ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection

Summary

This study reports a successful method of endoscopical adipose tissue-derived stromal cell (ADSC)-sheet transplantation for esophageal stricture prevention after an extended endoscopic submucosal dissection (ESD) in a swine model.

Abstract

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The development of diversified technologies combining adipose tissue-derived stromal cells (ADSCs) with various biomaterials has led to the construction of numerous types of tissue-engineered substitutes, such as bone, cartilage, and adipose tissues from rodent, porcine, or human ADSCs. Extended esophageal endoscopic submucosal dissection (ESD) is responsible for esophageal stricture formation. Stricture prevention remains challenging, with no efficient treatments available. Previous studies reported the effectiveness of mucosal cell-sheet transplantation in a canine model and in humans. ADSCs are attributed anti-inflammatory properties, local immune modulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages. This original study describes the endoscopic transplantation of an ADSC tissue-engineered construct to prevent esophageal stricture in a swine model. The ADSC construct was composed of two allogenic ADSC sheets layered upon each other on a paper support membrane. The ADSCs were labeled with the PKH67 fluorophore to allow probe-based confocal laser endomicroscopy (pCLE) monitoring. On the day of transplantation, a 5-cm and hemi-circumferential ESD known to induce esophageal stricture was performed. Animals were immediately endoscopically transplanted with 4 ADSC constructs. The complete adhesion of the ADSC constructs was obtained after 10 min of gentle application. Animals were sacrificed on day 28. All animals were successfully transplanted. Transplantation was confirmed on day 3 with a positive pCLE evaluation. Compared to transplanted animals, control animals developed severe strictures, with major fibrotic tissue development, more frequent alimentary trouble, and reduced weight gain. In our model, the transplantation of allogenic ADSCs, organized in double cell sheets, after extended ESD was successful and strongly associated with a lower esophageal stricture rate.

Introduction

The management of superficial esophageal tumors has changed with the development of new endoscopic techniques. Nowadays, endoscopic resection is the first-line treatment. Indeed, it is associated with lower morbidity and mortality rates than a surgery with equal oncological results^1,2,3. Endoscopic mucosal resection (EMR) and endoscopic submucosal resection (ESD) are the most widely-used techniques. In the case of an extended superficial tumor, ESD is preferred. Compared to EMR, ESD allows en bloc resectioning, regardless of lesion size and shape^4,5,6. The main delayed complication of ESD is esophageal stricture formation, which generally occurs between one and two weeks after resection. Recent published studies have shown that stricture formation is correlated to the size of the resection. The Japanese Endoscopic Society recommends avoiding ESD sizes larger than ¾ of the esophageal circumference because they are associated with stricture development in more than 90% of cases and are responsible for severe feeding troubles and major deterioration in quality of life.

The prevention of esophageal stricture remains challenging. Mechanisms involved in stricture formation are only partially known. Stricture formations seems to result from the association of two different mechanisms: (1) pro-inflammatory cellular recruitment and (2) excessive fibrosis development⁷. Several preventive treatments have been proposed. However, results were unsatisfactory, with little benefit and severe side effects^8,9. Recently, a Japanese team, Ohki et al., proposed to transplant a single-layer cell sheet of autologous oral mucosal cells into the esophageal scar. Transplantation was performed immediately after ESD^10,11. They demonstrated the effectiveness of this innovative approach, first in a canine model and then in patients.

Adipose tissue-derived stromal cells (ADSCs) are promising in regenerative medicine. Their application in several fields has shown interesting results, especially in the wound-healing process. ADSC therapy offers several advantages, because the cells are easily isolated and are associated with anti-inflammatory properties, local immunomodulating effects, neovascularization induction, and differentiation abilities into mesenchymal and non-mesenchymal lineages^12,13,14.

In a previous study, our team demonstrated the effectiveness of double ADSC-sheet endoscopic transplantation for esophageal stricture prevention after extended ESD in a swine model¹⁵. In this article, reports of ADSC-sheet construction and endoscopical transplantation technique are presented.

Protocol

All animals were treated according to the Animal Research Ethics Committee (guidelines of the French Ministry of Agriculture). The protocol received the approval of the local ethics committee authorized for animal experimentations at the Paris Descartes University (registered number MESR 2035.02; Faculty of Medicine Paris Descartes, Paris, France).

1. ADSC Culture and Labeling

1. Obtain confirmed ADSCs from a private institution. Culture allogenic ADSCs at 37 °C and 5% CO₂ with alpha minimum essential medium including 10% fetal veal serum and 1% antibiotics (penicillin and streptomycin).
2. Obtain around 24 x 10⁶ ADSCs per animal using standard cell expansion procedures (37 °C and 5% CO₂). Harvest the ADSCs using a standard trypsin solution (10 mL for a T150 dish). Perform cell counting with a hemocytometer.
3. Perform cell labeling the day before transplantation using the PKH67 staining procedure described below.
   NOTE: The goal of the staining procedure is to allow cell tracking after transplantation.
   1. In order to label 2 x 10⁶ ADSCs, prepare Solution A (2 x 10⁶ cells with 100 µL of Diluent C) and Solution B (4 µL of PKH67 with 1 mL of Diluent C).
   2. Incubate Solutions A and B for 2 min. Then, add 100 µL of fetal veal serum for 1 min.
   3. Rinse the solution with 300 µL of citrated RPMI (298.5 µL of RPMI and 1.5 µL of citrate) and centrifuge at 1,500 x g for 5 min. Repeat this operation 2 times. Maintain the labeled cells and protect them from light until sheet construction.

2. Double ADSC-sheet Construct

1. The day before transplantation, prepare a 12-well temperature-responsive cell culture dish with 4 mL per well of the cell culture medium, described above, before PKH-labeling. Seed the dish with 1.5 x 10⁶ PKH-labeled cells per well and incubate for 12 h (37 °C and 5% CO₂).
2. On the day of transplantation, detach the confluent cell sheet by incubating the dish at room temperature for 30 min.
3. Gently aspirate one ADSC sheet with a pipette and layer it on a hydrophobic paper (1.5-cm diameter). Use this paper as a support membrane.
4. Grasp another ADSC sheet and gently deposit it on top of the other one to obtain a double-layer construct.
5. Repeat these procedures to obtain as many ADSC-sheet constructs as needed (4 per animal).

3. Esophageal Endoscopic Submucosal Dissection (ESD)

1. On the day of transplantation, pre-medicate the animals with 10 mg/kg of intramuscular ketamine and induce them with 8 mg/kg of intravenous propofol. Then, perform endotracheal intubation and maintain the anesthesia with isoflurane 2.5% inhalation.
2. Once the animal is under general anesthesia, perform ESD with a gastroscope, a videoscope, and an electrosurgery unit.
   1. Use an endoscopic knife and soft coagulation to obtain hemi-circumferential dorsal marks ranging from 40 cm to 45 cm from the dental arch.
   2. Inject a glycerol solution containing indigo carmine dye into the submucosal layer for the separation of the mucosal layer from the muscular layer.
   3. Use an endoscopic knife with endocut I mode to obtain circumferential incisions.
   4. Use an endoscopic knife with forced coagulation mode to perform the submucosal dissection, moving from the proximal incision to the distal incision.
3. At the end of the ESD procedure, observe the hemi-circumferential, 5 cm-long esophageal scar exposing the muscular layer.

4. Endoscopic Transplantation

1. Immediately after the ESD, endoscopically transplant animals with 4 double ADSC-sheet constructs.
   1. Grasp an ADSC-sheet construct with endoscopic forceps and protect it during transportation to the wound site by using a large, transparent endoscopic cap.
   2. Apply the entire surface of the ADSC-sheet construct to the ulcer bed.
   3. Gently apply the ADSC-sheet construct for 10 min to obtain a complete and stable engraftment. For application, use endoscopic forceps or an endoscopic cap and apply the whole surface of the ADSC construct onto the esophageal submucosal layer.
2. Repeat this procedure four times for each animal in order to obtain four transplanted and adherent double cell-sheet constructs.
   NOTE: The goal is to cover approximatively half of the wounded area.

5. Postoperative Evaluation and Follow-up

1. For each animal, ensure post-ESD analgesia with the intramuscular injection of 0.2 mg/kg of morphine three times a day for the first day and with a 28-day anti-acid treatment of esomeprazole (40 mg/day). Perform antibiotic prophylaxis with amoxicillin (1 g/day) for 7 days. Authorize liquids on day 1 and solids on the following day.
2. Follow up with animals for 28 days. Obtain daily clinical evaluations using Mellow Pinkas dysphagia scores¹⁶ and weight variation measurements.
3. Multimodal stricture evaluation: under general anesthesia, on scheduled days 3, 14, and 28, perform a multimodal evaluation of stricture occurrence.
   1. Perform an endoscopic evaluation using scar description, stricture measurement, and paper support membrane detection. Measure the stricture using the gastroscope to cross through the stricture (the diameter of the gastroscope is 8 mm). Observe the scar, paying attention to the inflammatory aspect (mucosal erythema, mucosal edema, and mucosal spontaneous bleeding) and the absence or presence of the paper support membrane within the scar.
   2. Apply pCLE green probe through the gastroscope operator canal directly into the scar bed. Search for a spontaneous and organized green signal compatible with PKH67-labeled ADSC-sheet construct engraftment.
   3. Perform 2 orthogonal incidences of baryte esophagography with a radiological hoop (front and left-sided incidences). Keep the tightest incidence for stricture evaluation (degree of stricture (%) = [1-(length of the short axis under stenosis/length of the normal axis under stenosis) x 100)])^17,18.
4. Animal sacrifice.
   1. On day 28, at the end of the follow-up period, perform the animal sacrifice. While the animal is still under general anesthesia, inject 100 mg/kg of intravenous phenobarbital. Ensure animal death using clinical examination for the absence of a heartbeat and of breathing movement.
   2. After a sternotomy and organ exposure, remove the esophagus from each sacrificed animal (with 10% buffered formalin fixation, paraffin embedding, and 4 µm-thick sections). Stain the slides with Hematoxylin and Saffron¹⁵. Digitalize the slides for computerized analysis and compare the animal groups¹⁵.

Results

The culture of ADSCs and the procedure to obtain the ADSC sheet is shown in Figure 1. Figure 2 shows the construction of the graft, composed of two ADSC sheets layered upon each other on their paper support membrane. ADSCs were previously labeled with the PKH67 fluorophore to allow in vivo graft monitoring with pCLE. Figure 3 shows the different steps of extended esophageal endoscopic submucosal dissection, resulting in a 5-cm an...

Discussion

In this pig model, the ADSC-sheet transplantation was technically successful, and the in vivo pCLE evaluation allowed for cell engraftment monitoring. Clinical, endoscopical, radiological, and histological evaluations demonstrated the effectiveness of the endoscopic ADSC sheet at esophageal stricture prevention after extended ESD.

The endoscopic transplantation of an ADSC glycerol solution containing an indigo carmine dye sheet is an innovative approach in regenerative medicine. Ohki ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Transparent endoscopic cap Q180 compatible	Olympus Optical Co
GIF-Q180 gastroscope	Olympus Optical Co
Videoscope System Exera II	Olympus Optical Co
Injection needle 18 G	Olympus Optical Co
Electrosurgery unit ERBE ICC 350	ERBE Technology
Indigo carmin 1%	Life
Endoscopic hybrid knife	Life
Minisonde Z pCLE green probe	Mauna Kea Technology		You must learn how to use the probe. The manipulation could be difficult.
Fetal bovine serum	Sigma Aldrich	12105C
Trypsin	Sigma Aldrich	T146
Alpha minimum essential medium	Thermo Fisher	22561-021
Phosphate-Buffered Salines	Thermo Fisher	10010-023
PKH67 dye kit	Sigma Aldrich	Mini67-KT
12-well temperature responsive cell culture dish	Upcell Thermo Scientific	174900	Feel the weel with 4 mL standard medium culture 30 min before seeding cells
Esomeprazole 40 mg	Biogaran
Moprhine sulfate 50 mg/mL	Lavoisier
Amoxicilline 1 g	Biogaran
Ketamine 250 mg/5 mL	Panpharma
Propofol 10 mg/mL	Fresenius
Hydrophobic paper	Carrefour