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 shape⁴

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....

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