Thermoresponsive gel embedding extracellular vesicles from adipose stromal cells improves the healing of colonic anastomoses following irradiation in rats

ADSC culture and characterization

Subcutaneous inguinal adipose tissue was removed from green-fluorescent-protein Sprague Dawley (SD) rats, finely minced, and enzymatically digested at 37 °C in minimum essential media (MEM) containing 0.1% collagenase type I (Sigma-Aldrich) for 30 min, 3 times. The digested tissue was filtered through a 100 µm filter. Collagenase was then neutralized with culture medium containing 10% fetal bovine serum (FBS). After centrifugation (1200 rpm for 3 min), cells were suspended in MEMα containing 20% FBS, penicillin– streptomycin and L-glutamin (all from Invitrogen) plated at 1000 cells by cm² and cultured at 37 °C in humidified 5% CO₂. The phenotype of amplified ADSC was verified by flow cytometry. The percentage of CD90 (clone OX-7; BD Biosciences) and CD73 (clone 5 F/B9; BD Biosciences) positive cells were analyzed, and the absence of hematopoietic cells was verified with CD34 (clone ICO115, Santa Cruz Biotechnology) and CD45 (clone OX-1; BD Biosciences) markers. Isotype identical antibodies served as controls.

EVs production

Microcarriers of 200 µm (Cytodex 1, GE Healthcare) were diluted in Phosphate Buffered Saline (PBS) and sterilized using an autoclave. PBS was then replaced by MEMα and stored at 4 °C. Before seeding the cells, microcarriers were incubated in 37 °C complete MEMα (Dulbecco) at a 19 g/L concentration during 3 h. Cells were seeded with a cell to microcarrier ratio of 20/1 and submitted to 24 cycles of 45 min of rest followed by 3 min of a gentle mixing at 60 revolutions per minute (RPM) in 1 L bioreactor. After cell adhesion, bioreactor was supplemented with complete MEMα (Dulbecco) and incubated at 37 °C and 60 RPM until confluence. Every 2 days 50% of the medium was changed. Once cells were confluent on microcarriers, turbulence triggered EV production was launched. Cells on microcarriers were washed 5 times with serum-free MEMα (Dulbecco). EV production was carried out as previously described³⁰. In brief, the spinner flask bioreactor containing a magnetized paddle was placed on a magnetic stirrer to obtain a smaller vortex diameter of 35 µm (Kolmogorov length) for 4 h to generate a shear stress allowing high yield production of EVs. After that, cellular debris of the supernatant were removed by centrifugation at 2000 × g for 10 min. The supernatant solution was then ultra-centrifuged at 150,000 × g for 1.5 h to obtain an EV pellet.

EV characterization

Suspensions containing EVs were analyzed using a NanoSight LM10-HS (NanoSight, UK) with a 405 nm laser or a NS300 (NanoSight, UK). A total of 10 videos of 30 s duration were recorded, and EVs Brownian movement was tracked by nanoparticle tracking analysis (NTA) software in a frame-to-frame basis. The two-dimensional Stokes−Einstein equation was used to compute particle size from the velocity of EVs movement. The optimization of NTA post-acquisition settings was performed and these parameters were kept constant for replicate analysis. EVs size distribution, concentration, mean, and mode were obtained.

Nano-flow cytometry

Nano-flow cytometry analysis was performed utilizing the Flow NanoAnalyzer (NanoFCM Co., LTD) in accordance with the manufacturer’s guidelines. To calibrate the system, a concentration standard (200 nm PS QC beads, NanoFCM) was employed for measuring particle concentration, while a cocktail of silica beads (S16M-Exo, NanoFCM) was utilized to construct a calibration curve, facilitating the conversion of side scatter intensities to particle size. The laser configuration involved a 488 nm and 640 nm laser at 20 mW, 10% ss decay, with lens filters set at 525/40 and 670/30. The antibodies for staining were Alexa Fluor® 647-conjugated monoclonal mouse antibodies directed against CD63 (clone MX49.129.5, sc-5275 af647, Santa Cruz Biotechnology) and/or anti-CD9 (clone C-4, sc-13118 af647, Santa Cruz Biotechnology) and/or anti-CD81 (clone 5A6, sc-23962 af647, Santa Cruz Biotechnology) antibodies or isotype control (sc-516609, Santa Cruz Biotechnology). The staining process was carried out overnight at 4 °C and isotype control antibodies were utilized at identical concentrations and incubation times. Labeled vesicles were then resuspended in 50 µL of PBS and diluted 1:200 times. Positivity was defined using buffer alone (PBS), unstained vesicles, isotype controls, and auto-thresholding. Prior to staining, samples were initially acquired to determine particle concentration and were subsequently diluted for optimal staining of EVs to a concentration of 5.10⁹ particles/mL. Fluorochrome-conjugated antibodies were prepared for use by centrifuging at 10,000 × g for 15 min or filtering through a 0.22 µm syringe filter, and were diluted 10 folds for incubation with EVs. Before measurement, the samples underwent an additional 100-fold dilution in PBS. Blank controls included PBS and antibodies diluted 1000-fold in PBS. To establish the threshold between positive events and background noise in the fluorescence channel, signals from both unstained Extracellular Vesicles (EVs) and EVs incubated with the isotype control were considered. Each measurement was conducted in triplicate to ensure accuracy. The acquired data were processed using the NF Profession 1.0 software.

TEM

EV preparations for TEM samples were submitted to negative staining protocol. In brief, 4 µL EVs either suspended in PBS or combined with 20% PF-127 were incubated on copper grids F/C (300 mesh) at room temperature for 5 min. The excess was removed with a filter paper then the samples were stained with 1% uranyl acetate for 15 s at room temperature. After removal of excess uranyl acetate, the samples were dried and analyzed using a Hitachi HT 7700 120 kV transmission electron microscope.

Pluronic ®F127 preparation

PF-127 (Sigma-Aldrich) was dissolved in sterile PBS at a 20% concentration and stirred at 4 °C for 24 h. After solubilization, it was filtered with a 0.2 µm filter at 4 °C. A tube reversal test was applied to assess the solubilization at 4 °C and the viscosity at 27 °C.

Preparation of Pluronic ®F127 containing EVs (gel + EVs) and EVs alone for local administration

PF-127 (Sigma-Aldrich) was dissolved in sterile PBS at a 25% concentration and stirred at 4 °C for 24 h. After solubilization, it was filtered with a 0.2 µm filter at 4 °C. A tube reversal test was applied at 4 °C and at 27°Cto assess the thermoresponsiveness. Then ADSC EVs suspended in PBS were added to the gel, whose final concentration was 20% PF-127 and 1.10¹¹ EVs/mL.

On the day of surgery, ADSC EVs were dissolved in sterile PBS at 4 °C. The final concentration was 1.10¹¹EVs/mL.

In vitro assessment of EV pro-angiogenic properties by HUVEC scratch test

Human Umbilical Vein Endothelial Cells (HUVEC) were cultured at 37 °C and 5% CO₂, using complete Endothelial Cell Growth Medium (EGM). Cells were cultured in 175 cm² flasks until confluence. Pro-angiogenic properties of EVs were tested using a scratch test. Monolayers of confluent HUVEC cells in 96-well plates were scratched using 96-Well WoundMaker (Essenbioscience) and the medium was replaced to discard detached cells. HUVEC scratched monolayers were incubated for 24 h in IncuCyte live cell imager (Essenbioscience) with serum-free EGM medium. We tested increasing doses of EVs (5.10⁹ to 1.10⁶ EVs/mL) on the one hand and increasing doses of gel-encapsulated EVs (1.10⁹ to 1.10⁶ EVs/mL) on the other hand. Complete medium and serum-free medium were used as positive and negative controls respectively in the EVs alone experiment. Complete medium and PF-127 alone were used as positive and negative controls respectively in the EVs + PF-127 experiment. The percentage of migration was assessed via the gap width between wound edges measured using IncuCyte software (Essenbioscience) at 6, 12, and 18 h time points.

In vitro assessment of EV immunomodulatory properties by MLR

Lymphocytes were isolated from the thymus of SD/CD and Wistar strain rats. Lymphocytes from both strains were mixed in culture medium at a 1:1 ratio in 96 U-bottom well plates (5.10⁵ per well in triplicate) and stimulated with PHA-M (Gibco®) at 1% and ConA (Sigma) at 5 µg/ml during 48 h. The cells were then treated for 24 h with rat ADSC EVs suspended in PBS at doses of 0 (negative control), 8.7.10⁹, 1.7.10¹⁰, 3.5.10¹⁰, or 7.0.10¹⁰ particles per well. ADSCs (0,5 million per well) were used as a positive control. Lymphocyte proliferation was quantified using the commercial assay Kit Roche Diagnostic ELISA BrdU (#11 647 229 001) according to the manufacturer’s instructions. In brief, the cells were incubated with BrdU (1/100). After washing, fixation, and denaturation steps, intercalated BrdU was revealed by anti-BrdU antiboby, then TMB substrate. After adding stop solution, the ELISA plate was read by spectrophotometer at 450 nm.

Experimental protocol overview

All experiments were performed at the animal facilities of the Institut de Radioprotection et de Sûreté Nucléaire (Fontenay-aux-Roses, France, registry n°C92-032-01) in strict compliance with European directives (86/609/CEE) and were approved by local ethical committee of the Institute of Radioprotection and Nuclear Safety in Fontenay-aux-Roses. The experimental protocol was submitted to the French national authorization platform and after approval was registered under the APAFiS permit number #14843-201804241155405 v2 P18-03. We have complied with all relevant ethical regulations for animal use.

Forty SD rats (7 weeks-old male rats of 250 to 300, Janvier-labs, France) were randomized into 4 groups of 10 rats. They were housed in a temperature-controlled room (21 ± 1 °C) and were exposed to a 12-h light/dark cycle and fed with standard pellets with food and water available ad libitum. After an acclimatization period of a week, each rat received a fractioned irradiation of 3 × 12.5 Gray (Gy) in 1 week. Surgery was performed 3 weeks later with a low colo-colonic anastomosis during which the treatment of interest was applied (control, PF-127 alone, EVs alone or PF-127 containing EVs). Eight weeks after the end of the irradiation, colo-colic anastomoses were evaluated by colonoscopy. Immediately after colonoscopy, the rats were sacrificed and an autopsy was performed. The colonic anastomosis was then extracted for histological analysis (Fig. 1). The physical condition of the rats was monitored on a daily basis. In case of reaching the ethical limit points defined in the protocol, the rats were euthanized under general anesthesia and an autopsy was performed.

Irradiation protocol

Rats submitted to radiotherapy were anesthetized by isoflurane inhalation, and a 12.5 Gy dose was delivered three times in 1 week through a 2×3 cm window centered on the colorectal region using a medical accelerator (Elekta synergy_4MVp X-rays with a mean photon energy of about 1.5 MeV; 30 kA).

Surgery protocol and treatment procedures

3 weeks after the colorectal irradiation, colonic surgery was performed under general anesthesia by isoflurane inhalation (induction 5%, maintenance 3% in ambient air at 0.4 L/min) and prior analgesia with buprenorphine 0.5 mg/kg. All surgeries were performed by a single experimenter. The rat was maintained in a dorsal decubitus position under sterile condition on a heated operation table. The abdomen of the rat was shaved just before surgery and disinfected with povidone-iodine. A 3 cm low median laparotomy was performed. The regions of interest were the distal colon and the rectum with the hip as the lower limit and the middle colonic artery as the upper limit. The cecum and the small bowel were protected and kept out of the surgical field. The abdominal wall was exposed with two monocryl 4/0 traction points (Ethicon, France). A 5 mm portion of colon in the center of the irradiated area, ~4 cm from the anus, was resected using dissecting scissors. After an initial anchor point was made with 6/0 PDS (Ethicon, France), the anti-mesocolic side was sutured with separate 6/0 PDS stitches every 2 mm. After rotating the colon clockwise to expose the mesocolic portion of the anastomosis, the treatment was administrated. Thus, rats in the control group were injected at the upper and lower poles of the anastomosis with 50 µL of PBS and received an enema with 100 µL of PBS at the upper and lower poles of the anastomosis. Rats in the PF-127 group were injected with 50 µL of PBS at the upper and lower poles of the anastomosis and received an enema with 100 µL of PF-127 covering both the upper and lower poles of the anastomosis. Rats in the EVs group were injected at the upper and lower poles of the anastomosis with 50 µL of EVs each for a total dose of 1.10¹⁰ EVs, and received an enema with 100 µL of EVs for a total dose of 1.10¹⁰ EVs covering both the upper and lower poles of the anastomosis. Rats in the PF-127 containing EVs group received an injection of 50 µL of EVs in PBS at the upper and lower poles of the anastomosis for a total dose of 1.10¹⁰ EVs, and an enema of 100 µL of EVs embedded in PF-127 for a total dose of 1.10¹⁰ EVs covering both the upper and lower poles of the anastomosis. The injections were performed with a 25 G needle and the application of the substance of interest by enema using a flexible feeding tube. Then, the mesocolic part of the anastomosis was sutured. The abdominal cavity was then washed with warm saline and closed with a 3/0 vicryl suture (Ethicon, France). The skin portion of the incision was closed with 6 to 7 surgical staples (5.7 × 3.9 mm).

Postoperatively, the rats were evaluated daily for physical and behavioral performance. A subcutaneous injection of 0.25 mg/kg buprenorphine was performed 4–6 h after surgery and the following morning. Upon awakening, the rats were provided with food and water ad libitum. The staples were removed 15 days after surgery during a short general anesthesia with 2% isoflurane.

Colonoscopy protocol

Eight weeks after colorectal irradiation, a colonoscopy was performed to evaluate the anastomosis healing. Under general anesthesia with 2% isoflurane, rats were placed in dorsal decubitus on a heated operation table. A COLOVIEW murine endoscopy system (Karl Storz, Germany) was used. The colon was dilated during the examination by continuous injection of saline through the operating channel using a 20 mL syringe. The anastomosis was located ~4 cm from the anal margin. The feces were washed out with saline injected through the working channel and by abdominal mobilization. The anastomosis was filmed and pictures were taken. The images were then read by an experienced gastro-enterologist blinded to the treatment received by the specimens. The presence of anastomotic ulceration and its characterization was scored according to its severity (1: no ulcer, 2: superficial ulcer, 3: deep ulcer, 4: deep circumferential ulcer)¹⁴. Bleeding or stenosis of the anastomosis was also reported. An endoscopic inflammation score from 1 to 6 (ENDOSCORE) was then calculated by adding the grade of the ulcer (1-4), the presence or absence of bleeding (0-1), the presence or absence of anastomotic stricture (0-1).

Autopsy protocol

Immediately after colonoscopy, the rats were killed by cardiac puncture under 2% isoflurane anesthesia, and an autopsy was performed. The occurrence of intra-abdominal leak, peritoneal adhesion, and the number of structures adherent to the anastomosis (seminal vesicles, bladder, small bowel) was investigated before performing a total colectomy removing the anastomosis. Peritoneal adhesions were assessed by the Zuhlke score, commonly used in preclinical studies⁵¹. The colonic specimen was then centered on the anastomosis, washed with saline, embedded in a cassette, and placed in 4% formaldehyde. The autopsy was performed by a digestive surgeon blinded to the treatment received by the rats.

Histological analysis and fibrosis quantification

After 24 h in 4% formaldehyde, the colon blocks were embedded in paraffin after preparation with Tissue-Tek VIP6 (Sekura, France). The embedded colon blocks were then cut with a rotary microtome (Leica, Germany) into 5 sets of 10 slides of 5 µm thickness. The series were spaced 150 µm apart. One slide from each series was stained with Hematoxylin-Eosin-Safran (HES), Masson’s Trichrome (TM) and Sirius Red (SR). The slides were then scanned at 20× magnification using a slide scanner Lamina (Perkin Elmer, USA). The slides were read by a single experimenter blinded to the treatment received by the rats using CaseViewer software (3DHISTECH, Hungary).

The mean size of the ulcerated area (epithelial denudation) were reported after reading the HES.

Using the FIBER-ML program developed from MATLAB by our team, we quantified collagen at the anastomotic level by machine-learning⁵². The SR stained slides were selected and centered on the anastomosis and exported at ×100 magnification in TIFF format. After manual reading of 5 slides and by delimiting the collagenous zones (red), matrix zones (yellow) and the background (gray), we launched the program to analyze all the selected slides. The collagen surface per slide was calculated. We then performed a quality control on 25% of the sample.

Immunohistochemistry

For PMN detection by immunohistochemistry, sections were dewaxed and then placed in antigen retrieval solution (0.01 M citrate buffer, pH = 6 (DakoCytomation, France) for 3 × 5 min at 350 W). Endogenous peroxidases were inhibited by incubation with 3% H₂O₂ in methanol at room temperature for 10 min. After saturation (X0909; DakoCytomation), 35-fold diluted rabbit anti-rat MPO (Abcam, France) was applied to the slide for 1 h at 37 °C. Staining was developed with Histo-Green substrate (E109; Abcys), and the slides were counterstained with Fast Nuclear Red (S1963; DakoCytomation, France), dehydrated and mounted. Isotype controls were used as negative controls. The number of neutrophils (MPO positive) at the anastomosis was reported for a defined area.

For CD68 immunostaining (macrophage analysis), tissue sections were treated with proteinase K (DakoCytomation) at room temperature and quenched for endogenous peroxidases described above. After saturation, 200-fold diluted mouse antirat CD68 (AbD Serotec) was applied to the section for 1 h at 37 °C. Staining was developed with Histo-Green substrate (E109; Abcys), and the slides were counterstained with Fast Nuclear Red (S1963; DakoCytomation, France), dehydrated and mounted. Isotype controls were used as negative controls. The number of macrophages (CD68 positive) at the anastomosis was reported for a defined area.

Statistics and reproducibility

Normal continuous variables were described by their means and standard deviations. Non-normal continuous variables were described by their medians and interquartile ranges (Q1–Q3). Categorical variables were described by their numbers and percentages. Fischer’s exact test was carried out for comparisons between categorical variables and the nonparametric Mann–Whitney test was used for non-paired continuous variables.

Comparison between more than two-groups of non-normal quantitative variables was performed using a non-parametric Kruskall-Wallis test, if a statistical difference was found, the samples were compared in pairs by a Dwass-Steel-Crtichlow-Fligner multiple weighted test. Comparison between more than two-groups of normal quantitative variables was performed using an ANOVA, if a statistical difference was found, the samples were compared in pairs by a Tukey multiple weighted test. All tests were two-tailed with a significance level of p < 0.05. All analyses were performed with R software version 3.6.0. or GraphPadPrism (GraphPad Software, La Jolla, USA).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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• Source: https://www.nature.com/articles/s42003-024-07364-2