the intestine
	3	Lesions affecting 2/3 of the intestine
	4	Lesions affecting the whole intestine

RNA extraction and quantitative real-time PCR

Total RNA was extracted from colon tissues, followed by reverse transcription and quantitative real-time polymerase chain reaction (qPCR) analysis to quantitatively evaluate the transcriptional levels of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and the transcription factor complex nuclear factor kappa B subunit 1 (NF-κB1) in all groups. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels were used for normalization. The comparative Ct (2^–ΔΔCt) approach was used to determine relative mRNA transcription levels. The sequences of the primers used are shown in Supplementary Table 2.

Serum biochemical analysis

Following the experimental period, whole blood samples were collected from all groups via the abdominal aorta. Serum samples were obtained through 10 min of centrifugation (3000 ×g, 4 °C) before hepatic (alanine aminotransferase, ALT; alkaline phosphatase, ALP; aspartate aminotransferase, AST; albumin, ALB; total bile acids, TBA) and renal (urea; uric acid, UA; creatinine, CR) function parameters were analyzed.

Statistical analysis

All the data are presented as the means ± standard deviations (SDs). Before all the statistical comparisons, the normality of the data distribution was assessed using the Shapiro-Wilk test. The homogeneity of variance was evaluated using the Brown-Forsythe test or F test, as appropriate. For data that met both assumptions of normality and homogeneity of variance, parametric tests were applied. For data that violated either assumption, corresponding non-parametric tests were used instead. For comparisons across several groups, one-way ANOVA plus Tukey’s multiple comparison test was conducted. All differences were considered statistically significant at P < 0.05. GraphPad Prism 8.0 software (San Diego, CA, USA) was used for statistical analysis.

Results

Characterization of MSC-secreted EVs

In this study, EVs were isolated from the supernatant of human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) and characterized. As depicted in Fig. 1A, most UC-MSCs had fibroblast-like morphological characteristics, and a few were spindle-shaped or irregularly triangular. Under specific induction conditions, UC-MSCs can differentiate into adipocytes, chondrocytes, and osteocytes (Fig. 1B). UC-MSCs presented positive expression of standard mesenchymal markers (CD44, CD73, CD90, and CD105), but lacked hematopoietic and endothelial markers (CD11b, CD19, CD34, CD45, and HLA-DR). These results indicate that the UC-MSCs isolated and used for the downstream purification of EVs have the typical characteristics of MSCs. Next, the EVs were enriched by TFF and ultracentrifugation, with the number of EVs ranging between 5000 billion and 9000 billion per harvest. We determined the morphology, particle size distribution, and expression of marker proteins in EVs via detection methods established in previous studies. Most EVs obtained via our method were disc-shaped or cup-shaped (Fig. 1D). The results of the WB analysis confirmed that these particles specifically expressed hallmark proteins of small EVs, including CD9, CD81, and ALIX, with no detection of GM130 (Fig. 1E). The median diameter of the MSC-EVs was 76.80 nm (Fig. 1F). The median particle size was within the expected range for small EVs, further supporting their classification.

Fig. 1.

Identification of UC-MSCs and EVs released from UC-MSCs. A Cytomorphology of UC-MSCs (scale bar = 200 μm). B Multidirectional identification of UC-MSCs. The results of oil red O (scale bar = 200 μm), alizarin red (scale bar = 50 μm), safranin O (scale bar = 50 μm), and alcian blue (scale bar = 50 μm) staining are shown from left to right. C Immunophenotypes of UC-MSCs, including CD44, CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR, were identified via flow cytometry. D Representative TEM micrographs of MSC-EVs (bar = 500 nm). E WB analysis was conducted to measure the levels of CD9, CD81, ALIX, and GM130 in MSCs and MSC-EVs. The full-length blots are presented in Supplementary Fig. 1. F The size distribution of MSC-EVs was analyzed via nanoFCM

Establishing a freeze-drying method suitable for MSC-EVs

We developed a lyophilization method suitable for MSC-EVs, which contained 6% (w/v) trehalose and mannitol. First, the above mixture was frozen at − 35 °C to convert the water into ice. Next, the pressure was gradually reduced, and the temperature was carefully increased over three days to facilitate sublimation without causing structural collapse. Using this method, a dry product with an intact structure was obtained. Lyo MSC-EVs produced using our method presented a white cake-like appearance without shrinkage or collapse across three separate batches (Fig. 2A). The initial freezing temperature of the sample measured by DSC was − 19.99 ± 3.22 °C, and the collapse temperature measured via freeze-drying microscopy was − 37.93 ± 2.39 °C (Fig. 2B). We next conducted a characterization analysis of the Lyo MSC-EVs. TEM revealed that Lyo MSC-EVs maintained their classic central depression shape (Fig. 2C). The results of the WB analysis confirmed that Lyo MSC-EVs expressed hallmark proteins of small EVs, including CD9, CD81, and ALIX (Fig. 2D). Additionally, nanoFCM analysis revealed that neither the mean nor median particle size changed significantly before and after freeze-drying, with a single peak in the 40–200 nm range (Fig. 2E). The fluorescence mode of nanoFCM was used to analyze the EV subgroups before and after freeze-drying. The results revealed that the percentages of CD9-positive MSC-EVs were 58.6% and 56.3%, respectively (Fig. 2F). These findings indicate that our freeze-drying method had a negligible effect on the properties of the MSC-EVs.

Fig. 2.

Characterization of lyophilized MSC-EVs. A Images before lyophilization (Before Lyo), after lyophilization (Lyo MSC-EVs), and reconstituted lyophilizate. B The collapse temperature was measured via a freeze-drying microscope (scale bar = 100 μm). C-F Morphological characteristics (scale bar = 500 nm), protein markers, particle size, and CD9 positivity were analyzed before lyophilization (Lyo) and after lyophilization (Lyo MSC-EVs). The full-length blots are presented in Supplementary Fig. 2

Stability studies on Lyo MSC-EVs

In this study, we established a freeze-drying process for MSC-EVs and tested their long-term storage stability at 4 °C. The results revealed that the Lyo MSC-EVs maintained a moisture content of 6–9% after storage for 3, 6, 9, and 12 months at 4 °C (Fig. 3A). Additionally, at all time points, the particle concentration (Fig. 3B) and size distribution (Fig. 3C, D) of the Lyo MSC-EVs did not significantly change. These findings indicate that our freeze-drying method has a minimal effect on the properties of MSC-EVs. These results suggest that Lyo MSC-EVs can be stored for up to 12 months at 4 °C, making them suitable for transportation and storage.

Fig. 3.

The stability of Lyo MSC-EVs was evaluated at different time points (0, 3, 6, 9, and 12 months) at 4 °C. A Analysis of moisture content. B Analysis of particle concentration. C Analysis of median particle size. D Analysis of the mean particle size. “M” represents “Month”

Effect of freeze-drying on MSC-EV function

The scratch assay results revealed that the migration ability of HUVECs was significantly enhanced after the addition of Lyo MSC-EVs at 18 h (Fig. 4A). After 18 h of culture, the wound closure rate in the Lyo MSC-EV-treated group reached 76.1 ± 5.6%, whereas that in the non-lyophilized MSC-EV group was 75.3 ± 2.0%, with no statistically significant difference between the two groups (Fig. 4B). In this study, Lyo MSC-EVs were coincubated with PBMCs from healthy donors, and flow cytometry analysis was performed to determine the regulatory effect of Lyo MSC-EVs on populations of Th1, Th17 and Treg cells. The results showed that Lyo MSC-EVs inhibited the proliferation of Th1 and Th17 cell subsets, while promoting the proliferation of the Treg cell subset (Fig. 4C, D). Moreover, their activity was comparable to that of MSC-EVs, indicating that the lyophilization process does not cause functional impairment in MSC-EVs.

Fig. 4.

Functional evaluation of Lyo MSC-EVs in vitro. A Enhanced effect of MSC-EVs or Lyo MSC-EVs on the migration ability of HUVECs; images showing cell migration in HUVECs at 18 h (scale bar = 200 μm). B Quantification of the migration rate of HUVECs. C Regulatory effect of Lyo MSC-EV treatment on Th1 (CD3 + CD8- IFN-γ+)/Th17 (CD3 + CD8- IL-17 A+)/Treg (CD4 + CD25+ FOXP3+) cells in PBMCs. D Inhibition rate or promotion rate of Th1/Th17/Treg cells following MSC-EV or Lyo MSC-EV treatment. All the data are presented as the means ± SDs; n = 3 per time point and group. Statistical analysis via a t test revealed the following: * P < 0.05, ** P < 0.01, **** P < 0.0001 versus the negative control

Characterization of the Lyo MSC-EV enteric capsules

To shield Lyo MSC-EVs from damage caused by gastric acid during oral administration, we encapsulated them in enteric-coated capsules (Fig. 5A). As shown in Fig. 5B, the enteric capsules used in this study maintained good morphological and structural integrity when the pH was 2.0, similar to the acidity of gastric acid. However, when the pH was 7.4, similar to the acidity of the intestinal fluid, the capsules rapidly disintegrated and released a large amount of gas due to the reaction between sodium bicarbonate and citric acid.

Biological distribution of Lyo MSC-EVs in rats orally

NanoFCM analysis demonstrated that 27.5% of Lyo MSC-EVs were successfully labeled with DiD (Fig. 6A). The median diameter of the stained Lyo MSC-EVs was 74.20 nm, and the average particle size was 83.70 nm (Fig. 6B). The results revealed significant fluorescent signals in the gastrointestinal tract (GIT) 6 h after oral administration, which markedly decreased by 24 h. To further characterize the distribution of MSC-EVs across major organs, fluorescence imaging using IVIS revealed no detectable signals in the heart, liver, spleen, lungs, or kidneys (Fig. 6C-F).

Fig. 6.

Biodistribution in rats after oral administration of Lyo MSC-EVs. A The labeling efficiency of DiD-labeled Lyo MSC-EVs was evaluated using NanoFCM. B Size distribution of DiD-labeled Lyo MSC-EVs. C Distribution of DiD-labeled Lyo MSC-EVs in the stomach, small intestine, and colon of rats after oral administration. D Imaging of the heart, liver, spleen, lungs, and kidneys of rats after the oral administration of DiD-labeled Lyo MSC-EVs. E Statistical analysis of the relative fluorescence intensity in the gastrointestinal tract of rats after the oral administration of DiD-labeled Lyo MSC-EVs. F Statistical analysis of the relative fluorescence intensity in the livers of rats after the oral administration of DiD-labeled Lyo MSC-EVs. * P < 0.05 versus the PBS control

In vivo therapeutic effects

Lyo MSC-EVs alleviated DSS-induced colitis in rats

The therapeutic effect of MSC-EVs on IBD was investigated in a rat model of DSS-induced colitis. In DSS-induced colitis, the DAI score of rats treated with MSC-EVs (DSS + EV group) was significantly lower than that of rats treated with PBS (DSS + PBS group) on day 10 (Fig. 7A and B). Supplementary Table 3 summarizes the statistical parameters for the DAI scores. On day 11, the rats in the DSS + PBS group had a shorter colon length than did those in the DSS + EVs group. (Fig. 7C and D). The results revealed that after the model was established, many inflammatory cells invaded the mucosal layer of the rat colon; such cell infiltration was also observed in the submucosa of some tissues (Fig. 7F), accompanied by a significant increase in the histological score. However, these pathological changes improved considerably after treatment with MSC-EVs; compared with the DSS + PBS group, the DSS + EVs group had markedly lower histological scores (Fig. 7E). These findings suggest that MSC-EVs mitigate structural damage to colonic tissue, suppress the infiltration of inflammatory cells, and prevent crypt loss.

Fig. 7.

Lyo MSC-EVs reduce the severity of DSS-induced acute colitis. A A schematic illustration of the experimental methodology used to assess therapeutic efficacy. The rats were continuously administered 5.0% DSS for 10 days to induce colitis, were orally administered Lyo MSC-EVs or PBS on day 5, and were sacrificed on day 11. B DAI scores were recorded daily from day 0 to day 10. Macroscopic images of the colon C, colon length D, histological scores E, and H&E staining F of the control, DSS + PBS, and DSS + EV groups on the 11th day. The scale bar specification of the panoramic images is 500 μm, and that of the local images is 100 μm. Serum biochemical results of ALP G, AST H, ALB I, ALT J, CR K, TBA L, UA M, and urea N in the three groups. n = 6 in each group. H&E staining images from all 6 rats in each group are provided in Supplementary Fig. 3. *P < 0.05, **P < 0.01, and ***P < 0.001

After model establishment, the rats presented impaired liver function, characterized by significant increases in the serum AST, ALT, and ALP levels. Moreover, renal function was impaired, as indicated by high serum urea and CR levels. After treatment with MSC-EVs, the abovementioned liver and kidney function-related indices decreased, with a significant reduction in liver function indices (Fig. 7G-N). The descriptive statistics from the animal experiments are provided in Supplementary Table 4. Our findings indicate that MSC-EVs mitigate DSS-induced colonic injury and inflammatory responses, leading to secondary improvements in liver and kidney function. These results highlight the colon as the primary therapeutic target, with systemic organ protection likely resulting from the mitigation of intestinal inflammation.

Lyo MSC-EVs enhanced intestinal barrier function and reduced inflammation in DSS-induced colitis rats

The immunohistochemistry results revealed that the ZO-1 and occludin protein levels in the DSS + EV-treated rats notably increased relative to those in the DSS + PBS-treated rats (Fig. 8A). These findings indicated that the permeability of the intestinal barrier increased after modeling and subsequently improved after treatment. Compared to the control, the DSS + PBS group showed a substantial increase in the mRNA levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and NF-κB1, but a significant decrease in the anti-inflammatory cytokine IL-10, as determined by qPCR. Compared with those in the DSS + PBS group, the pro-inflammatory factor levels in the DSS + EV group were considerably lower, and there was a marked increase in IL-10 transcription (Fig. 8B-F).

Fig. 8.

Lyo MSC-EVs enhanced intestinal barrier function and reduced inflammation in DSS-induced colitis rats. A Immunohistochemistry results for ZO-1 and occludin in the control, DSS + PBS, and DSS + EV groups. Scale bar = 100 μm. The qPCR results show IL-1β B, IL-6 C, NF-κB1 D, TNF-α E, and IL-10 F mRNA transcription in the three groups; n = 6 in each group. The results are presented as the means ± SDs. *P < 0.05, **P < 0.01, and ***P < 0.001

Discussion

In this study, we isolated and characterized MSC-EVs and developed a lyophilization protocol to increase their stability for long-term storage. We also demonstrated the therapeutic potential of MSC-EVs in a DSS-induced rat model of colitis, highlighting their ability to mitigate intestinal inflammation, restore gut barrier integrity, and improve the systemic function of the liver and kidneys.

Maintaining stability during storage is a major challenge in EV-based therapeutics. Our lyophilization protocol, which involved incorporating 6% trehalose and mannitol as cryoprotectants, preserved the structural and functional integrity of the MSC-EVs. The cryoprotectants used in this study, trehalose and mannitol, are widely recognized as safe and biocompatible agents for pharmaceutical and biomedical applications. Both compounds are approved by regulatory agencies, such as the U.S. Food and Drug Administration (FDA), for use in both injectable and oral formulations. Trehalose, a naturally occurring disaccharide, has an excellent safety profile and is commonly used as a stabilizer in lyophilized biologic products due to its non-reducing and non-toxic properties [32]. Similarly, mannitol, a sugar alcohol, is extensively employed as an excipient in oral drug delivery because of its low reactivity and minimal toxicity [33]. Importantly, the selected concentrations of trehalose (58 mM) and mannitol (220 mM) in this study fall well within the established safety ranges for oral administration, as documented in previous preclinical and clinical studies [34, 35]. Therefore, their use in the present lyophilized EV formulation is not expected to introduce significant safety concerns. More importantly, the freeze-drying process did not significantly affect the size distribution, morphological characteristics, expression of surface markers, or certain primary functions of MSC-EVs. The Lyo MSC-EVs remained stable for 12 months at 4 °C, while the particle content or size distribution did not change significantly. The residual moisture content (6–9%) was within an acceptable range, further supporting the robustness of this method for long-term storage [36]. The high stability of lyophilized exosomes may be attributed to their dry environment, which minimizes interaction with the environment and reduces potential reactions and aggregation.

As EVs are susceptible to degradation in the harsh gastric environment, we encapsulated Lyo MSC-EVs in pH-sensitive enteric capsules. These capsules remained intact at gastric pH (2.0) but rapidly disintegrated under intestinal conditions (pH 7.4), ensuring targeted release in the intestine —a critical factor for treating IBD. This approach may increase the bioavailability of orally administered MSC-EVs. It has been reported that intravenously administered extracellular vesicles (EVs) are primarily cleared by the reticuloendothelial system and accumulate mainly in the liver, spleen, and lungs, with relatively less distribution in the kidneys. This “first-pass effect” significantly reduces their delivery efficiency to specific target sites [37–39]. In fact, research on the in vivo distribution and metabolic processes of orally administered extracellular vesicles, particularly lyophilized forms, remains extremely limited. In this study, we employed DiD fluorescence labeling, combined with an in vivo imaging system, to confirm the presence of specific and significant fluorescent signals in the gastrointestinal tract of rats following oral administration. These findings demonstrate that orally delivered Lyo MSC-EVs can directly act on the gastrointestinal tract. For our inflammatory bowel disease model, this local targeting effect may be crucial. In contrast, oral administration bypasses these major clearance organs, offering a safer and less invasive route of delivery. For chronic conditions requiring long-term treatment, such as inflammatory bowel disease, the convenience of oral administration is unmatched by intravenous injection, significantly increasing patient compliance and quality of life [22].

Our results indicated that the oral administration of MSC-EVs significantly ameliorated DSS-induced colitis. The reduction in DAI in the experimental animals indicated improvements in clinical symptoms and the stability of the colon length, indicating a decrease in inflammation-induced tissue damage. Histopathological improvements, including a decrease in inflammatory cell infiltration and alleviation of crypt damage, were observed. T cells play a central role in the pathogenesis of IBD, and their functional imbalance directly drives the development of intestinal inflammation. Overactivation of Th1 and Th17 cells leads to the secretion of pro-inflammatory mediators, thereby triggering inflammation, whereas a reduction in the number or functional impairment of Tregs results in a loss of immunosuppressive balance [40]. Studies have shown that mesenchymal stem cell-derived extracellular vesicles can ameliorate experimental colitis by modulating Th1, Th17, and Treg cell responses [41]. In this study, coculture of lyophilized MSC-EV powder with healthy human peripheral blood mononuclear cells suppressed the proliferation of Th1 and Th17 cell subsets and promoted the proliferation of Treg subsets, providing stronger support for related mechanistic research. Growing evidence suggests that these effects are attributed primarily to the cargo of bioactive molecules, such as anti-inflammatory microRNAs (e.g., miR-21-5p, miR-146a-5p, and let-7b), which can suppress pro-inflammatory signaling pathways, including the NF-κB and STAT3 pathways [42–46]. Although this study confirmed the partial immunomodulatory effects of freeze-dried MSC-EVs, future work will focus on elucidating the specific contributions of these individual components to the observed effects. Additionally, MSC-EVs can repair damaged intestinal epithelial cells (IECs), inhibit the apoptosis of IECs, maintain the balance of oxidative stress, reduce intestinal permeability, and attenuate colon fibrosis [47]. Our study further confirmed that MSC-EVs enhance intestinal barrier function by upregulating tight junction proteins, such as ZO-1 and occludin. Moreover, MSC-EVs can modulate inflammatory cytokines, decreasing the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and increasing the levels of an anti-inflammatory factor (IL-10). The study also revealed that MSC-EVs can alleviate DSS-induced liver and kidney dysfunction, as indicated by decreases in the serum levels of AST, ALT, ALP, urea, and CR. These findings suggest that MSC-EVs primarily modulate intestinal inflammation, with secondary systemic benefits that may involve immune regulatory mechanisms.

Compared with certain conventional drugs and cell-based therapies used for treating IBD, orally delivered lyophilized MSC-EVs may offer distinct advantages [3–7]. Specifically, as a cell-free product, it avoids risks associated with whole-cell therapies, such as infusion-related adverse events and potential immunogenicity [48]. Several studies have systematically evaluated the systemic and immunotoxicity of MSC-EVs in non-human primates and mice. The results demonstrated that even with high-dose single intravenous administration, the exosomes did not induce significant toxic reactions and exhibited good biocompatibility. These findings provide non-clinical safety data supporting the clinical translation of EVs [49, 50]. Preliminary safety assessments in our animal models indicate good tolerability without observed systemic toxicity; however, comprehensive toxicological and immunogenic evaluations in higher-order models are still warranted. While this route of delivery has the potential to reduce long-term treatment costs by simplifying administration logistics, formal health economic analyses are needed to substantiate this claim.

The innovation of this study lies in combining lyophilization technology with an oral delivery strategy using enteric-coated capsules, providing a practical translational research solution for cell-free IBD therapy. This study has several limitations, which are reflected in the following aspects. While the DSS-induced acute colitis model employed in this study provides a well-established and reproducible system for evaluating anti-inflammatory efficacy, it primarily reflects acute mucosal injury and inflammation. It does not fully recapitulate the chronic, relapsing-remitting immune dysregulation characteristic of human IBD. This limitation may affect the direct extrapolation of our results to long-term clinical outcomes. The promising therapeutic effects observed here, particularly barrier restoration and immunomodulation, warrant future validation in chronic or relapsing-remitting models to better assess the sustained efficacy and potential disease-modifying effects of Lyo MSC-EVs. Although the sample size used in this study (n = 6 per group) is consistent with many preliminary investigations in the field, it remains a limitation that may affect the statistical power to detect smaller effect sizes. Nonetheless, future studies with larger cohorts will be valuable for confirming these findings, enabling more robust subgroup analyses and better assessing dose-response relationships. Second, the depth of the mechanistic investigation was insufficient. While this study focused on phenotypic observations and partial mechanistic assessment, it did not comprehensively elucidate the key bioactive molecules (e.g., specific miRNAs) within the exosomes or their downstream signaling pathways. Notably, the MSC-EVs used in this study were derived from a human source, whereas their in vivo therapeutic efficacy was evaluated in a rat model of colitis. Although significant therapeutic effects were observed, potential species-specific differences in EV uptake, biodistribution, or biological activity cannot be entirely ruled out. Future studies utilizing species-matched models would help to further validate the translational relevance of these findings. Furthermore, the current colitis model provides supportive functional data, and dedicated GLP toxicology studies serve as the primary basis for our safety conclusions. These gaps highlight areas that require investigation to elucidate the effects of Lyo MSC-EVs in the treatment of IBD.

Conclusion

In this study, we developed a freeze-dried powder of Lyo MSC-EVs with excellent stability. Oral administration of Lyo MSC-EVs effectively alleviated DSS-induced experimental colitis in rats, significantly improving clinical symptoms, reducing pathological damage to colon tissue, inhibiting excessive inflammatory responses, and restoring compromised intestinal barrier function. Preliminary safety assessments revealed that Lyo MSC-EVs were well tolerated in vivo. Oral delivery of Lyo MSC-EVs represents a potentially convenient administration route that could improve patient compliance and reduce treatment costs, pending clinical validation. Owing to their ability to modulate inflammation and support intestinal barrier integrity, Lyo MSC-EVs show promising therapeutic potential.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

IBD

Inflammatory bowel disease

UC

Ulcerative colitis

CD

Crohn’s disease

MSCs

Mesenchymal stromal cells

MSC-EVs

Mesenchymal stromal cell-derived extracellular vesicles

EVs

Extracellular vesicles

TFF

Tangential flow filtration

TEM

Transmission electron microscopy

NanoFCM

High-sensitivity flow cytometry analysis

DSS

Dextran sulfate sodium

H&E

Hematoxylin and eosin

NF-κB1

Nuclear factor kappa B subunit 1

qPCR

Quantitative real-time polymerase chain reaction

DAI

Disease Activity Index

5-ASA

5-Aminosalicylates

FMT

Fecal microbiota transplantation

HIV

Human Immunodeficiency Virus

HBV

Hepatitis B Virus

HCV

Hepatitis C Virus

GMP

Good manufacturing practice

MCB

Master cell bank

PPCB

Post-production cell bank

MWCO

Molecular weight cut-off

PBS

Phosphate-buffered saline

WB

Western blotting

DSC

Differential scanning calorimeter

Lyo MSC-EVs

Lyophilized MSC-EVs

HUVECs

Human umbilical vein endothelial cells

FBS

Fetal bovine serum

PBMCs

Peripheral blood mononuclear cells

PHA

Phytohemagglutinin

PMA

Phorbol 12-myristate 13-acetate

BFA

Brefeldin A

Th1

T helper 1 cell

Th17

T helper 17 cell

Treg

T regulatory cell

ARRIVE

Animal research: reporting of in vivo experiments

SD

Sprague–dawley

SPF

Specific pathogen free

DID

1,1’-dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt

IVIS

In vivo imaging system

AAALAC

Assessment and Accreditation of Laboratory Animal Care

ZO-1

Zonula Occludens-1

TNF-α

Tumor necrosis factor-α

IL-1β

Interleukin-1β

IL-6

Interleukin-6

IL-10

Interleukin-10

GAPDH

Glyceraldehyde-3-Phosphate Dehydrogenase

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

AST

Aspartate aminotransferase

ALB

Albumin

TBA

Total bile acids

UA

Uric acid

CR

Creatinine

UC-MSCs

Human umbilical cord-derived mesenchymal stromal cells

GIT

Gastrointestinal tract

FDA

Food and Drug Administration

IECs

Intestinal epithelial cells

Author contributions

SY and TZ contributed to the conceptualization, methodology, and original draft writing. SC and YX contributed to the data analysis and review, editing, writing of this manuscript and investigation. WZ and QW contributed to the software, formal analysis and supervision. YT and WH contributed to the project administration and funding acquisition. All the authors have read and approved the final manuscript.

Funding

The present study was funded by the Ministry of Science and Technology of China [grant numbers 2021YFA1101500], the Natural Science Foundation of Fujian Province (2023J011616) and Investigator-Initiated Clinical Research Fund Project of The First Affiliated Hospital of Xiamen University (XMFHIIT-2023SL060).

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

All procedures involving human participants and animals were carried out in full compliance with established ethical standards. The protocol for the collection of human samples was reviewed and approved by the Medical Ethics Committee of Yantai Yuhuangding Hospital (Approval No. 2021003) on November 19, 2021. The approved study, titled “Collection of Human Umbilical Cord Tissue for the Implementation of Registered Stem Cell Clinical Research Projects,” included the acquisition of umbilical cord tissue from three healthy donors. Written informed consent was obtained from each donor or from legally authorized representatives before participation. The animal studies were reviewed and approved by the Animal Research Ethics Committee of Xiamen University (Approval No. XMYY-2025KYSB112) on March 3, 2025. The approved project, entitled “Mechanisms of the IOD–Intestinal Microbiota–AhR Axis in Regulating Tryptophan Metabolism in Inflammatory Bowel Disease,” was conducted in accordance with all applicable institutional and national guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.