Name: ADSC-sheet Transplantation to Prevent Stricture after Extended Esophageal Endoscopic Submucosal Dissection (Video) | JoVE Journal
Uploaded: 2017-02-10T13:00:00.000+00:00
Duration: 5 min 57 s
Description: Learn ADSC-sheet transplantation to prevent esophageal stricture after extended endoscopic submucosal dissection in a porcine model. This technique demonstrates improved healing outcomes and reduced fibrosis.

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5 minutes

This study was supported and funded by the Avenir Foundation (Fondation de l'Avenir, 255 rue de Vaugirard, 75719 Paris cedex 15, Paris, France). This study would never have been conducted without the precious help of the veterinary team of the Laboratory of Biosurgical Research from the Alain Carpentier Foundation.

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
<ol>
	<li>Obtain confirmed ADSCs from a private institution. Culture allogenic ADSCs at 37 &#176;C and 5% CO2 with alpha minimum essential medium including 10% fetal veal serum and 1% antibiotics (penicillin and streptomycin).</li>
	<li>Obtain around 24 x 106 ADSCs per animal using standard cell expansion procedures (37 &#176;C and 5% CO2). Harvest the ADSCs using a standard trypsin solution (10 mL for a T150 dish). Perform cell counting with a hemocytometer.</li>
	<li>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.
	<ol>
		<li>In order to label 2 x 106 ADSCs, prepare Solution A (2 x 106 cells with 100 &#181;L of Diluent C) and Solution B (4 &#181;L of PKH67 with 1 mL of Diluent C).</li>
		<li>Incubate Solutions A and B for 2 min. Then, add 100 &#181;L of fetal veal serum for 1 min.</li>
		<li>Rinse the solution with 300 &#181;L of citrated RPMI (298.5 &#181;L of RPMI and 1.5 &#181;L 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.</li>
	</ol>
	</li>
</ol>
2. Double ADSC-sheet Construct
<ol>
	<li>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 &#176;C and 5% CO2).</li>
	<li>On the day of transplantation, detach the confluent cell sheet by incubating the dish at room temperature for 30 min.</li>
	<li>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.</li>
	<li>Grasp another ADSC sheet and gently deposit it on top of the other one to obtain a double-layer construct.</li>
	<li>Repeat these procedures to obtain as many ADSC-sheet constructs as needed (4 per animal).</li>
</ol>
3. Esophageal Endoscopic Submucosal Dissection (ESD)
<ol>
	<li>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.</li>
	<li>Once the animal is under general anesthesia, perform ESD with a gastroscope, a videoscope, and an electrosurgery unit.
	<ol>
		<li>Use an endoscopic knife and soft coagulation to obtain hemi-circumferential dorsal marks ranging from 40 cm to 45 cm from the dental arch.</li>
		<li>Inject a glycerol solution containing indigo carmine dye into the submucosal layer for the separation of the mucosal layer from the muscular layer.</li>
		<li>Use an endoscopic knife with endocut I mode to obtain circumferential incisions.</li>
		<li>Use an endoscopic knife with forced coagulation mode to perform the submucosal dissection, moving from the proximal incision to the distal incision.</li>
	</ol>
	</li>
	<li>At the end of the ESD procedure, observe the hemi-circumferential, 5 cm-long esophageal scar exposing the muscular layer.</li>
</ol>
4. Endoscopic Transplantation
<ol>
	<li>Immediately after the ESD, endoscopically transplant animals with 4 double ADSC-sheet constructs.
	<ol>
		<li>Grasp an ADSC-sheet construct with endoscopic forceps and protect it during transportation to the wound site by using a large, transparent endoscopic cap.</li>
		<li>Apply the entire surface of the ADSC-sheet construct to the ulcer bed.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
5. Postoperative Evaluation and Follow-up
<ol>
	<li>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.</li>
	<li>Follow up with animals for 28 days. Obtain daily clinical evaluations using Mellow Pinkas dysphagia scores16 and weight variation measurements.</li>
	<li>Multimodal stricture evaluation: under general anesthesia, on scheduled days 3, 14, and 28, perform a multimodal evaluation of stricture occurrence.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Animal sacrifice.
	<ol>
		<li>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.</li>
		<li>After a sternotomy and organ exposure, remove the esophagus from each sacrificed animal (with 10% buffered formalin fixation, paraffin embedding, and 4 &#181;m-thick sections). Stain the slides with Hematoxylin and Saffron15. Digitalize the slides for computerized analysis and compare the animal groups15.</li>
	</ol>
	</li>
</ol>

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</ol>

The authors have nothing to disclose.

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 ...

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 results1,2,3. 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 shape4,5,6. 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 &#190; 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 effects8,9. 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 ESD10,11. 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 lineages12,13,14.
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.

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

adsc-sheet transplantation to prevent stricture after extended esophageal endoscopic submucosal dissection

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

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.

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...

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.

In past years, the cell-sheet construct has spurred wide interest in regenerative medicine, especially for reconstructive surgery procedures. The ...

In clinical practice, esophageal stricture occurrence is a major limitation for extended endoscopic resection of esophageal superficial neoplasia. This video presents an ADSC-sheet transplantation technique that prevents such strictures, making extended endoscopic resection possible in a porcine model. This method can advance a key question in the field of esophageal stricture prevention after endoscopic extended resection.The main advantage of this technique is that endoscopic ADSC sheet transplantation is meaning-lative, effective, and not time consuming in order to prevent such esophageal stricture. Demonstrating this procedure will be Gabriel Rahmi, a physician from my laboratory, and myself. Collect around 24 million ADSCs per pig using standard cell expansion procedures and T150 culture flasks.Harvest the ADSCs using 10 milliliters of standard trypsin solution per flask. Then, count the cells using an automatic cytometer. Load 1.5 million cells into each well of a 12-well temperature responsive cell culture dish.Now, top up each well to four milliliters with culture medium. Then, incubate the plates for 12 hours overnight for the transplantation on the following day. On the day of the transplantation, detach the confluent cell sheets in the plate wells by incubating them at room temperature for 30 minutes.After half an hour, gently pipette one ADSC sheet and layer it on a 1.5 centimeter diameter hydrophobic paper within a plastic dish. Use this paper as a support membrane. Be patient and take caution when handling the ADSC sheets.They are very fragile. Next, grasp an ADSC sheet and gently layer it over another to make a double layer construct. Make four double layer constructs per animal.Under general anesthesia, perform an ESD on the pig. Use a video gastroscope and an electrosurgery unit. First, using an endoscopic knife with soft coagulation, create hemicircumferencial white marks 40 to 45 centimeter dorsal from the dental arch to create a virtual lesion.Then, separate the mucosal layer from the muscular layer by injecting glycerol solution containing indigo carmine dye into the submucosal layer. Now, make a circumferential incision of the mucosal layer around the dorsal marks, using an endoscopic knife with forced coagulation mode. Next, perform the submucosal dissection from the proximal incision to the distal incision.Then, complete the resection by ESD, a hemicircumferential, five centimeter long esophageal scar exposing the muscular layer. Now, endoscopically transplant the four double ADSC sheet constructs. Grip the construct with endoscopic forceps and cover it with a large transparent endoscopic cap to protect it during transportation to the wound site.Then, spread the entire surface of the ADSC sheet construct onto the ulcer bed. Keep the graft in position for 10 minutes to ensure complete and stable engraftment. Continue to apply the ADSC sheets so the four cover about half of the wounded area.Transportation can be difficult in an animal under general anesthesia breathing and swallowing, so take great care to avoid damaging the sheets. After performing the described procedure on several animals, a PCLE evaluation was conducted three days later. The positive results confirmed the success of the transplantations.The follow-up period allowed for a multimodal analysis. Compared to control animals, the transplanted animal group showed less frequent alimentary trouble and greater weight gain on day 28. Endoscopic and radiological findings at the end of the follow-up period did uncover one animal from the transplanted group that did develop a severe esophageal stricture.Histological analysis showed that compared to the transplanted animals, the control animals developed significant fibrotic tissue. We show that an ESD followed by ADSC sheet transplantation can be performed in approximately one and a half hours. While attempting this procedure, it's important to remember that ADSC sheets are fragile and need to be manipulated with caution.The key of success is to take the necessary time for each step of the procedure. After exhibitment, this technique paves the way for research showing the field of cellular therapy To treat endoscopically other digestive disease.

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Research

JoVE Journal

Medicine

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Learn ADSC-sheet transplantation to prevent esophageal stricture after extended endoscopic submucosal dissection in a porcine model. This technique demonstrates improved healing outcomes and reduced fibrosis.

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

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